key: cord- - qo krxv authors: wilcox, bruce a.; gubler, duane j. title: disease ecology and the global emergence of zoonotic pathogens date: - - journal: environ health prev med doi: . /bf sha: doc_id: cord_uid: qo krxv the incidence and frequency of epidemic transmission of zoonotic diseases, both known and newly recognized, has increased dramatically in the past years. it is thought that this dramatic disease emergence is primarily the result of the social, demographic, and environmental transformation that has occurred globally since world war ii. however, the causal linkages have not been elucidated. investigating emerging zoonotic pathogens as an ecological phenomenon can provide significant insights as to why some of these pathogens have jumped species and caused major epidemics in humans. a review of concepts and theory from biological ecology and of causal factors in disease emergence previously described suggests a general model of global zoonotic disease emergence. the model links demographic and societal factors to land use and land cover change whose associated ecological factors help explain disease emergence. the scale and magnitude of these changes are more significant than those associated with climate change, the effects of which are largely not yet understood. unfortunately, the complex character and non-linear behavior of the human-natural systems in which host-pathogen systems are embedded makes specific incidences of disease emergence or epidemics inherently difficult to predict. employing a complex systems analytical approach, however, may show how a few key ecological variables and system properties, including the adaptive capacity of institutions, explains the emergence of infectious diseases and how an integrated, multi-level approach to zoonotic disease control can reduce risk. the growing problem of globally emerging infectious diseases (eids) has prompted a substantial effort by the biomedical research establishment to identify the causes and recommend action. as reported in the most recent of a series of volumes ( ), a main finding is that the current episode of global infectious disease emergence is the result of a convergence of factors involving complex interactions among numerous variables. this includes genetic, biological, social, economic, political, ecological, and physical environmental factors, and calls for an interdisciplinary research agenda. it is also concluded that "human development and large scale social phenomena are closely associated to infectious disease threats at a global level," which points to the need for research focused on "social and ecological factors affecting infectious disease emergence" ( ) . the phenomenon of globally emerging infectious diseases requires understanding biological systems in the broadest sense and dealing with their extraordinary complexity. this includes processes operating at the level of transmission and evolution of a pathogen within and among host species and humans. it extends to and includes processes involving ecosystems and regional environmental change occurring on a global scale ( ) . in fact the scale and magnitude of anthropogenic activity has reached a point of virtual co-dominance with natural processes of energy and material flows globally ( ) . understanding these kinds of processes traditionally has been the domain of classical ecology, or natural history, plus systems ecology ( , ) . adding to this are recent ecological perspectives and models applied at the molecular, cellular level, and organismal levels ( ) , and others addressing the complexity, multiple variables, cross-scale influences, and dynamic behavior at the level of natural ecosystems ( ) , and social-ecological systems ( ) . along with the research at the organismal level and below, that aimed at the level of social-ecological (coupled humannatural systems) is critical to the development of the comprehensive scientific framework necessary for understanding zoonotic infectious disease emergence in particular. not only does this new area address the dynamic behavior of complex, large scale systems, but also bridges theory from the traditionally separate biological and social science disciplines, thus contributing to the interdisciplinary research agenda also called for in the above reports. the purpose of this paper is to consider how regional and global zoonotic disease emergence trends might be explained on the basis of current thought in biological ecology including the very recent developments new to the field of infectious disease ecology. here we draw on ecological science as broadly defined as a basis for identifying causal mechanisms of zoonotic disease emergence, the ultimate goal being to enhance disease prevention and control programs. several authors have categorized causal factors of infectious disease emergence, including explicitly citing 'ecological' ones involving land use change ( ) ( ) ( ) ( ) or 'land use drivers' ( ) , human movement ( , ) , encroachment and wildlife translocation ( , ) , rapid transport ( , ) and climate change ( , ) . most recently, the institute of medicine ( ) described these along with others ( categories of factors in all) and a model stating the major categories of factors have historically converged to bring about the current global emerging infectious disease crisis. ecological factors are described as one of four major categories of factors that have converged with social, political, and economic factors; genetic and biological factors; and physical environmental factors. we take a different approach to understanding the interaction of the above factors and their causal relationships by focusing on disease emergence as an ecological-evolutionary phenomenon influenced by human factors. our interest is in how human factors interact with natural processes and, in particular, how mechanisms operating at levels meaningful to understanding pathogen transmission and evolution can result in regional and ultimately global phenomena (i.e., regional endemism, epidemics, or global pandemics). as a first step we distinguish between the two broad categories of human factors, 'demographic and societal' and 'disease intervention and policy' suggested previously by gubler ( ) ( table ) . this categorization differentiates between factors associated with specific kinds of environments or ecosystems and those involving biological and policy factors not so associated. however, both can be described as part of a single ecological framework involving interaction of systems of essentially natural versus human design, respectively. our focus is on the first category from the standpoint of how disease emergence is explained by ecological concepts and principles. this includes some relatively new models and theory not previously used to explain the current trend in increasing emerging infectious diseases. we present a general model of zoonotic disease emergence on this basis. we also discuss recent explanations based on complexity theory for how human behavior and ecosystems interact to contribute to disease emergence. classical ecology, or natural history, has been the basis and mainstay of infectious disease research since its origins with 'koch's postulates' and subsequent development of microbiology and zoonotic disease epidemiology during the th and th centuries ( , ) . throughout much of its early history, zoonotic disease research involved this descriptive, empiricallybased ecology: identifying the life cycle, transmission, incidental and natural hosts of pathogens, along with demographic, life history, dispersal, and habitat attributes of reservoirs and vectors. a substantial theoretical dimension has developed, beginning with ross's mathematical analysis of malaria transmission ( ) and extending to anderson and may's ( ) recent synthesis infectious diseases of humans. although essential to designing effective prevention and control programs, empirical field based, disease ecology has been neglected in recent years ( ) . fortunately, theoretical disease ecology, stimulated largely by the work of anderson, may and others has flourished and led to a significant syntheses involving application of ecological-evolutionary biology to the study of infectious diseases ( , ) . parallel to this, systems ecology has begun to extend its domain by applying complexity theory to emerging infections with at least initial suggestions of its implications ( ) . this development in particular, along with observations from several decades of applications of systems ecology to natural resources and economic development ( , ( ) ( ) ( ) , have resulted in important insights of significant potential in understanding zoonotic disease emergence as a cross-scale process. this area uses complex systems theory applied to coupled, human-natural systems to explain how processes such as local phenomena can result in a cascade of effects ultimately reaching global proportions. the finding suggests this crossscale behavior is controlled by relatively few variables, and is mitigated by social and ecological resilience. the loss of this resilience in ecological systems is observed to lead inevitably to unpredictable events or the 'surprise' characteristic of complex systems generally. this combination of socialecological systems and resilience theory helps explain the unpredictability of disease emergence events. it represents another potentially useful area of application to understanding emerging infectious diseases along with those areas generally considered within the domain of ecology mentioned above: population ecology and genetics, community ecology, and systems ecology. population ecology, genetics and disease emergence of particular relevance to disease emergence is the explanation provided by theoretical population biology, already mentioned, of how host (including human) population size determines whether or not a pathogen can persist in a population. the accumulated findings demonstrate thresholds exist, depending on the type of pathogen and host population, below which a pathogen cannot be sustained. considered in light of the exponentially increasing size of human and domestic host and vector populations in the world, the breaching of thresholds of pathogen persistence can explain much of the increase in emerging infectious diseases. this can be explained as follows. although zoonotic disease emergence is not entirely a tropical phenomenon, it is mostly associated with tropical developing regions undergoing the most rapid population growth and ecological changes. prior to the post-wwii economic era, most regional ecosystems in the tropics consisted of relatively scattered human settlements, and only a few large cities (> , ) ( ) . these were separated by large expanses of cropland and pastureland and relatively undisturbed forest. since then, in what has been the most rapid period of large scale ecological transformation in human history, the pattern has essentially reversed ( ) . the once scattered settlements and few large cities have coalesced into expansive megacities and surrounding periurban settlements with only remnant patches of undisturbed forest remaining in a sea of cropland, scrub, and ecologically degraded lands. dacca, bangladesh, which grew from a population size of , to million from to , is one of many examples. thus the existence of population density-dependent thresholds for disease emergence is particularly relevant ( , ) . this explains the abrupt transitions of urban diseases between non-persistence to endemic and endemic to epidemic behavior as population densities of susceptible humans, hosts, and vectors reach critical densities. the classic illustration is that of measles which, given its particular transmission rate, requires human settlements with population sizes in excess (> , ) of what historically existed in most pre-industrial states and geographically isolated populations even today ( ). thus, for example, many infectious diseases endemic on continents have not become established on islands despite their occasional introduction and the occurrence of local outbreaks. the same mathematical ecology that explains why measles and virtually all diseases have threshold densities, explains the much lower thresholds existing for vector-borne diseases such as arboviruses ( ) . particularly noteworthy is the theoretical demonstration that the pathogen 'reproductive rate' increases with the square of vector population density. this indicates threshold densities can fast be breached as domestic and peridomestic hosts and vectors expand (or re-expand) their geographic ranges (once they are introduced or re-introduced) and increase their densities. this helps explain the explosive re-emergence of dengue and dengue hemorrhagic fever in the american tropics as vector populations responded to relaxed controls and new breeding habitats associated with urbanization ( ) . this phenomenon can be likened to the gradual build-up of 'dead and down' wood across a forested landscape with a history of fire suppression. the build-up of fuel, like that of host or vector populations, becomes an 'accident waiting to happen' when a single ignition event in one locality, similar to a single infection event, spreads to the entire region. another consequence of the dramatically increased densities of humans, host reservoirs, and vectors is the increased number of pathogen genomes. the resulting increased levels of genetic variability can accelerate microbial adaptation, including evolution of pathogenesis, and antimicrobial resistance. genetic variability increases with population size and density through a variety mechanisms including mutation. the probability of producing more virulent variants not only increases with host population size but also with crowding and co-mingling of different host species ( ) . in general, parasite (pathogen)-host relations naturally constitute a co-adaptive/ evolutionary 'dance' along the pathogenicity threshold, which is likely to be crossed with greater frequency due to unnatural anthropogenic disturbances ( ) independent of increasing population sizes and pathogen genetic diversity. the study of ecological communities and the 'community ecology' theory it has yielded includes a number of principles and mechanisms that describe how human disturbances as well as natural environmental variation can contribute to any of the above population level factors ( ) . there are a number of implications to zoonotic disease emergence, although most have not yet been described in terms of disease emergence or in the medical, public health, or zoonotic disease literature. of critical significance from this area of ecology is the general principle of community assembly. research has demonstrated that communities of arranged predicably in terms of 'assembly rules' ( ) . this order, in terms of the spatial distribution, composition and the abundance of each species in an ecological community, is affected by interspecific interactions (predation, competition, and parasitism). density independent factors (e.g., weather, natural catastrophes) play an important, but a more ephemeral role in most ecosystems. the process of community assembly (and disassembly) has been particularly well demonstrated through studies of insular ecosystems, the so-called 'species area relationship,' and the phenomenon of faunal collapse ( ) ( ) ( ) . a principal outcome and the ecological significance of this body of research first described in terms of the process of 'habitat fragmentation', has been identified as the principal mechanism by which human land and resource use contributes to species extinction ( ) . although initially debated, a significant amount of evidence has since accumulated resulting in its general acceptance and applicability, particularly to tropical forest ecosystems ( , , ) . the critical significance of habitat fragmentation and related human disturbances to disease emergence stems from it contribution to the disassembly of orderly natural communities. for example, human activities such deforestation, use of pesticides, and various forms of pollution often result in the loss of predators. in fact, carnivorous mammals typically are the first species to disappear following forest fragmentation. their local extinction represents the loss of 'top down' natural control in ecological communities. this can in turn result is an increase in abundance, even 'hyper-abundance' ( ) , of species such as rodents and biting insects. community disassembly and the resulting loss of natural population control mechanisms for such species generally is associated with the conversion of natural landscapes to urban and agriculture landscapes. broad spectrum pesticide use and habitat simplification, along with habitat fragmentation, are important contributing factors. the reduction in species diversity can contribute to the phenomenon of 'ecological release' in remaining species, whose predators, competitor and parasites are reduced in numbers or eliminated. some of these may be already serving as zoonotic reservoirs or vectors. if so, ecological release may result in their proliferation. this helps explain why many emerging zoonotic diseases occur in areas where settlement and agricultural expansion recently have encroached into tropical forests. a similar phenomenon, associated with the regrowth of forests in developed regions, can lead to zoonotic disease emergence. the pattern of reforestation in eastern north america during the past half-century resulted in increased habitat favored by forest edge adapted species such as whitetail deer and white-footed mice. white-tailed deer, the principle host of the adult tick ixodes scapulari, reinvaded the area and with few predators and competitors the population exploded. this pattern of ecological change arguably has been a major factor in the emergence of lyme disease in this region. an extension of the concept of 'ecological release' has been suggested to explain the invasive species phenomenon in which super-successful introduced (alien) species have escaped from their natural complement of parasites ( ) , as well as predators and competitors. this may explain in large part our most successful invasive domestic species, rattus and aedes species, which are hosts and vectors of some of our current and historically most problematic urban zoonotic diseases. in sum, zoonotic infectious disease emergence can be explained in part as a consequence of the disruption of natural ecological communities, and the breakdown of naturally existing predator-prey, competitive, and host-parasite relationships that tend regulate and stabilize species' abundance. this can occur through the use of non-selective pesticides through changes in land use and land cover that affect the distribution and abundance of species. it should be noted that the disassembly of natural ecological communities that results from habitat fragmentation is a protracted process compared to exponential decay. depending on the extent of habitat loss and other factors such as the sizes, shapes and spatial relationships of remaining fragments, the process may require decades, centuries or longer as communities 'relax' toward a new equilibrium ( , , ) . it follows that the frequency of disease emergence can be expected to follow a similar path: highest rates at first, followed by gradual decline as an ecosystem 'stabilizes'. the application of systems thinking to ecological communities centers on the ecosystem concept and has been the basis of significant research activity in ecological science since the 's ( ) . much of the focus has been on describing and understanding energy and nutrient fluxes across different kinds of ecosystems, and more recently emphasizing the human dimensions of global environmental change ( ) . although global scale ecosystem change has been considered in reference to human and infectious diseases ( , ) , no systematic framework has yet been presented in this regard. the evidence presented has been anecdotal. however, the development and application of systems theory independently of and largely outside the realm of mainstream systems ecology ( ) shows significant promise in providing a basis for more systematic interpretation of the role of ecosystem change in disease emergence. recent thinking draws in particular on the application of the theory of 'complex adaptive systems' to ecological systems in which ecosystems are represented as scaled, self-organizing, far from equilibrium, evolutionary, and non-linear ( ) . organization, diversity, and resilience are important 'emerging' properties of complex systems-often equated with a 'healthy' state. 'surprise', or qualitative disagreements of system behavior with a priori expectations, is another property ( ) . their association with loss of system resilience and increased vulnerability applies both to increasing inflexible social systems 'managing' and attempting to 'control' natural variables (i.e., vectors, pathogens) and that of the ecosystems whose component variables (e.g., vector and pathogen populations) are targeted for management or control. alteration of natural disturbance regimes (via control of natural variation via flood control projects for example) reduces resilience, while secondary disturbance events (wildfires, storms, floods, and earthquakes) precipitate events caused by crossscale influences (e.g., a thunderstorm igniting a fire locally that spreads regionally as a result of fuel build-up from years of artificial fire suppression). regional environmental change such as that associated with population growth development often does not accommodate the need to maintain resilience ( ) . such ecosystems have been described as 'over-connected' or 'brittle' and, as stated above, 'accidents waiting to happen' ( , ) . floods, often associated with waterborne disease outbreaks, occur more frequently and with greater severity as a result of lost resilience in natural systems due to attempts to 'control' (in contrast to manage) natural variation. conversion of upland forests to plantations or cropping systems for more 'efficient' natural resource production is another, as is agriculture generally. both result in a reduction of heterogeneity in ecosystems that tend to 'buffer' against disease outbreaks, which can spread more readily across the more uniform landscape including immunologically vulnerable domesticated species. gunderson et al. ( , ) describe this as general pattern of institutional behavior. it begins with the targeting of a natural variable for control, followed by increasing institutional efficiency and inflexibility in the control methods used. as the target variable and ecosystem changes and initial signs of failure are ignored, the ultimate result is a crisis. this 'pathology of regional resource and ecosystem management' and is apparently applicable to infectious disease management as well. the above body of ecological theory and observations involving specific emerging infectious disease cases suggests a causal schema that links ecological phenomena on the scale of pathogen transmission and evolution to regional and global transformations. this is represented by figure , focusing on the role of demographic and societal factors in disease emergence (shown in table ). this schema is constructed using the generally adopted view of human-environment interaction in which the impact of human population and technology is taken as the driving force, or ultimate cause. clark et al. ( ) elaborate on this in their seminal treatment on global and regional change. here, the combination of population, technological capacity, and sociocultural organization act as the system drivers of regional environmental change. these and 'mitigating' forces are in turn influenced by 'human behavior', referring to patterns of actions and the rationales giving rise to them. broadly speaking, these forces and their affects on ecosystems represent the 'ecological factors' in social-ecological systems, while human behavior represents the 'social factors' for the purposes of discussion. this schema represents zoonotic disease emergence from the perspective of the ecosystems within a regional environment, the large scale processes involved, and the associated ecological effects and processes involved in disease emergence. thus, referring to table , the factors under 'demographic and societal changes' can be identified in reference to particular ecosystems and processes (i.e., urban and urbanization, agriculture and agricultural intensification, forest and forest alteration), and associated factors operating on regional and global scales. these are unprecedented population growth, unplanned and uncontrolled urbanization, non-biodegradable packaging of consumer goods, and modern transportation, all contributing to conditions that promote pathogen transmission and persistence. for zoonotic diseases, which by definition involve the jumping of a pathogen from its natural host to humans, and in some instances extension of its host cycle to include humans, conditions can be described for this simple schema as follows. the likelihood or frequency of transmission events change when the natural host or pathogen changes, humans change, or the ecosystem supporting both changes. thus, fundamentally the processes influencing transmission of zoonotic pathogens can be described as a consequence of one or a combination of three possible kinds of change: expansion of the habitat or geographic range of a host, of a pathogen or both; expansion of human's habitat or geographic range; or change in the habitat or ecosystem occupied by both humans and the natural host. evolutionary adaptation of pathogens is omitted from the figure. however, it can be assumed any factor contributing to increased likelihood of transmission, as well as increased population size of hosts and pathogens will contribute to the potential that new, increasingly pathogenic, infectious, antimicrobial resistant variants will emerge. anthropogenic climate change, while not incorporated in the diagram, can be considered to potentially contribute to disease emergence through its contribution to habitat alteration. our review of ecological theory and the resulting complex model described here demonstrates the limitation of classical disease ecology and natural history. for example, without incorporation of population genetic theory in ecology as 'evolutionary ecology', along with the concepts of pathogen spillover and cross-scale ecosystem dynamics, classical disease ecology cannot explain recent emergence events like those involving hiv/aids, sars, avian influenza, e. coli : , dengue, malaria, west nile virus, and nipah virus, among others. the resurgence of vector populations, having acquired pesticide resistance and lost predators and competitors as natural controls after a regimen of inappropriate pesticide use, similarly is explained by modern ecology and evolutionary biology. this has direct application to control and intervention policies and practices. for example, it points to the need to adopt control strategies that integrate landscape and habitat management with careful rotation of targeted chemical and biological agents ( ) . developing such strategies requires detailed field studies, built on traditional natural history and classical disease ecology, but supplemented with advanced molecular techniques, as well as ecological research that takes into account the community and systems level dimensions of a particular pathogen-host complex. the continual expansion of human populations since prehistoric times, and particularly since the advent of settled agriculture with its associated domesticated animals exposing humans to their pathogens as well as those spilling over from wild animal populations, has incrementally added to the pathogen load through successive invasions by different organisms over time ( , ) . well established principles of population ecology, applied via mathematical epidemiology as anderson and may and others so aptly have done, readily explain why, ceteris paribus, infectious disease incidence should generally be increasing with human population size, as it has in the world's poorest and most populous regions. yet in spite of what are in general commonly understood principles, and warning signs that went unheeded in the 's and 's ( , ), biomedical and public health institutions were unprepared for the recent surge in emerging infectious diseases ( , , , ) . not until the 's, prompted in part by the hiv/aids pandemic and the failure of 'quick-fix' solutions based on drugs, vaccines, and pesticides ( ) , did the biomedical science and public health communities begin to launch a significant response. in light of the complexity of the factors involved, this lack of preparedness should perhaps not be surprising. as explained by the infectious disease ecology described here, zoonotic disease emergence involves biological processes operating on the scale of molecules and cells to that of coupled, regional scale human-natural systems. the latter involve political economic factors and policies driving regional environmental change, spreading geographically across the globe. it is this process of globalization-its ecological underpinnings and consequences-to which the current eid global trend of zoonotic disease emergance can be largely attributed. lack of awareness of the ecological implications of fig. diagram depicting infectious disease ecology causal schema. the aggregate variable at the top of the diagram, representing population and consumption, along with mitigating socio-cultural attributes, is the driver or 'forcing function' responsible for land use and land cover change characteristic of a particular region. the result is varying degrees (and types) of urbanization, agricultural intensification (including food production and distribution), and alteration of natural habitat. these changes at the level of landscapes and habitat produce conditions influencing the ecological and evolutionary dynamics of vector and host species and vector/host-pathogen dynamics. in turn, these conditions facilitate the geographic expansion, novel appearance, or increased epidemic activity of a disease. regional environmental transformations, and of their synergy with the ecological and evolutionary consequences of inadequate or inappropriate policies or methods of vector control and disease prevention, have unwittingly promoted disease emergence. the 'ecological' changes taking place as a result of public health agencies' actions (or inaction) involve pathogen biology and are small scale in time and space: selection for vector pesticide and antimicrobial resistance. however, the cumulative effect of micro-scale processes involving pathogen adaptation and host range expansion (or re-expanding) can ultimately produce regional and even global consequences. however, policy action and inaction outside the domain of biomedical or public health agencies has produced ecological changes at a historically unprecedented rate and scale. urbanization, agricultural and food production intensification, alteration of natural habitat, along with concomitant loss of ecosystem functions, have transformed entire regional ecosystems during the past years. the role of urbanization cannot be underestimated. the direct land use changes associated with urbanization effectively concentrate human, animal reservoir and vector populations at unprecedented densities. but urbanization also is strongly tied to socio-economic and cultural factors, along with human migration dynamics, affecting agricultural and food production intensification, as well as rural and natural ecosystems. in the case of food production and distribution, dramatically increased contact with and transport of wildlife (e.g., bush meat trade in africa), and increased ruralurban transport and concentration of wild species for the exotic food market (e.g., civet cats in guangdong, china), is another contributor to increasing disease emergence. it can be assumed these impacts on wildlife populations also contribute to changes in the composition of ecological communities on a regional scale ( ) , and often result in a hyper-abundance of small mammal species low in the food chain, which are likely to serve as human disease reservoirs. similar effects of ecological disruption appear to generally apply to invertebrate commu- whether a disease 'jumps' to the regional population and the global human population scale is determined by quite different processes. these depend on demographic and transport patterns related to processes, like urbanization, influencing pathogen transmission and behavior operating on the regional and global scales. physical environmental processes such as climate variability (on the right side of the figure), which are episodic by nature, include short term and small scale variations in the form of seasonal storms (e.g., monsoons), for example, as well as larger time and space scale variations. these include, for example, decadal and regional scale changes in weather patterns such as el niño. these climate changes and weather events can precipitate floods and droughts. these act as cross-scale mediators that directly affect disease reservoir and vector populations, or pathogens (e.g, dispersal via flood waters). they cause the disease to jump from a smaller demographic unit to a larger one (e.g., from a single village to a district). human factors such as land use and land cover changes (table and fig. ) produce ecosystem changes contributing or magnifying the cross-scale processes. for example, urbanization and deforestation increase the magnitude of floods and droughts resulting from natural or anthropogenic climate variation. nities ( ) , the kinds of organisms most commonly serving as human disease vectors. the estimated acceleration of natural species extinction rates by orders of magnitude over nonanthropogenic extinction rates ( ) gives an indication of the scale and magnitude of change in natural communities, particularly in the tropics. in these regional ecosystems, the original extent of tropical forest has declined to a fraction of its original area with concomitant affects on biodiversity ( ) . large scale agricultural expansion (a common proximate cause of tropical deforestation) has decelerated and all but ended in many regions. this suggests that further contributions from what historically has been a main factor in natural habitat alteration, ecosystem disturbance and biodiversity loss, will decline. yet agricultural intensification has replaced expansion with technologies that further impact native biodiversity ( ) . the reduction in plant species richness that accompanies agricultural intensification leads to changes in the community composition of the pest complex-herbivorous insects, their natural enemies (predators and parasites), and the microbial community attacking crops ( ) . the schema described here demonstrates how regional environmental change, involving ecological dynamics such as demographic or landscape transformations on the scale of decades, interact with change on the scale of host-parasite/ pathogen dynamics. how these cross-scale mechanisms produce regional or global scale disease emergence patterns is beyond the realm of conventional epidemiology, or analytical approaches generally. this may explain why such changes either are not apparent or their implications in terms of disease emergence are a low priority to biomedical and public health agencies. such cross-scale processes are however characteristic of ecosystems, whose dynamics involve interaction of variables and operate on vastly different time and space scales, involving natural processes that are discontinuously distributed as shown in figure . change in such systems is difficult to understand employing conventional analytic approaches alone, but require new thinking and analytical methods drawn from complex systems theory. the discontinuous character of the processes or variables makes their interaction intuitively improbable except for the mediating effect of 'cross-scale influences' as illustrated in figure . examples include the eruption of hantavirus in the american southwest, thought be associated with weather events precipitated by el nino southern oscillation. storms events are similarly episodic, and represent a discontinuous form of variation transporting pathogens via flood waters in domestic and natural environments. on the other hand, massive environmental events, like the recent southeast asian tsunami, are potentially capable of producing epidemics across an entire region, and may episodically extinguish some zoonotic diseases by temporarily destroying reservoir and vector populations and habitat. the roles of resilience and vulnerability in determining the severity of such events, both in terms of social systems and ecological systems is another critical aspect of social-ecological systems behavior ( ) . the recent worldwide upsurge of zoonotic infectious diseases, involving the resurgence of a growing number of diseases previously believed under control or the emergence of newly recognized diseases, has been attributed to a list of global factors characterized in terms ranging from microbial adaptation and land use to changing ecosystems, breakdown of public health, and poverty ( , ) . the categorization provided by gubler ( ) , elaborated here based on an expanded view of infectious disease ecology, provides the basis for a schema describing the causal relations involving factors previously identified, along with hypothesized mechanisms. the complicated nature of this problem, which obviously entails numerous interacting variables operating on different time and space scales pose a significant challenge to biomedical science and epidemiological research as well as public health intervention. however, the current trend of increasing global emerging infectious diseases is linked with another issue of 'global governance', sustainable development, with which disease control and prevent strategies must be integrated. here too, the problems of politically stability, population growth, unplanned urbanization and economic development, income disparity, and environmental degradation, are all integral to the solution. this interdisciplinary imperative challenges what historically has been an increasing disciplinarily focus in infectious disease research. greater investment in research has succeeded in revealing more detail about diseases within specific disciplines. yet this arguably has been at the cost of greater disintegration rather than integration among disciplines. the division is widest between the genetic and biological aspects of disease and health on the one hand and the social, economic, political, and physical environmental factors on the other. the disciplinary distinctions within infectious disease research of course belie the true transdisciplinary nature of this and most problems in the global heath and environment arena ( ). the above disciplinary divisions largely reflect the gap between disciplines focusing on systems at or below and above the level of the organism or species. the former involve disciplines addressing the genetics, pathogenesis, or immune response within particular organisms (humans, vectors of pathogens), for example, or disease as a statistical phenomena at the species population level (the mainstay of epidemiology). the latter deal with social or ecological phenomena, essentially representing higher levels of biological organization, within which the organism-level processes operate but that also involve many other variables or processes operating on a wide range of biological scales (from genes to the biosphere) characterized by cross-scale influences, and interactions at multiple societal and ecological levels. it follows that for intervention to be globally effective, in addition to rebuilding public health infrastructure based on the comprehensive view of infectious disease ecology presented here, at least three elements are required: . a multilevel ecosystem approach, involving cross-scale integration. . incorporated ecological theory and data for the specific disease system, . local scale intervention using a participatory approach that matches pathogen management with sustainable 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change: research pathways for the next decade ecosystem change and public health: a global perspective systems thinking, systems practice adaptive dancing: interactions between social resilience and ecological crises plagues and peoples human frontiers, environments and disease emerging infectious diseases: a cdc perspective a global perspective on habitat disturbance and tropical rainforest mammals species richness-decomposition relationships depend on species dominance extinction rates agricultural intensification and ecosystem properties biodiversity and ecosystem processes in tropical forests emerging infections: microbial threats to health in the united states transdisciplinarity: recreating integrated knowledge. mcgill-queen's university press we thank shannon bennett, kristin duin, mayee wong, and yuko chiba for assisting with the research for this paper and its critical review. key: cord- - mx thr authors: neuwirth, rostam j. title: gaia —a ‘glocal agency in anthropocene’: cognitive and institutional change as ‘legal science fiction’ date: - - journal: a post-wto international legal order doi: . / - - - - _ sha: doc_id: cord_uid: mx thr to assess a future scenario of the world without a wto, the present chapter projects the reader into the distant future of the year by which time the global community is aiming to establish gaia, the so-called “glocal agency in anthropocene”. gaia is designed as the first truly integrated global institution with a universal character with the aim to tackle the complex and multiversal governance challenges of humanity and the planet as a whole. this chapter marks both a legally and a scientifically fictitious account of the years from until , from a dystopian and a utopian perspective, with the aim of highlighting the importance of cognition for legal and institutional change. the need for cognitive change is driven by changes in the environment, and by the challenges resulting from a perceived acceleration of the pace of change and the unprecedented levels of technological complexity. both change and complexity increase the relevance of cognition, as laws and policies adopted in one area are more likely to affect their success or failure and that of the global governance system as a whole. thus, this chapter predicts that the foremost necessity for law in the future is to build on novel and enhanced modes of human cognition to deal better with complexity and rapid change. why do we remember the past and not the future? we are writing at the end of the year , and marking a period of efforts towards the establishment of a new and comprehensive global governance mechanism, known, for short, as "gaia ". gaia stands for "glocal agency in anthropocene", and describes a project for a novel institutional framework to be set up with the objective of tackling humanity's most urgent problems and glocally governing and sustainably developing human affairs in the future. the future is what matters the most right now, as the world is hoping to recover from the devastating effects of the time period known as the "digital dark age". this is the era from until when-as the result of an internet blackout (outage)-all prior digital resources, including records and archives, became inaccessible and, because of the wide prevalence of the internet of things (iot), the same happened to most related technical applications and utilities. in short, this dark period wiped out all digitally or electronically stored data. as a consequence, it not only paralysed all technical facilities and caused millions of casualties, but also led to a collective loss of humanity's memory, which, furthermore, caused a loss of identity. many still speculate about what triggered the meltdown of the former information society based on the internet, with the suggested causes ranging from scientific, economic, and political to cognitive and, notably, legal reasons. for instance, scientific explanations range from a cyberattack using malware to a nuclear blackout caused by the detonation of an atomic warhead in outer space, an accident at cern creating a black hole in geneva following a malfunction of the large hadron collider ii, or a failure of the g network in combination with a data overload on the world wide web caused by states' geopolitical struggles and the monopolistic tendencies of various tech giants. other people invoke politicoeconomic explanations, simply citing the second global financial crisis of , which was caused by trade and currency wars that, inter alia, eroded the finances needed for the maintenance of the internet's sophisticated infrastructure. environmental factors are also often named, such as floods and rising sea levels, increased volcanic activity and earthquakes, combined with heatwaves and global pandemics, which disrupted or paralysed the energy supply by damaging its essential facilities. politics also played a role, as populism and racism increased, further fragmenting society into ever smaller units, down to its most vulnerable minority, the individual. constitutional democracies came under threat from changes that were, at first, undetectably small but that eventually combined and posed a serious threat. in legal circles, the fragmentation of international law, the rise of norm conflicts due to bad regulation, overregulation and the automation that took over several fields of law are mentioned as the prime causes of the inability of humanity to halt the dangers of the conditions it had created for itself. it again became obvious that the rule of law needed not just legal texts but also stable institutional guarantors at both the national and the global levels. it is more likely that the blackout was due to a combination of interrelated factors in an environment characterized by an increasing complexity and ever faster pace of change. since time immemorial, humanity has struggled with change or, notably, with its perception of change, since this perception may itself have been subject to change. however, since the last millennium many observers have noted an apparent effect of an acceleration of change or so-called "time shrinking". this trend for change to accelerate started to pose a major threat to lawmakers and policymakers and, in particular, to law, as it became more difficult to "preserve its integrity over time, while managing to address the newly emerging circumstances that continually arise throughout our history". another threat to law came from unprecedented levels of technological innovation, which culminated in a situation in which the regulation of specific industries and technologies, such as those of artificial intelligence, nanotechnology, big data and genetics, failed to find a successful balance between their unknown risks and their expected benefits. in short, excessive levels of specialization, not met by an adequate general understanding, led technological progress to spin out of control. thus, by the year the international legal order had witnessed the disastrous effects of a gradual loss of control caused by decades of neglecting first subtle and later obvious signs of multiple law and policy failures. these policy failures culminated in the failed attempts to reform the most important international institutions, the united nations (un) and the world trade organization (wto), in a holistic and comprehensive manner. there was a failure to bridge the rift that was caused when the international trade organization (ito) did not materialize; the ito would have complemented the institutional balance between the three bretton woods sisters, the ito, the imf and the world bank. in other words, the institutional failure of the ito to materialize also meant that greater coherence, or to integrate trade and non-trade concerns, was renounced as an ideal for global governance. the separation between the un on the one hand and the gatt on the other, can also be regarded as a further strong manifestation of a dualistic mode of thinking based on binary logic. most of all, it was the beginning of a failure of cognitive modes of thinking to keep pace with technological changes in the context so as to provide a stable and coherent global institutional framework for the governance of global affairs. historically, the gatt was characterised as having found "itself without an inadequate legal and constitutional base and required to fill a vacuum created by the failure of the ito". the situation also meant that attempts to reconcile trade and non-trade concerns in both substantive and institutional terms was postponed to a distant day. this delay later led to the emergence of the "trade linkage debate", by which different pairs of "trade and . . ." problems were supposed to be reconciled. lacking their reconciliation, criticism of the wto also became louder and culminated in the protests accompanying the seattle wto ministerial conference. since then, the failure to address non-trade concerns and to achieve greater policy coherence continued and provided further momentum to the procedural crisis affecting the wto dispute settlement body. in sum, a lack of political will as much as outdated cognitive modes framed by dualistic thinking led to the law being inadequate to meet the institutional and substantive challenges. to exemplify the impact of cognition on whether the law meets the demands put upon it, the present chapter divides the time between the year and into two opposite scenarios. first, sect. traces the lost years by drawing up a dystopian johnson ( ), p. . harari ( ), p. . jackson ( ), p. . dimatteo ( scenario, in which cognition remained largely static and dualistic, such that changes did not keep pace with the speed of technological and scientific innovation. section is more utopian, in the sense that it relies on a newly acquired cognitive mode of thinking as exemplified by synaesthesia, which not only allows for the development of actions as causes of change but also includes a review and evaluation of the consequences. the conclusion finds that an entirely new mode of cognition, triggered by linguistic changes that are reflected in a recent rise of oxymora and paradoxes, opens a new perspective on how lawyers can help to both predict and shape the future. fail' in the world of theory, there are many dichotomies. in the real world, there are many divisions and divides. in the world of power, all too often, these divisions, divides, and dichotomies serve to maintain and reinforce existing imbalanced and skewed power relations between individuals, communities, governments, and nation-states. in the world of power, it is indeed divide and rule. in attempts to describe the evolution of the multilateral trading regime from until , so-called "digital historians" have argued that december marked the decisive moment and unofficial date of the decline of the world trade organization. although aggressive regionalism, unilateral measures and resulting trade wars as well as inertia in addressing the trade linkage debate and interinstitutional linkages between the wto and the un had been eroding the multilateral trading regime for some time before, it was on that day that the wto's dispute settlement system became dysfunctional following the expiration of the terms of two of the three remaining members of the appellate body (ab). this left the ab with fewer than the three members required by art. . dsu to serve on appeal cases. as with many international organizations, nation states and other legal constructs before it, the official decline of the wto and the multilateral trading regime as a whole started slowly and then ended abruptly. the decline was probably the result of a chain of missed opportunities for the reform and adaptation of the relevant system to changes in the context, which notably saw a strong trend towards greater convergence of various industries, technologies and products. this convergence further increased the need for the consideration of non-trade concerns and greater policy coherence. for some time, the system lingered on and ministerial conferences were merely held without achieving tangible results, continuing the deplorable tradition that began with the launch of the doha development round in . most wto members engaged in either a relapse into unilateral measures adopted at the domestic level or a rat race to join numerous regional trade agreements. however, in both cases, the most important challenges faced at that time, in the form of institutional gaps, regulatory fragmentation and overregulation, were not tackled, either nationally or regionally, let alone globally. numerous calls and suggestions for institutional reform to achieve greater policy coherence, under the aegis of the so-called "trade linkage debate" discussing various "trade and . . . problems" or how to better link trade with non-trade issues, were ignored. in this regard, more importantly, the cognitive and conceptual dimension of trade policy was not duly considered, as "transformational change in the institutions and politics of international trade" were found to go "hand in hand with cognitive change" as andrew t. f. lang wrote. he also found a nexus between cognitive change and institutional reform being linked to the criticism of the wto and notably the failure to address the trade linkage debate. as a result, from this time on, the international system, or, more accurately, the remaining "international systemic chaos", was described as having moved "away from an assembly of distinct, territorial, sovereign, legally equal states toward different, more hierarchical, and in many ways more complicated structures". even though an attempt was launched in to reform the united nations organization in order to streamline the coordination of its many specialized organizations and to enhance the coherence of global policymaking by "delivering as one", the management of this reform eventually proved to be an oxymoron before it failed altogether. for a long time, the un's work in its core areas, from development to the environment, was described as fragmented, and its inefficient governance structure contributed to "policy incoherence, duplication and operational ineffectiveness across the system". what could be framed as a "trade and technology" problem, the earlier us-china trade disputes ( - ), also caused frictions in the innovation of new technologies and especially in the realm of telecommunication and information technologies. these frictions led to a further fragmentation of the internet and the market for information and communication technologies (ict). russia initiated the creation of an alternate web, which was later also used by the remaining brics countries, pauwelyn ( ) with other countries following suit. moreover, it was common for the governance gaps and frequent legal inconsistencies between the various fragments of laws governing international trade and commerce to be abused by artificial intelligence and unregulated algorithms, those lacking a kind of lex algoritmica, meaning that global business generally operated in a "black box" inaccessible to consumers and lawmakers. from a commercial perspective, the "world wide web" (www), which was intended to become a "wireless world wide web" (wwww) by virtue of the transition to the g network, became further fragmented by national and regional firewalls as well as restrictive measures known as geolocation measures. other conflicts, in the form of regulatory paradoxes framed as dichotomies, such as those of "goods versus services", "free trade in data versus data localization", or "regulatory fragmentation versus technological convergence", were also ignored. in the same way, in other sectors covered by the gatt, gats and trips agreements, the multilateral trading rules were further eroded by trade disputes in which different claims were met by defences made on the basis of national security concerns. known as the "trade and security" problem, questions about the selfjudging nature of the security exceptions widened the institutional gap opened by the split between the un system and the gatt/wto system caused by the failure of the international trade organization (ito) to materialize. this institutional rift had, since , left the world with a conflict of norms in the form of a "catch xxi" or "trade and security" dilemma. after the wto ruling in russia -measures in transit in , in which the panel held that it had jurisdiction to determine whether the requirements of article xxi of the gatt were satisfied, other countries simply refused to respond to requests for consultations and boycotted the relevant meetings. in the end, the invocation of the security exceptions further eroded belief in the wto and accelerated its demise. the broader conditions in the global economy also grew dimmer and were closely related to the "trade and energy" problem. from as early as , the tipping point in the production of oil was reached, meaning that conventional crude oil production pasquale ( ) . yu ( ), pp. - . smith and woods ( ) , pp. - . burri ( ) was not rising to match increasing demand. the "all-oil peak" meant that, after decades of controversy over its arrival, there was a drastic dampening in economic growth and, actually, the inauguration of an era of global depression. notably, for oil-producing and other resource-rich countries, the so-called "paradox of plenty" aggravated, and the ensuing economic disaster also led to, the collapse of their constitutional system, and also triggered new waves of political violence. since human civilization was a sensitive "complex adaptive system", other countries too, and the globe as a whole, also were drawn into a downward spiral of economic recession and political instability. institutionally, the proliferation and fragmentation of energy organizations and the failure to address the "global energy governance gap" by reforming existing energy agencies or creating a global energy agency, further contributed to the resulting disaster. the complexity of the global economy could also be seen in the connection between trade, the environment and energy. in parallel, numerous efforts towards achieving greater sustainability in economic development based on renewable energies were also unable to offset the devastating effects of the disruptive consequences of the "all-oil peak" and the ensuing global energy crisis on economies and societies around the world. the result was a global energy crisis in that disrupted most global trade in both goods and services and caused stock crashes through inflationary pressures, and later accelerated the eventual collapse of the international monetary system as it had done during the oil crisis. renewable energies were also incapable of averting the crisis, even though the impact of the paris agreement, adopted with the objective of responding to the threat of climate change, had no negative effect on the global economy or on countries' welfare gains. because of the absence of a coherent global regulatory framework, renewable energies remained expensive and investments often did not materialize. in this context, it was also a failure when the wto membership addressed the "trade and environment" problem by, inter alia, reforming the wto subsidies agreement, which resulted in various trade disputes challenging green policies. the lack of global competition rules, as a result of the abandonment of a project in that was one of four "singapore issues", led to the emergence of various multinational tech giants, which widely abused their dominant positions, explored tax loopholes, and competed unfairly in courts, patent offices and markets. they were also crucial in meddling in elections and were accused of undermining democratic institutions. no efficient dispute settlement system under the multilateral trading regime, and a lack of legal standing for private parties, both natural and legal murray and king ( ) , pp. - . hall and klitgaard ( ) , p. . ahmed ( ) . downie ( ) , p. . cosbey and mavroidis ( ), pp. - . demedts ( ), pp. - . ginsburg and huq ( ), p. . persons, in domestic or supranational courts caused further havoc and greater inequality among global citizens. as early as , inequality was reported to be standing at unprecedented levels, and it was claimed that "just eight men own the same wealth as the poorest half of the world". these inequalities, dividing societies around the world, not only perpetuated themselves but even increased, with devastating effects on the global economy and the environment. another aspect of inequality was found in the "trade and development" link, another complex and cross-cutting "trade and . . ." problem. cutting a long story short, the development discourse continued along the "developing versus developed country" dichotomy, despite calls to the contrary and even its abandonment by the world bank in . even though the definition of "official development assistance" (oda) evolved, it continued to be understood as resource flows from developed to developing countries. with the mindset unchanged, the language use of "developed-developing countries" also remained the same. as a result, the us and the eu continued, on the one hand, to subsidize their agricultural producers and to reap excessive benefits from royalties on various patented and copyrighted products, while on the other, they kept granting support through official development assistance programs to a large number of developing countries. this practice continued to impede the development of local industries in the targeted countries. moreover, the uncertainty surrounding the meaning of "development status" further eroded the wto's status after the us began to challenge it in . at the same time, the trade and development problem was closely tied to the "trade and finance" problem. generally, the trade and finance link suffered from the failure of the ito mentioned above, and the absence of a coherent framework for both trade and finance became manifest in the problem of so-called "currency manipulation", that is, the depreciation of a country's currency relative to other currencies so as to develop a large global and bilateral trade surplus. despite the three organizations, the wto, the imf and the world bank, having pledged to enhance their policy coherence, their inter-institutional dialogues did not yield tangible results and the use of policy coherence was criticized instead as a way to introduce policy conformance. an economy for the %. oxfam briefing paper, january . https://www-cdn.oxfam.org/ s fs-public/file_attachments/bp-economy-for- -percent- -en.pdf. neuwirth ( ), pp. - . hynes and scott ( ) . moyo ( ) thomas ( ) , pp. - . staiger and sykes ( ) , pp. - . grabel ( ), pp. - . put briefly, the "trade and finance issue" also remained unsolved, and the two respective regulatory regimes of trade and of finance continued to evolve side by side in dramatically different directions in spite of being mutually interdependent systems. this had a serious impact on the world as the regulatory gap between them grew even more intense while the technological reality saw them increasingly converge notably with the emergence of the blockchain technology that underlies cryptocurrencies. cryptocurrencies, from bitcoin to altcoin, proliferated and gradually disrupted business and financial services as well as the global economy. this meant that, in parallel to the third currency war between the world currencies (the dollar, the euro and the yuan), which began in , a digital currency war also began to be waged. in this digital currency war, the traditional world currencies fought against emerging digital currencies backed both by state and non-state actors. the ensuing chaos of currency wars brought about a loss of state control over financial markets and a rise of a global underground economy thriving on tax evasion and criminal activities. it all culminated in a collapse of global trade and finance and ended with the second global financial crisis of the twenty-first century, which began on "black wednesday" of october . together with military conflicts, global health pandemics, like the large-scale outbreak of the novel coronavirus (ncov) infectious disease, and various natural disasters, like large scales rainforest wildfires and bushfires, caused by climate change and other anthropogenic factors, the total disruption of the global economy created a dangerous dynamic, causing a severe migratory wave that increased the number of forcibly displaced people from about million people in to . billion less than years later. the crisis of migratory flows was aggravated by national governments and courts, who denied the affected persons the status of climate refugees. at the same time, after an erosion of the rule of law in the majority of countries around the world, ever more restrictive national policies regarding citizenship in general, and dual citizenship in particular, also increased the number of stateless persons, who were thus deprived of all fundamental rights within and across state borders. the european union, paralysed by endless brexit debates and the united kingdom's final exit from the eu on january , made little progress in reforming gadbaw ( ) , p. . tapscott and tapscott ( ) . rickards ( ) weissbrodt and collins ( ), pp. - . its institutions, the single market, the single currency or its taxation system. on the contrary, past achievements in the four freedoms were scaled down as the result of nationalist policies in many member states. the eu's so-called "luxembourg treaty", negotiated under the eu presidency of luxembourg, added nothing that substantively remedied the lost opportunity of creating a constitution for europe in . the eu's failure to become the world's most competitive market (the lisbon strategy) extended the lost decade of the eu, which began in , when, to a lost near-half century. many more regional integration projects worldwide also stagnated. for instance, the brics, representing % of the world's population in and initially set up as a "cooperation and dialogue forum" between brazil, russia, india, china and south africa in order to defy differences and make a difference in global governance, saw no progress, such as by seeking greater policy coherence based on more closely coordinating institutionalizing their cooperation. similarly, the positive effects on infrastructure development based on the belt and road initiative (bri), initiated by the chinese government in , were largely neutralized by opposition from the us and several european countries. the african union also failed to prevent the food security crisis that hit the african continent in and that pushed the achievement of the objectives enshrined in agenda into an even more distant future. in sum, despite the emergence of a creative economy, which was initially fostered by the potential of new and innovative technologies, the sustainable development goals were still not realized by . across the globe, political disagreements prevented action for greater regulatory harmonization, while centrifugal forces and fragmentation intensified. legally, a patchwork of isolated areas of law continued to grow, as did the number of regional trade agreements, which were violated or withdrawn from faster than they were negotiated. put simply, the so-called "global governance paradox", or the logical loop that a global platform was needed in order to create a global platform, proved a problem that was too difficult to tackle in conceptual and cognitive terms. with no consistent global legal order, fragmentation prevailed and led to the aggravation of the negative effects of "trade and . . ." problems on global peace and welfare. neuwirth ( ), p. . neuwirth et al. ( ) and neuwirth and svetlicinii ( ) . onyutha ( onyutha ( ), pp. onyutha ( - african union ( ) progress report on the implementation of agenda first ten-year implementation plan. available at: https://archives.au.int/handle/ / . the limits of my language mean the limits of my world. in , historians are still pondering the reasons for the collapse of the wto. some attribute it to isolationist us trade policies and the political turmoil around brexit, while others tend to focus on the rise of first asian and subsequently african countries. however, a different view identifies this failure as the beginning of the end of "end of . . . stories" or a drastic paradigm shift in cognition. cognition had reached a tipping point in human evolution following two centuries of rapid technological innovations, which "shaped consciousness directly". linguistic changes also confirmed this cognitive shift, mostly through the rise of essentially oxymoronic concepts. as a result of these external factors, epigeneticists found biological and cognitive changes occurring in parallel. new organs of cognition hence emerged, as humans were evolving biologically and consciously. as a visible result, even by % of new-born children displayed conditions of synaesthesia, i.e. the ability to better connect stimuli received from different sensory organs, which improved the skills to foresee developments and solve complex problems as well as enhanced their abilities of abstract thought. related changes in educational policies were implemented, with the goal of creating a "world brain", or "an enhanced educational system through the whole body of mankind", designed to "replace our multitude of uncoordinated ganglia, our powerless miscellany of universities, research institutions, literatures with a purpose, national educational systems and the like". this goal was to be achieved by fostering oxymoronic learning methods and giving training in both bivalent and polyvalent logic, complemented by training in multilingualism and oxymoronic thinking. for the trade and health link, the novel coronavirus (ncov) pandemic underscored once more the strong links between international trade, public healthcare and the global economy. as a result, the global healthcare system was integrated with a future trading system and backed by a consistent set of global innovation rules consisting of competition rules, intellectual property laws and fiscal incentives, which prevented price discrimination in pharmaceutical products and secured global access to affordable medicines. additionally, universal healthcare was provided freely by a combination of measures that combined both traditional and conventional medicines. based on further changes in perception, such as through the development of technologies for the visualization of auras, medical diagnosis improved and conventional, traditional and alternative medicines as well as mental health strategies were all integrated into a coherent set of laws and policies. further changes in healthcare were influenced by discoveries in so-called "life after life studies", which transcended the life-death dichotomy as the result of a greater awareness of the missing link in a globally coherent health policy. research on near-death experiences scientifically confirmed popular and religious beliefs about life after life. overall, a new cognitive mindset emerged in parallel with technical tools, leading to a new conception of the areas of economics, politics and law. for instance, the global economy became more sustainable, based on the spirit of "degrowth", which rejected the illusion of endless economic growth by advocating for a "democratically-led shrinking of production and consumption with the aim of achieving social justice and ecological sustainability". at the same time, new and cleaner energy resources were derived from nuclear fusion developed by the iter project, helping to decarbonize the world's energy system. various new and renewable energies complemented this. since a g initiative formulated in , the global community embraced an ambitious tax agenda to improve cooperation and transparency on the basis of a strategy for a global taxation system. this initiative originally included a global financial transaction tax to fund the new global governance system. it later included a digital tax on the world's tech giants, which levied taxes in the places where the goods and services were sold rather than the places where the company was based. it also introduced a global minimum tax rate to prevent a company from shifting its sales to a country with lower taxes. finally, a robotic tax, aided by the creation of a single global cryptocurrency (sgcc), covered all activities based on automation to compensate for the disruptive effects of automation on the global labour market. together, these measures helped to contain the outbreak of currency wars and related trade/finance disputes. on the other hand, the changes in the taxation system also helped to reverse the former trend of higher income taxes as opposed to lower corporate taxes. the new system achieved an optimal balance, with around % income tax, % corporate tax (including on digital activities) and % robotic tax, as measures towards greater global tax justice. these and other fiscal policies were introduced in coherence with other incentives to end the poverty trap. the cognitive shift also remodelled the international multilevel governance system towards a "glocal" and holarchic system in which local, largely selfgoverning, entities were complemented by various regional regimes often organized into mega-regional blocks, with those of an inclusive global system enshrined in the gaia charter of . holarchy here means a system in which various selfregulating entities function as autonomous wholes in supra-ordination to their parts but, at the same time, also as dependent parts in subordination to controls on higher levels their local environment. the foundation is a polyvalent logic by which the links between stakeholders at different levels are supported by a dynamic system of subsidiarity and an open method of coordination. it is based on a set of different criteria that the best possible level of regulation is determined without any prior bias towards either the local or the global level of governance. the same method is also applied to territorial questions, such as national sovereignty is no longer fixed but where territorial boundaries are drawn based on a similar set of criteria. thus, like in the quantum world, different political entities, and even national governments' competences, can overlap and even be superimposed without being perceived in conflict. changes in cognition facilitated the adoption of the gaia charter, as the global community finally found the "common language" to tackle the "global governance paradox" successfully. the paradox was that, in order to create a global legal order, the world community needed a global governance platform that had not previously existed, confirming that linguistic and cognitive changes are quintessential to institutional change. structurally, the gaia was based on an institutional setting in which every "trade and . . ." problem was coordinated by a "coherence committee", with the competence to avoid conflict between different policy goals. the functioning of the institutional framework was aided by an e-governance system, which used intelligent algorithms to consolidate redundant norms and detect incoherent measures. however, artificial intelligence was considered not only to be hype but also to be an oxymoron, and, in relation to law and policy, artificial intelligence measures were strictly bound by the requirement inherited from the former eu general data protection regulation that they could not be implemented and enforced "without any human intervention". brock and pogge ( ), pp. - . thang dao and edenhofer ( ), pp. - . koestler ( ), pp. - . neuwirth ( halpin and roeben ( ) in substantive terms, existing sources of global law were codified into a single legal document, the gaia code, a global constitutional text that reduced the previous disarray of international laws. in this way, former conflicts between norms that arose because of dichotomies that were too simplistic to account for real complexity, be it between electronic and traditional modes of consumption of goods (gatt) and services (gats), between ip law and competition law, or between international trade law and various subfields of general public international law in the name of "self-contained regimes", were no longer unavoidable and irresolvable. in this context, global competition rules were integrated and enhanced so as to not only guarantee the sustainable economic development of the global market but also "solve social problems ranging from unemployment to income inequality and indeed to improve the functioning of democracy itself". other necessary changes simply came with changes in cognition and understanding, and did not require a change in language as they occurred earlier when a higher court reconsidered its established case law. for instance, in the field of global economic law, the national treatment provisions of gatt article iii, gats article xvii and article of trips were now no longer merely interpreted as prohibiting discrimination between domestic and imported goods or services but also as encompassing "all measures having an equivalent effect on consumers in covered markets". this became necessary to cover personalized price discrimination made possible by the data economy. these cognitive changes, plus a seamless global wireless web, also ended the western failure of the territorial national state. as a result, the "four freedoms" related to the free movement of goods, services, persons and capital, became recognized as ubiquitous civil right and a global reality. the various systemic changes resulting from cognitive changes were supported by, and helped to establish, an effective and inclusive global dispute settlement mechanism, which recognized the right to bring an action and to be heard in court (locus standi) of not only international organizations, states and multinational corporations but also natural persons. it effectively aligned the former wto dispute settlement system with investor-state arbitration. both systems were elevated to a "world trade court" embedded within a fully-fledged global judicial body, the gaia tribunal, that was vested with various constitutional powers to sanction and enforce the rulings it issued. overall, the new institutional design based on these cognitive changes was said to have helped to "improve and eventually overcome the perceived lack of legitimacy of international courts and tribunals in the eyes of the governments, the legal community and civil society". conclusion: 'dystopian utopia' or oxymora to predict the future by creating it my project was retarded by laws of nature. the world was not prepared for it. it was too far ahead of time. but the same laws will prevail in the end and make it a triumphal success. a dystopian or a utopian future, does it matter? after all, from the "perspective of the brain, there's a thin line between a good decision and a bad decision". it seems, though, that what matters to everyone is the future, because the future is "where we are all going to spend the rest of our lives". however, perhaps this must also not be the case as, paradoxically, most (or all) dichotomies have a limitation in that they not only trade accuracy for simplicity, but also provide an invisible barrier to a vision of the bigger picture. this is where subtle linguistic changes may gradually trigger cognitive changes and eventually bring in legal and institutional changes, in the same way as dripping water hollows out stone, even though the cognitive habits of binary or dualistic thinking are said to die hard. easy or difficult, it is a source of encouragement for everyone, as we may, à la longue, be able to bring about the change we desire, given that institutional change is possible once a cognitive change has occurred. the recent rise in the number of oxymora and paradoxes, however, seems to indicate this "creative" possibility. for instance, various concepts implicitly reflecting the tensions between prediction and destiny within the confines of past and present, such as science fiction or free will, have been qualified as oxymora. in their context (and possibly in the context of all paradoxes), time therefore appears as the clue, or the key we need to find to unlock the limitations inherent in our current linear perception of time. the fact is that the grammar of many modern languages, conjugating verbs in the "present," "past," and "future" tenses, was said to not be "well-adapted for speaking about the real temporal structure of reality, which is more complex". various "nostalgic visionaries", dimitropoulos ( ) lang ( ) , p. . csicsery-ronay jr. ( ), p. ("as its name implies, science fiction is an oxymoron."). crewe ( ) , p. . rovelli ( ), p. . like herbert g. wells or jules verne, however, transcended the past-future dichotomy in their writings, thereby anticipating many future inventions. lawyers, too, can act in this way, as their work is often similar to that of science fiction authors, given that they can help in translating fiction into legal fact or turning dreams into reality by combining "the law as it is" (lex lata) with "the law as it should be" (de lege ferenda) perspectives. for lawyers to achieve this creative goal and to synthesize the two competing scenarios of the future beyond the wto outlined in this chapter, it will be necessary, first, to work actively towards a cognitive change. applied to legal reasoning, it means to transcend an exclusively dualistic mode of reasoning, which means to solely rely on the "law of the excluded middle" (i.e. "everything must either be or not be."). applied to the regulation of global trade, this kind of "either/or thinking" or that something either belongs to the sphere of trade or is classified as a "non-trade concern" must be complemented by the law of the included middle. the law of the included middle can be achieved through oxymoronic thinking by way of which, for instance, trade and non-trade concerns are not opposed to each other but where their apparent contradiction can be resolved at a higher level of reality or complexity. more concretely, lawyers must be able cognitively to transcend the iron law of binary logic by also being versatile in reasoning based on polyvalent logic-a kind of multivalued logic in which there can be more than two truth values. a good way to remind ourselves of this is given in the following quote: "laws may differ but they do not conflict: the only possible conflict is in the mind of the judge". in this regard, legal education seems the best place to begin. thus, addressing problems by legally expressing them solely using dichotomies is no longer apt to deal with their underlying complexity. it also means that a purely static approach should be complemented with a more dynamic one to ensure that laws are adopted in a way that embraces change ex ante and makes them fit for the future. in institutional terms, a more dynamic approach is also needed, as otherwise we merely observe their initial rise and subsequent demise. in this respect, discursive institutionalism provides an excellent complement, as it regards norms as "dynamic, intersubjective constructs rather than static structures". ultimately, it is submitted here that, based on this cognitive change, novel models of global institutions, like gaia , can emerge. second, the cognitive changes of polyvalent or oxymoronic thinking, when combined with new organs of perception as symbolized by synaesthesia, will enable another important insight, which lies in the realization that the best way to predict the future is by creating (and regulating) it. this insight puts the theory of a selffulfilling prophecy into a new light, one which proves that theory and practice are intimately linked and that sociological predictions are "products of an era, co-determinants of what they assert". when applied to humanity as a whole, predicting the future by creating it (and regulating it accordingly) means that if we plan something and act upon it coherently and persistently, it is more likely to happen, eventually. this, however, first requires a cognitive change in the understanding of humanity as a divided amalgam of different peoples or nations instead of an organic whole united in its diversity. only then can an adequate global governance mechanism capable of putting the necessary global legal order into place be expected to be brought forth. in short, the global governance paradox and other apparent contradictions can only be successfully addressed by expanding our reasoning from bivalent to polyvalent modes of thinking, as reflected in synaesthesia or in new optimized ways to receive and channel information through our different senses or, in legal terms, different institutions. applied to the dichotomy of the past versus the future, a new cognitive mode may one day enable humans to recall the fundamental importance of the present, that is, the magical power of the present to rewrite both the world of yesterday and the world of tomorrow. by the same token, humans can then eventually use the same magic to control the outcome between the two apparently opposite scenarios, the dystopian and the utopian one. for now, the two scenarios outlined in this chapter cannot answer the questions of whether or not, in our perception, linear time exists, or whether or not we have free will or all fate (including the fate of the wto) is already written in a comprehensive book of destiny. this chapter is equally unable to say whether a post-wto legal order will be able to avoid global disaster and humanity's as well as nature's demise. however, the hope is that the chapter shows that the many "scientifically fictitious" developments described in both the dystopian and the utopian scenarios concur in essence or are-if at all-divided by a very thin line, one merely drawn in our minds. nevertheless, this thin line determines how we will deal with the most urgent challenges in the anthropocene, or at any given point in time (when no longer understood as the linear process from the past to the future), because it clearly proves that today we humans cannot say that "we did not know (about the dangers and inherent consequences of our actions)". on the contrary, we can only say "we knew but did not care". neurath ( ), pp. - . g ( ) ministerial statement on trade and digital economy should robots pay taxes? tax policy in the age of automation failing states, collapsing systems: biophysical triggers of political violence cryptocurreny & robots: how to tax and pay tax on them artificial intelligence and legal analytics: 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examining the neonatal synaesthesia hypothesis international relations and international law: from competition to complementarity the doha declaration and beyond: giving a voice to non-trade concerns within the wto trade regime investor-state dispute settlement reform and theory of institutional design global energy governance in the g- : states, coalitions, and crises nuclear energy and sustainability: understanding iter belief in life-after-death, beliefs about the world, and psychiatric symptoms legal fictions systemic regulation of global trade and finance: a tale of two systems how to save a constitutional democracy choice of logic and choice of law policy coherence or conformance? the new world bank-international monetary fund-world trade organization rhetoric on trade and investment in developing countries energy and the wealth of nations: an introduction to biophysical economics theorising the global legal order homo deus: a brief history of tomorrow the age of transition: trajectory of the world-system the evolution of official development assistance: achievements, criticisms and a way forward world trade and the law of gatt. the bobbs merril company irresolvable norm conflicts in international law: the concept of a legal dilemma the culture of time and space - the ghost in the machine fuzzy thinking: the new science of fuzzy logic reflecting on 'linkage': cognitive and institutional change in the international trading system can the trading system survive us-china trade friction? convergence of wto dispute settlement and investor-state arbitration: a closer look at umbrella clauses ) the appellate body of the wto and its reform auras in mysticism and synaesthesia: a comparison dead aid: why aid is not working and how there is another way for africa oil's tipping point has passed the civil right we are not ready for: the right of free movement of people on the face of the earth global market integration and the creative economy: the paradox of industry 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revolution: how the technology behind bitcoin and other cryptocurrencies is changing the world. penguin, new york ten hoopen g et al ( ) auditory isochrony: time shrinking and temporal patterns my inventions and other writings on the fiscal strategies of escaping poverty-environment traps towards sustainable growth balance-of-payments crises in the developing world: balancing trade, finance and development in the new economic order a proposal for international monetary reform big data and personalized price discrimination in eu competition law technology and progress in jules verne, or anticipation in reverse a propos de la lex ferenda the human rights of stateless persons felt time: the psychology of how we perceive time requiem for a paradox: the dubious rise and inevitable fall of hipster antitrust currency wars: offense and defense through systemic thinking a hater's guide to geoblocking key: cord- -itkj o o authors: moser, susanne c.; hart, juliette a. finzi title: the long arm of climate change: societal teleconnections and the future of climate change impacts studies date: - - journal: clim change doi: . /s - - -z sha: doc_id: cord_uid: itkj o o “societal teleconnections” – analogous to physical teleconnections such as el niño – are human-created linkages that link activities, trends, and disruptions across large distances, such that locations spatially separated from the locus of an event can experience a variety of impacts from it nevertheless. in the climate change context, such societal teleconnections add a layer of risk that is currently neither fully appreciated in most impacts or vulnerability assessments nor in on-the-ground adaptation planning. conceptually, societal teleconnections arise from the interactions among actors, and the institutions that guide their actions, affecting the movement of various substances through different structures and processes. empirically, they arise out of societal interactions, including globalization, to create, amplify, and sometimes attenuate climate change vulnerabilities and impacts in regions far from those where a climatic extreme or change occurs. this paper introduces a simple but systematic way to conceptualize societal teleconnections and then highlights and explores eight unique but interrelated types of societal teleconnections with selected examples: ( ) trade and economic exchange, ( ) insurance and reinsurance, ( ) energy systems, ( ) food systems; ( ) human health, ( ) population migration, ( ) communication, and ( ) strategic alliances and military interactions. the paper encourages further research to better understand the causal chains behind socially teleconnected impacts, and to identify ways to routinely integrate their consideration in impacts/vulnerability assessment and adaptation planning to limit the risk of costly impacts. electronic supplementary material: the online version of this article (doi: . /s - - -z) contains supplementary material, which is available to authorized users. teleconnections are a widely recognized phenomenon in the physical sciences, made most famous by the el niño-southern oscillation (enso). the globally most significant physical teleconnections are generally those that originate in the ocean or atmosphere (e.g., enso). however, teleconnections can occur between and across land, ocean, freshwater and atmosphere. beyond the direct physical impacts, sometimes extensive and expensive environmental and societal impacts also ensue in their wake. for instance, an el niño event can have a devastating impact on the peruvian fisheries. in the western u.s., there is strong evidence that winter and spring snow cover extent over north america impacts monsoonal variables in the desert southwest, such as the total amount and frequency of precipitation, hail, wind and severe weather (hawkins et al. , see also supplementary material # for a more extensive discussion on societal impacts from physical teleconnections). but teleconnections are in no way constrained to the physical world alone. the phrase bteleconnections^has been adopted by some social scientists to help convey the idea that vulnerabilities and climate change impacts do not just originate and unfold in one place but can also result from long-distance relationships (adger et al. ). the idea of bsocietal teleconnections^thus is analogous to that of physical teleconnections, but is focused on the human-created linkages via people, structures, institutions and processes. for instance, floods in thailand in (driven by the strongest monsoon in years) killed more than people locally, disrupted local agriculture, and forced some , computer supply and electronic car and camera part manufacturing factories to close and lay off , employees (garside ) . in addition to the economic impact locally, it also had rebounding impacts globally. before the floods, one facility owned by western digital (a silicon valley-based corporation) produced % of the company's hard-drive production and % of the world's supply of bsliders,^a prime component of hard-disk drives. post-floods the cost of hard drives doubled (acclimatise ). the event not only caused about $ billion in damages to the thai economy, and weakened the economic recovery throughout asia, it also had far-reachingand expensiveimpacts on a leading economic sector in california, usa. this example is now widely cited by the private sector and has spurred several fortune firms to begin to assess supply chain vulnerabilities-one possible societal teleconnection-through the lens of climate change (gledhill et al. ) . this example of an economic teleconnection illustrates how local climate vulnerabilities can and do originate in far-away places. this adds a layer of risk that is currently not fully appreciated in most climate change impacts assessments and on-the-ground adaptation planning. yet being unprepared for these additional risks can cost millions of dollars and, sometimes, lives. in short, teleconnections point to a risky gap in both research, policy and planning. this paper proposes a systematic, but pragmatic and simple conceptual framework that helps researchers and practitioners identify how what happens afar can affect them. it then remarkably, however, the recognition of physical teleconnections is not at all new: the southern oscillationthe atmospheric component of ensowas first characterized by the british mathematician and meteorologist, sir gilbert walker. in he wrote: bby the southern oscillation is implied the tendency of pressure at stations in the pacific … to increase, while pressure in the region of the indian ocean … decreases.^a few years later, angstrom ( ) was the first to use the term bteleconnections^in reference to the north-south dipole atmospheric anomaly pattern now referred to as the north atlantic oscillation. highlights and conceptually explores a range of societal teleconnections that have arisen out of societal interactions and the globalization of our world and which create, amplify, and sometimes attenuate climate change vulnerabilities and impacts in regions far from those where a climatic extreme or change occurs. our goal is to encourage further research to better understand the causal chains behind socially teleconnected impacts, and to identify ways to routinely integrate their consideration in impacts research and adaptation planning. to achieve the latter, the inclusion of long-distance relationships in locally-focused assessments must remain a manageable task for adaptation practitioners in the private and public sectors who neither have easy access to nor the capacity for complex systems modeling, but whose assets are nonetheless at stake. societal teleconnections . societal teleconnections: a simple but systematic framework the notion of societal teleconnections applies the idea of long-distance interactions and connectivity to the social realm. while the basic idea of spatial interactions in the physical/ natural environment or in societal activities is as old as the discipline of geography, and therefore has a rich theoretical and empirical basis in that field, the idea of societal teleconnections was first explicitly discussed by adger et al. ( ) . societal teleconnections link activities, trends, and disruptions across large distances, such that locations spatially separated from the locus of an event can experience a variety of impacts from it nevertheless. the concept of teleconnections broadens place-based discussions of climate change impacts to also include the dynamic, process-based implications of long-distance connections across the globe (seto et al. ; liu et al. ). there is a broad body of literature on the impacts of globalization and related activities on social, economic, and coupled human-natural systems (e.g., young et al. ; leichenko and o'brien ) . to pick just one example, several studies have examined the impacts of deforestationdue primarily to global demands for increased cropland and local slash and burn agricultureon communities located near the sites of deforestation (aide and grau ; lambin and meyfroidt ) . such studies clearly bring into focus the interconnectivity across space, but these connections have not been fully accounted for to date in climate impacts and vulnerability studies. moreover, while globalization is one of the main processes creating and perpetuating societal teleconnections, it is by far not the only important way in which locations and events get connected across long distances. others have developed the related but broader notion of btele-coupling^as an overarching conceptual framework to link complex, coupled human-environment systems across spatial distances (e.g., liu et al. liu et al. , eakin et al. ) . while teleconnections as conceptualized here (and more commonly used in the climate change literature) focuses on the structural and functional connection itself, tele-coupling extends the integrative systems research (particularly in land use and land change science) that emerged over the past two decades on coupled natural-human systems to account for long-distance influences on the functioning of these systems. the influence of biofuels production, such as ethanol from corn, on land clearing and thus carbon emissions is a good example (e.g., melillo et al. ; youngs and somerville ) . our framework resembles that of tele-coupling, but aims to keep it as simple as possible, yet as complex as necessary, to uncover distal vulnerabilities via a distinct focus on the connection itself. societal teleconnections require at minimum three basic components in order to occur. if one or more are missing, the teleconnection is interrupted (fig. ) : & a natural system or human-built construct that forms the conveying or transmitting physical structure (metaphorically, one may think of this as the bhardware^). it does not itself move but stays in place as a teleconnection is made. the finite set of types of physical structures include water, energy, transportation and communication-related infrastructure such as roads, ports, pipes, transmission lines, wires, or satellites. & the process explains the reason, manner or cause for the teleconnection (the bsoftware^). it establishes a functional exchange or relationship between distant entities. examples of an almost infinitely large set of social processes include the market, travel or migration, human needs and desires, or social and cultural ties. this process is enacted by actors, who in turn are enabled and constrained in their actions by applicable institutions, i.e., the social norms, cultural customs, laws, rules and regulations that govern the interaction. & a material substance or immaterial element that is moved from one location to another in the course of a teleconnection. it is the only part in a teleconnection that physically moves (the bdata^). again, the finite set of substance types include money, energy, goods or materials, biological agents, people, and information or ideas. the combination of structure, process and substance result in connection and movement over long distances, often in complex ways and through combinations of mechanisms. importantly, there is a finite number of physical structures and of types of substances, but a large number of types of processes, and with them come a wide variety of actors and institutions. the latter are involved in establishing, maintaining and sometimes disrupting the physical structure of a teleconnection. they also enact important services as agents of change and as guardians of the causal interconnectivity (and thus as producers, consumers or movers of the substance being conveyed). actors are also crucial as those who can increase or decrease a community's vulnerability or resilience in the face of disruption originating elsewhere. this basic way of organizing and thinking of teleconnections helps explain how each component exerts its effects, and how climatic changes and climate-driven disruptions can impact communities and sectors across long distances. in essence, climate change and its impacts can: & interrupt, modify, reroute or create structural connections; & alter, establish, disrupt or eliminate (with the help of actors and/or changed institutional arrangements) the causal processes; and & transport at lower or greater quantity, different quality, or speed certain substances or elements (both familiar, new or unknown). the resulting wide range of long-distance relationships and interactions can either attenuate or amplify the impacts of far-away events on local circumstances (pidgeon, kasperson, and slovic ; renn ) . thus, societal teleconnections should not be understood as inherently good or bad (although the same teleconnection can lead to impacts perceived as positive at one scale, and as negative at another): how exactly they play out will depend on a number of factors, including some that have nothing to do with the teleconnection itself, but with the ability of local communities, sectors, and actors to deal with variable circumstances in their specific context (i.e., preparedness, robustness, and resilience). of greatest importance, however, may be those far-away events that can exert economic, social, public health or political harm in local contexts. using the systematic conceptualization of teleconnections introduced above, we have delineated a few crucial teleconnections ( table ) . several of these connections and how climate change impacts will buse^them to cause far-reaching impacts (i.e., the blong arm of climate change^) are discussed below with their main causal relationships as well as key agents and institutions involved. the sample of teleconnections listed in table is incomplete, several are interrelated and some can be nested within or may result in other teleconnections mentioned. we would argue nonetheless that the ones listed are teleconnections that fundamentally affect the resilience of local communities or even larger regions as many current examples of crises attest (weatherrelated disasters, the ebola epidemic, social and military unrest in the middle east). below, we discuss some of these societal teleconnections through the lens of our conceptual framework and discuss what communities might consider to begin planning for these teleconnections. one of the most important long-distance relationships in society results from global economic relationships. a quick look at the labels in clothing or the stickers on food indicates the origin of what we consume locally. while perceived as necessities, these economic ties in a globalized economy make consumers, and intermediaries involved anywhere along the trade chain vulnerable to impacts occurring far from a product's origin. teleconnections can derive from disruptions to the production process, supply chain, or critical transportation routes and communication channels. impacts on the perceived desirability of sites or shifts in the labor market due to climate related impacts can ultimately increase production costs and consumer prices. in the example of the thai floods, examining the impacts to one sector specifically, e.g., hard drives sold by a company such as western digital, the structure that allowed this teleconnection to occur were the transportation routes and communication channels that established the manufacturing linkage between silicon valley and thailand. the process that drives the teleconnection is the desire for cheap labor and the globalization of manufacturing. the substances that should have been transported were hard drive components and subsequent financial transactions based on a completed work flow. the key actors directly involved include regulators in thailand allowing the hard drive producing subsidiary to locate in the floodplain and those at western digital (and/or their intermediaries) deciding from whereand where aloneto purchase key equipment for their silicon valley firm. land use plans and regulations, building standards, and disaster preparedness policies and measures are the institutions most directly affecting the thai location, but the blaw of supply and demand^, shareholder agreements, the drive to ever larger profit margins, indirect economic drivers, and organizational policies and norms (or lack thereof) to downplay safety and disaster preparedness across the trade system in question also contributed to the ultimate impact. the thai floods resulted in $ - billion in insurance claims (garside ) , thus bringing in yet another set of actors, and rippling through the insurance industry with its own set of institutional arrangements. the example highlights the interconnection of trade and economic interests with insurance interests, which is why we integrate them here. importantly, this example of supply chain teleconnections should not be confined to the private sector. local governments also have their own supply chains, both for their normal operations (e.g., materials and equipment needed in water treatment, fire safety, local infrastructure maintenance) and even more so in the case of emergencies (e.g., food and water, heath services, building materials). another subset of economic activity, singled out because of its importance in all societal and economic activities, is energy production, transmission and use. pathways are both direct and indirect, and disruptions can have local, regional, national, and sometimes even international ripple effects. energy production in one location can be disrupted by climate change impacts and thus directly (through immediate interruptions of supply through brown-or black-outs) or indirectly (through increased energy prices for energy purchased from elsewhere) affect consumers in faraway places. the spatial extent depends on the area served by the affected utilities and on supply redundancies in the system. climate change impacts on supply may result from extreme heat (e.g., affecting nuclear power plant operations), inland or coastal flooding as sea level increases (power plant operations), or drought (availability of and competition among various users for scarce water resources for hydropower generation, nuclear power, fracking, etc.). similarly, energy transmission can be disrupted by climate change-related extremes (wildfire, heat, storms, ice). again, the spatial extent depends on the area served by the affected utilities and on supply redundancies in the system. in september , a transmission line tripped due to high heat in yuma, arizona, usanotably a location already adapted to high heatwhich led to a chain of approximately additional events over an -minute period culminating in the shutdown of the san onofre nuclear power plant in california. the power loss impacted arizona, california and mexico and resulted not only in long-distant impacts along the interconnected energy system, but also cascading impacts on other sectors, such as sewage spills and water distribution disruptions in the city of san diego, usa, impacting more than seven million people (wilbanks et al. b ). using our teleconnection conceptualization described above, the structures that initiated this teleconnected event are the transmission lines, transmitters and power stations that connect power generation and use, in this instance, across multiple states. the processes underlying the linkage involved the energy market, driven on one level by supply and demand, yet more directly by the decisions of energy producers and traders, and the energy policies and market regulations that affect utilities' decisions. the primary substance moved (or rather: no longer moved) was electricity, but as the event unfolded also sewage. as the cascade of events proceeded, different actors came to be involved, such as water treatment plant operators, emergency responders and, of course, the affected consumers and businesses. with reduced redundancies in the supply system, due to higher electricity demands during heat waves, the most immediate impacts of such a disruption may be only regional or maybe statewide, but energy markets often go beyond state boundaries, thus potentially having farreaching ripple effects through direct losses and increases in energy pricing (sathaye et al. ) . the role of cascading impacts due to interdependencies and a better understanding of the behavioral, ecological, and technological coupling and feedbacks were identified as critical gaps in knowledge in a recent report informing the third u.s. national climate assessment (wilbanks et al. a ). in the case of energy transmission and distribution, feedbacks and impacts will be exacerbated in urban environments due to the higher population and increased number of infrastructural interdependencies; what is necessary for efficiency in large urban centers may in turn contribute to a greater risk of cascading impacts (wilbanks et al. b ). migration of people is among the more widely recognized societal teleconnections. migration always has multiple reasons. people tend to not wish to migrate away from their ancestral or cultural home unless faced with complex and extreme conditions affecting their ability to maintain their security (o'brien and barnett ; birkmann et al. ; black et al. ) . climate change in this context has been conceptualized as a bthreat multiplier^ (cna ) that may aggravate already challenging circumstances to the point where people will consider leaving home. such aggravated threats may come from climate change impacts such as reduced water availability with negative impact on the ability to sustain minimal subsistence requirements or grow food for external markets; sea-level rise and related repeated coastal flooding, progressive coastal erosion, permanent inundation, and saltwater intrusion undermining public safety and the ability to maintain land, communities, economic activities or agriculture in place; or any number of other direct and indirect impacts of a climate-altered environment. migration in the context of climate change is considered a transformative adaptation (kates et al. ) . a report by the international organization for migration (iom) noted the distinction between migrations due to sudden, extreme events versus slow-onset environmental impacts (laczko and aghazarm ) . for instance, after hurricanes katrina and rita, a large proportion of the city of new orleans was evacuated and , homes were either destroyed or severely damaged, resulting in several hundred thousand city residents without a place to return to. by october ,~ % of evacuees had returned to their pre-hurricane addresses and % had returned to the counties in which they lived prior to the hurricanes (groen and polivka ) . in contrast to sudden events, migration from slow environmental degradation, which is expected to increase in the future, may have larger and longer lasting impacts and affect more people. between and , million people were affected by storms compared to . billion people affected by gradual environmental changes (em-dat ). the structures through which the teleconnection is established are the legal or illegal transportation routes, and the substance being moved are people, but the reasons for migrating, the causal processes, and thus the actors and institutions involved, can vary significantly. in the case of an extreme event, the causal process may be the need for temporary safety and shelter, involving evacuees, first responders, and those providing temporary refuge, while people migrating permanently away from an environmentally degraded home region may need relocation/housing assistance, help with finding new permanent work, assistance with sociocultural integration and so on. such help can take many forms, and can both help or hinder in maintaining community functioning and ecological integrity, which in turn increase local resilience in the face of other future stresses. for instance, hecht and saatchi ( ) describe how in el salvadore, outmigration remittances allowed relatives who stayed behind to be able to buy food, rather than utilize slash and burn agricultural practices to grow their own food, thus contributing to forest resurgence instead of further deforestation. in both instances of short-term and permanent relocation, migration has immediate and lasting impacts on the affected regions as well as permanent demographic, economic, health, and socio-cultural impacts on the source and destination regions (mcintosh ; hori and bowman ) . considering the teleconnection of people via migration in local adaptation planning requires that a local jurisdiction develop a better understanding of their community's demographics and socioeconomic conditions to assess who may be mobile, immobile and most at risk of being dislocated (e.g., black et al. ) and their community's capacity to absorb temporary or permanent new residents. for instance, sea-level risein combination with other complex, non-environmental processesis expected to cause outmigration over time from low-elevation island nations (e.g., piguet ; marino ; mcleman and hunter ). migrants may seek new homes in the u.s., australia or new zealand, where there are already established migration pathways, kin or cultural communities. for communities that already have sizable populations of former island residents residing there, it is reasonable to expect additional influx as sea level continues to rise (e.g., perch-nielsen et al. ) . anticipating this increase in immigration allows the jurisdiction to identify municipal and community needs and resources to adequately prepare. a slow-onset impact, such as sea-level rise, also illustrates that some teleconnections can play out in advance of the actual climate change impact manifesting, i.e., people may decide to abandon a location due to the anticipated impact prior to it actually becoming uninhabitable (kiribati's bmigration with dignity^plan, aimed at establishing expatriate communities off-island for i-kiribati people to join on their own terms instead of waiting for a refuge problem later is one example [wyett ] ). our final example focuses on health. human health and well-being are the result of complex interactions of social, economic, occupational, lifestyle, psychological, cultural and genetic factors. this is a clear case of individual to global interconnectivity. mcmichael ( ) discusses the correlation of climate change and globalization in human health. among those discussed, climate change can have impacts on human health through the spread of disease vectors; further spread of vectors due to migratory displacement; impacts on fresh-and coastal water quality; impacts on air quality; impacts on food production . the sars epidemic of highlights how a virus originating in one corner of the world (in this instance, the guangdong province in china) can quickly spread globally (with cases reported in canada), due to international trade and travel (adger et al. ). in this instance, the structures leading to the linkage were the existing ground and international air transportation infrastructure, andimportantlythe global communication infrastructure that carried both the threat and warnings ahead of the threat around the world. the causal processes driving the teleconnection were related to trade and travel and the substance transported was the sars virus (and secondarily warnings and information about the health threat). the actors involved included those affected by the disease, health care providers and local, national and international health system observers (who and national centers for disease control) under the international and intra-national agreements and guides for public health emergency response. the communication of these sorts of teleconnected, global pandemics is expected to increase with climate change. however, the enhanced flow of information also allows the world to respond quickly to potential outbreaks, epidemics and disasters and there is increased coordination in vaccination programs and systems to respond to infectious disease (mcmichael ) . thus, human health provides an excellent example of how teleconnections connect the local to the global and can both attenuate and amplify different situations. the types of teleconnections introduced above are easily exemplified, but have not yet been assessed systematically, neither globally nor nationally nor locally. while a range of studies have used the broader tele-coupling framework on questions of land use, ecosystem services, and sustainability more broadly (e.g., challies et al. ; liu et al. ; güneralp et al. ; seto et al. ) , few studies have attempted to do so in the context of climate change (e.g., perdinan and winkler ; adger et al. ). these global connections have been largely neglected in u.s., and other national and global climate assessments to date. the importance of formally assessing such long-distance linkages, however, has been recognized in the third u.s. national climate assessment (nca) and in working group ii of the ipcc's fifth assessment (e.g., oppenheimer et al. ; corell et al. ; wilbanks et al. a ) and is an important area for further improvement in future assessments. below we highlight some key areas ripe for further investigation. quantifying the impacts of teleconnections is actually quite complex. the case of the thai floods may be rare in that there was a fairly direct link between economic losses and companies whose manufacturing plants were flooded and ensuing cascading impacts. in many instances, however, societal teleconnections are more difficult to trace and/or are deeply interwoven, thus making analyses difficult. quantification of these interconnections requires a systems modeling approach that can work at both the local and globallyintegrated scale in order to catch the nuance of place-based repercussions of global linkages (wilbanks et al. b; perdinan and winkler ) . moreover, when dealing with impacts to trade, it can be difficult to get information since data typically are proprietary or considered trade secrets. for many societal teleconnections, it will be difficult to point to a clear or singular cause and effect. for instance, the arab spring cannot be solely attributed to wheat shortages in china or russia (sternberg ; werrell and femia ) . similarly, it may never be possible to pin a violent conflict singularly on climate change impacts, yet multiple, complex teleconnections between the threat multiplier of climate change and national security, regional stability and violent conflict are of growing concern to the military (e.g., cna ). in-depth studies of cases such as these can help sort out and better understand the significance of different societal teleconnections, informing the development of systematic assessment models. the linkages between and among different societal teleconnections are complex, but ubiquitous. it is therefore critical to identify bthe most important^linkages for different types of climate change impacts. some may dominate regardless of context whereas others may play out differently depending on context. under what circumstances do which of these linkages matter the most? this may not be a simple question to answer in light of the various components of vulnerability: exposure, sensitivity and adaptive capacity all affect how severely a climatic change or extreme event manifests locally, and each of these components of vulnerability is affected differently by societal teleconnections (e.g., global economic forces, cultural teleconnections via education and knowledge flows co-determine local adaptive capacity; legal and governance structures shape capacities and barriers to adaptation). the answer to these questions, however, will be of critical value to adaptation planners in public offices as well as in private firms who may otherwise be overwhelmed by the universe of potential teleconnections. with an increasing number of people choosing, or having no choice but, to live in urban areas, the impacts of societal teleconnections have the potential to be compounded. as in the example of the san onofre power plant shutdown in , seven million people were impacted by that -minute series of events. identification of urban interdependencies and redundancies, and how those could be impacted with increasing population, is critical for effective disaster preparedness and adaptation. how is local resilience enhanced or diminished through societal teleconnections and how can communities use this understanding in their efforts to prepare and plan for the long arm of climate change? in the thai floods, for instance, production and sales for silicon valley companies (and elsewhere) were temporarily crippled because there was no redundancy in their supply chains. building diversity into business operations may reduce economic efficiency in the short-term but buffer against major disruptions and economic losses over the medium to longer term. how can such diversification strategies be applied to local communities? how do legal, communication, social and cultural teleconnections simplify or complicate adaptation options? in summary, societal teleconnections are important considerations for locally-based climate change vulnerability assessments and adaptation planning in the public and private sector. while not necessarily understood in technical terms (bteleconnections^), the high-profile flooding in thailand has had a major awareness-raising impact on private firms, which now increasingly assess the impacts of climate change on their supply chains. by contrast, societal teleconnections are not yet included in local vulnerability assessments or adaptation planning, largely because they are not recognized or, if they are, they are considered too complex to assess or manage. work to date on tele-coupling has highlighted the complex natural-human systems interactions, yet most communities do not have resources or capacity to conduct such computationally-demanding studies. we have therefore proposed a simple and pragmatic but systematic framework for better identifying, understanding, and thus planning for, societal teleconnections. in our examples, we also focused on teleconnections that are crucial to basic community functioning and thus relate most easily to land use planning and resource management, basic service provisions, and emergency preparedness, which is where most adaptation planning currently resides. the research questions we have identified certainly do not exhaust the research agenda on societal teleconnections, but they delineate key elements of further examination that would significantly improve our understanding of local vulnerabilities to climate change in a deeply interconnected world. answers to these types of research questions would also support a needed addition in focus to adaptation planning in urban, business or resource management contexts that expands the perspective from the local to the relevant global. building our understanding of societal teleconnections, in this way, would help counteract some persistent but misleading slogans about vulnerability and adaptation: neither the context of vulnerability nor all adaptation is ever just local. it is glocal. infographic: in thailand was under water; but the ripples were felt across the world nested and teleconnected vulnerabilities to environmental change globalization, migration, and latin american ecosystems teleconnections of climatic changes in present time enhancing early warning in the light of migration and environmental shocks migration and extreme environmental events: new agendas for global change research what role for social-ecological systems research in governing global teleconnections? national security and the threat of climate change climate change impacts in the united states: the third national climate assessment significance of telecoupling for exploration of land-use change international emergencies disaster database thailand flooding costs lloyd's of london $ . bn. the guardian 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energy infrastructure from projected climate change urban land teleconnections and sustainability chinese drought, wheat and the egyptian uprising: how a localized hazard became globalized the arab spring and climate change: a climate and security correlations series climate change and energy supply use. technical report by the u.s. department of energy in support of the third national climate assessment climate change and infrastructure, urban systems, and vulnerabilities. technical report to the u.s. department of energy in support of the third national climate assessment escaping a rising tide: sea level rise and migration in kiribati the globalization of socio-ecological systems: an agenda for scientific research best practices for biofuels the authors wish to acknowledge the union of concerned scientists who provided support and funding for part of this work, as well as the feedback of several anonymous reviewers and the participants in a session on supply chain risk at the california adaptation forum in august . we have appreciated all their inputs, but take full responsibility for the views expressed here. key: cord- -r ii bu authors: butler, colin d.; corvalan, carlos f.; koren, hillel s. title: human health, well-being, and global ecological scenarios date: - - journal: ecosystems doi: . /s - - - sha: doc_id: cord_uid: r ii bu this article categorizes four kinds of adverse effects to human health caused by ecosystem change: direct, mediated, modulated, and systems failure. the effects are categorized on their scale, complexity, and lag-time. some but not all of these can be classified as resulting from reduced ecosystem services. the articles also explores the impacts that different socioeconomic–ecologic scenarios are likely to have on human health and how changes to human health may, in turn, influence the unfolding of four different plausible future scenarios. we provide examples to show that our categorization is a useful taxonomy for understanding the complex relationships between ecosystems and human well-being and for predicting how future ecosystem changes may affect human health. the interconnection between ecosystems and human activity is complex, important, and poorly understood. ecosystems support human health and well-being through their provisioning, regulating, cultural, and supporting services (butler and others ) . shortages of food, fiber, and other ecosystem products adversely affect human health via many direct and indirect pathways. the regulating functions of ecosystems that affect health include the purification of air and fresh water, the reduction of flooding and drought, and range limitation of certain vector-borne diseases. ecosystems also impact mental well-being through provision of cultural services, for example by providing totemic species and sacred groves, landscapes, and water bodies. these influence the aesthetic, recreational, educational, cultural, and spiritual aspects of the human experience. ecosystem changes have also altered the epidemiology of communicable and noncommunicable diseases, including through some pathways that would not be considered as arising from a reduced ecosystem regulating service. this article explores the impact that different plausible future scenarios (cumming and others ; raskin ) may have on health, and it also suggests how changes to health may feedback on and hence modify the course of the future. in doing so, we have categorized the effects of ecosystem change on human health into a useful taxonomy which can help predict which future ecosystem changes will impact human health and how. although positive scenarios are conceivable, in this article we are mainly concerned with how adverse ecosystem changes and reduced ecosystem services may harm future human health. until the very recent past, most human induced ecological changes have had favorable effects on human society and health. human health, judged by average life expectancy, has increased substantially, as has population size (riley, ; tuljapurkar and others ) . the reasons for these increases are well-known and include the mutually reinforcing and interacting elements of improved knowledge, technology, and social organization, including of public health (horiuchi ; szreter ) . a fundamental contributory factor has been the increased human capacity to modify ecosystems, for example, by increasing food supplies, by restricting populations of large carnivores, and by providing more fiber for fuel and shelter. this success is not unqualified. some adverse effects of our increased capacity to modify ecosystems on human health can already be seen. the transformation of ecosystems to provide certain benefits has reduced the scale and integrity of many ecosystems (pimentel and others ) . reduced ecosystem integrity decreases their ability to provide some ecosystem services, which can, in turn, have negative impacts on human health. this relationship is unlikely to be linear and may contain thresholds beyond which incremental loss of ecosystem services has a disproportionately negative effect on human-health and well-being. examples of the negative impacts of ecosystem change on human health abound. national life expectancy has fallen in several countries, including many parts of sub-saharan africa, haiti, russia, north korea, and ethiopia (farmer and others ; shkolnikov and others ; united nations population divisional ) . reduced ecosystem services may explain a part of these declines in national life expectancy and thus may be an underrecognized factor in the slowed rate of increase in global life expectancy. the problems of decreased provision of ecosystem services are often unequally distributed, with the majority of the burden falling on the poor. additionally, poor populations frequently lack the income and other means to substitute or partly compensate for reduced ecosystem services (for example, by boiling microbiologically contaminated water). in addition to the effect of ecosystem services on human health, human health itself influences access to critical ecosystem services and can modify the environmental impacts of human populations. for example, the aids epidemic in sub-saharan africa has reduced the provisioning ecosystem service of food supply (de waal and whiteside ) . the high prevalence of yellow fever and malaria delayed the construction of the panama canal, and sleeping sickness still limits human settlement and thereby affects human access to ecosystem services in parts of central africa (bhalla ) . a major challenge in this field is to apply real world data to conceptual models (miranda and others ) , to validate the models, and to develop approaches that can serve as a vehicle for generating hypothesis-driven research. although realization of these goals remains distant, the conceptual frameworks which will stimulate data acquisition and analysis in this field are developing (butler and others, ) . figure shows one such framework, linking natural and social systems with human wellbeing, of which health is an important component. here we introduce four categories of adverse effects on human health due to ecosystem change as a means to help understand the impacts of the different ecological scenarios on human health and well-being. in ascending order of scale, complexity, and lag-time, we call these adverse effects direct, mediated, modulated, and systems failure (see table ). we emphasize that, at their margins, these categories overlap because drivers may differ on temporal and spatial scales. figure graphically presents these concepts and provides a preliminary attempt to approximate the quantitative impact of the different categories. direct (adverse) health effects are manifested through the immediate impacts of the loss of a useful ecosystem service, such as the provision of sufficient food, clean water, fertile soil or the restriction of erosion and flooding. direct effects occur as the result of physical factors but do not include pathogens per se. miranda and others, ) climate change has recently been recognized as causing a substantial change in the lake tanganyika ecosystem. the fish catch has decreased due to a climate-related reduction in the nutrient supply (o'reilly and others ) . this reduction in ecosystem services places additional economic and nutritional stresses on an already poor and vulnerable human population. although data to measure the health effect of this reduced catch are unlikely to be available, the effect is probably adverse because it causes reduced income and reduced nutrition (verschuren ) . another example is the collapse of cod fishing in the north atlantic, which caused widespread unemployment, mental distress, and social dislocation, but little if any true under nutrition because the social mechanisms operating in canada were able to partially substitute for the lost provisioning services once supplied by the fishery. a third example of a direct health effect from a reduced ecosystem service is the disruption and physical injury caused by flooding. there is increasing recognition that floods are caused by the interaction of climatic and landuse changes (hellin and others, ; zhang and others, ) . there is also increasing evidence that mental and physical health is enhanced by contact with nature (friedman and thomas ) . reduction of the cultural services that ecosystems provide is likely to contribute to the already enormous burden of disease caused by impaired mental health. compared to direct effects, mediated effects have increased causal complexity and, in some cases, involve pathogens. some mediated effects have the potential for high rates of illness and death. there is also often a longer lag between the ecosystem change and the health outcome than for direct effects. however, by definition, mediated effects are insufficient in scale to cause the larger-scaled social collapse that we define as a modulated effect. many infectious and some chronic diseases fall in this category. the epidemiology of many communicable diseases is related to ecological factors. some major nonvector-borne diseases, including tuberculosis, measles, and influenza, are thought to have crossed into human populations because of close contact with domesticated animals (mcneil ; daszak and others, ; oxford and others, ) . changes to biodiversity may be associated with increased numbers of disease-transmitting insects. although contested, there are suggestions that malaria may also have become a significant human disease following the development of agriculture (pennisi ; joy and others ) . more recently, variant creutzfeld-jacob disease, nipah virus, and hendra virus illustrate novel infectious diseases that have entered human populations because of changed and more intensive animal feeding and farming practices (waltner-toews and lang ). the emergence of nipah virus may also have been related to bats fleeing from the intense drought and el niñ o-related fires in indonesia (epstein and others, ) . the long list of other infectious diseases related to ecosystem change (patz and others, ) includes schistosomiasis (li and others, ) , cholera (pascual and others, ) , and lyme disease (jones and others, ; blockstein ) . in many of these cases, the disease has emerged as a result of increased food-producing capacity of ecosystems-a provisioning ecosystem service-for example, by animal domestication, irrigation, dams, and other intensive farming practices. a tradeoff has been the unforeseen increase in the incidence and prevalence of many of these communicable diseases. some mediated health effects have also led to migration, while others have prevented the colonization of certain areas. for example, malaria has long restricted human settlement in lowland areas, including the terai in nepal, and many parts of equatorial africa. some chronic, noninfectious diseases can also be classified as mediated effects of ecosystem change, including allergies, asthma, and some forms of cancer and chronic lung disease. for example, lung cancer and pulmonary fibrosis have become particularly common in the region around the shrunken aral sea, as pesticide-contaminated dust from this human-made desert is inhaled (o'hara and others ). both long-distance dust transport and more localized air pollution are also related to ecosystem service change and have been linked with a number of diseases, including asthma and atopy (monteil ) . there is also increasing evidence that air pollution, often exacerbated by ecosystem change such as land clearing and fires, may aggravate heart disease (pyne ) . future ecosystem change, such as desertification, leading to a decrease in the ecosystem provisioning service of clean air, could thus alter the epidemiology of these diseases. these diseases are classified as mediated rather than direct because their connection to changed ecosystems is more complex than are direct effects. direct and mediated health effects are analogous to the direct and indirect health effects of climate change. in that classification, direct (adverse) health effects include phenomena such as heat stroke, while indirect effects include changes to certain vector-borne diseases because of altered patterns of temperature, humidity, and rainfall, and other effects secondary to extreme weather and adverse economic effects. it is possible that a novel emerging disease could escape from a remote ecosystem to enter the wider human population, as the plague and hiv probably did. however, at least in the case of hiv, its really major (modulated) impact depended on powerful social cofactors, including severe poverty, social practices and taboos, and poor governance (butler a (butler , b . the ecological factors that underlie the recent sars outbreak remain unclear (enserink and normile ) , but its origin and amplification in a region of china, characterized by extremely dense populations of humans and domesticated animals and by poor public health services (anonymous ) , is consistent with the view that human-dominated ecosystems today harbor more danger to population health than does the ''wild'' (oxford and others ) . a plausible example of this principle could be the widespread transmission of multi-or even omni drug-resistant tuberculosis emerging from a prison (tanne ; dye and others, ) . this scenario would have severe economic implications, especially for aviation and other industries perceived as increasing the probability of disease spread. we also identify a larger-scale, more lagged, and more causally complex adverse consequence of adverse ecosystem change, than either direct or mediated effects, which we call a modulated or tertiary effect (figure ). these effects include episodes of state failure, or of nascent or realized large-scale social and economic collapse. the role of environmental factors in the causation of large-scale conflicts, state failure, and social collapse is controversial (deudney ; gleick ; homer-dixon ; uvin ; cramer ) . we agree that causation for the phenomena is complex, but we assert that reduced ecosystem services and other adverse ecosystem changes are frequently a component of the causal webs that lead to these phenomena (butler and others ) . this may be of increasing significance in the near future as evidence accrues that ecosystems are being changed more frequently and at larger scales. there is compelling evidence that reduced ecosystem services were a causal factor for several large-scale social collapses and catastrophes, from both archeological sources (weiss and bradley ) and more recent history. two ancient cases are the collapse of the ancient mesopotamian and the mayan civilizations, contributed to, respectively, by increased salinity (jacobsen and adams ) and drought (haug and others ) . two more recent examples are the irish famine of the s and the rwandan genocide in . the irish famine was caused by the spread of a potato fungus (wilson ) interacting with a refusal by the british government to supply an effective substitute, such as famine relief (sen , pp - ) . the rwandan genocide also occurred as a result of the interaction of multiple factors, including poor governance, long-standing ethnic hatred, and rapid population growth. the violence was inflicted mainly by a large number of unemployed young men (mesquida and weiner ; potts ) , displaced from a livelihood in farming because of the shortage of fertile arable land, thus losing a key ecosystem service (andré and platteau ; butler- a) . in these examples adverse health effects are likely to be larger than those from mediated effects, although in some cases state failure may be limited to small populations, such as for the people of easter island or the norse in medieval greenland. inevitably, ecosystem service changes that contribute to modulated effects will be embedded in a mosaic of social, economic, and political cofactors. in turn, many of these cofactors are likely to have at least partial ecosystem change-dependent causation. depending on the knowledge, bias, and experience of the observer, the causal role of ecological factors in state failure may sometimes be underestimated, or even totally denied. for example, rotberg ( ) identifies the roots of state failure as based in ethnic, religious, linguistic, or other intercommunal enmity. he argues that state failure is ''man-made, not merely accidental nor-fundamentally-caused geographically, environmentally, or externally.'' we do not claim that reduced ecosystem services or other ecosystem changes that lead to adverse health effects are always a ''fundamental'' factor in state failure, but they are often important and usually identifiable. the enmity that rotberg refers to often arises over the distribution of diminishing per capita ecosystem services. there may be increasing recognition of this. for example, o'reilly and others ( ) concludes, in discussing the potential for further reduction in the ecosystem provisioning service of lake tanganyika, that ''the human implications of such subtle, but progressive, environmental changes are potentially dire in this densely populated region of the world, where large lakes are essential natural resources for regional economies.'' ecosystem services as a significant element in state failure may be underrecognized due to our tendency to discount the future possibility of thresholds or emergence. thresholds refer to sudden, nonlinear changes that result from a small increment and that are not intuitively predictable without prior experience (alley and others ; waldrop ; may ) . emergence refers to the new property that becomes apparent beyond the threshold. modulated and systems failure effects (described below) are emergent phenomena that become apparent when linked socioecological systems pass a threshold, caused by the interaction of numerous social, political, and ecological elements. we also describe an even larger scale phenomenon than state failure, as a result of coalescing, interacting modulated effects. we call this phenomenon ''systems failure.'' the increasing connections that insulate diverse human communities from scarcity also create large-scale vulnerabilities, magnifiable by feedbacks such as collapsed global trade, terrorism, technological breakdown, and radiating failure of institutions and governance. collapse could occur on a regional, continental, or even global scale. it is also possible, however, that a reverse state, ''systems success,'' could occur. large-scale epidemics exacerbated by chronic food insecurity, poor governance, and wide-scale conflict are plausible elements of this pathway. drug-resistant bacteria, in part driven by the excessive use of antibiotics in animal husbandry, could contribute to this, as could the emergence of new viruses. however, we stress that novel infectious agents are unlikely to lead to modulated or systems failure effects without significant cofactors. only modulated and systems failure effects are likely to be of sufficient scale to alter the unfolding of the various ecological and socioeconomic scenarios that are described elsewhere in this issue of ecosystems. cumming and others ( ) and raskin ( , this issue) review several socioecological scenarios that may unfold over this century. although all plausible futures are influenced by the same driving forces, these forces evolve in different ways in the different scenarios. demographic, economic, political, cultural, and social factors -including health -are codependent so that each factor will continually influence other factors. each scenario will influence and be influenced by ecological factors as well. just as in the past, the future world will contain a mixture of familiar and novel situations. scenarios seek to coalesce these myriad possibilities into a limited number of theme futures that have strongly plausible elements. although there are dozens of names for the existing environmental scenarios, most can be categorized into a surprisingly small group of core pathways, as described by cumming and others (this issue) . the names of the four scenarios considered in this article are market forces, reformed market, value change, and higher fences (see table ). it is difficult to succinctly describe these scenarios, but a number of axes can be identified along which they vary. for example, there is a spectrum identifiable between comparative economic deregulation (market forces) to a neo-keynesian model, here called reformed market. another spectrum can be identified between a concerted attempt to protect existing ecosystem services (the ''value change'' scenario) and a laissez faire approach to ecosystems and the nonliving environment, such as climate change (the ''market forces'' scenario). a third spectrum is identifiable between trade deregulation (''reformed market'') and continuing or even increased protectionism (''higher fences''). as well, the trend of global income distribution can be predicted from these scenarios, from a continuing increase (''higher fences'') to a marked decrease (''reformed market'' and ''value change''). the state of health for high-and low-income populations can be predicted, largely consequent to the anticipated change in income for each group. three of these scenarios, as very briefly described, are essentially optimistic, because they all assume an increase in income for high-and low-income populations. however, in the higher-fences world, it is conceivable that incomes will decline for populations that currently have a low income, and the increased global inequality in this scenario could exacerbate tensions between low and income populations. cumming and others ( ) examine the assumptions made about ecosystem resilience in each of four scenarios and find that the outcome of different scenarios is influenced by this resilience. table lists some key terms concerning health for both high-and low-income populations in these four scenarios. it also shows our estimation of the probability of changes in the ''health gap,'' that is, the gap between high-and low-income populations with respect to health. at present, for example, the burden of disease from diarrhea (of which nearly % can be attributed to unsafe water, poor sanitation, and hygiene) is over times higher in the least developed countries than in developed ones. the health gaps between high-and low-income populations have been systematically estimated using a measure called ''disability adjusted life years'' which accounts for total years of life lost because of disease and also for partial years of life lost because of disability (world health report ) . it is likely that in three of the four scenarios the health gap will decrease. this is because three of these scenarios assume gradual socioeconomic convergence between populations that are currently rich and poor. these scenarios postulate continued advances in science, technology, and the dissemination of information and expertise. the response to the sars outbreak illustrates the potential for a coordinated response to events that are perceived as sufficiently threatening. if convergence between rich and poor populations occurs, then coordinated responses to numerous health problems that continue to affect poor populations are likely. advances could improve new vaccines, attention to ''orphan'' diseases, and dis-tribution of the improved seeds and agricultural techniques needed to enhance food security. in these scenarios the health of high-income populations is also likely to improve, though not as rapidly as for low-income populations. in only one scenario -called ''barbarization'' (raskin, this issue) or ''higher fences'' (cumming and others, this issue) -is the health gap likely to increase. in this scenario, poor populations are increasingly ignored by the remaining population. food security of the poor is likely to further diminish, perhaps leading to positive feedbacks as malnutrition impairs cognitive development and further hinders education and the chance of skillful social and political responses. in this scenario the health of high-income populations is unlikely to be ideal: we identify obesity, diabetes, and anxiety as likely to increase, with their negative effects only partially countered by improved medical technology. modulated effects could sabotage even optimistic scenarios. table provides an estimation of the chance of systems failure, depending on assumptions concerning the resilience of ecosystems and the linked socioeconomic system. cumming and others ( ) have defined ecosystem resilience as the capacity to absorb anthropogenic impacts without the loss of essential structure or functions. we suggest that it is meaningful to assume that human populations are characterized by social resilience, which modifies their capacity to effectively deal with stress (carpenter and others ) . from a public health perspective, resilience may be defined as the capacity of society to respond to problems and challenges, over the short and long term, in ways that protect and advance public health, over the short and long term. affluence, education, social cooperation, technological capability, and flexibility are important determinants of the size of social buffers and human resilience. we suggest that systems failure effects are more likely to occur in the market-forces and higher-fences scenarios, because inequality between high-and low-income populations is likely to be the greatest in these scenarios. the most optimistic future for human health is likely to be if both ecological and social systems prove highly resilient. in this case both major ecosystem and social services are likely to be preserved even if ecosystem changes (currently perceived by many as adverse) continue to occur at a high rate. on the other hand, the chance of further modulated or even systems failure effects occurring is increased if ecosystem and social systems prove to be brittle. because the size of ecosystem service buffers is falling and the extent of ecosystem service resilience is uncertain, it is prudent to reduce fur- ther ecosystem damage as rapidly as possible. however, it is also prudent to conduct this in a way that minimizes harm to human health. continuing tradeoffs are likely to be required. for example, improving the health of populations in indonesia may require further clearing of forests to generate more income. but this process cannot be continued indefinitely. on the other hand, excessively strict protection of the surviving ecosystems could reduce the size of the socioeconomic buffer, thus perhaps increasing the longterm risk of a modulated effect. in reality, economic and social forces make an extremely rapid transition to full protection of surviving ecosystems unlikely, but it may be just as risky to delay protection in response to only short-term socioeconomic concerns. the causal relationship between ecosystem service change and impact upon human health remains incompletely understood. we have explored how ecosystem services impact human health and have proposed that adverse ecological changes can interact and feedback with dysfunctional social responses, leading to the development of states that we have termed mediated and systems failure. we have grouped the myriad possible interactions and cascading responses between ecosystem services and public health into four categories, a previously undescribed taxonomy which might help us better comprehend how ecosystem change and human health interact to affect the way the future unfolds. we believe that this is an important conceptual advance that will be useful for understanding the relationship between human health and changes in ecosystem services. continued analysis of cases studies, using both quantitative and qualitative methods, will advance our understanding of these relationships. several regions of the world, characterized by substantial ecological and social stresses, may be useful ''natural laboratories'' for this purpose, including sub-saharan africa, indonesia, and northern india. direct and mediated effects, although they may lead to some important changes to the path of the future, are unlikely to seriously compromise the development of regional or global civilization. despite occasional and localized setbacks, human health is likely to generally improve if future ecosystem changes result only in events such as occasional flooding, periodic disease outbreaks, and episodes of air pollution. on the other hand, modulated and systems failure effects, if they occur, have the power to alter the course of society in significant ways. if negative this will cause substantial harm to human health and well-being, and, by exacerbating poor governance, could also further erode ecosystem services. such events could derail even the most optimistic scenario. however, we do not deny the chance that positive systems effects could emerge, especially if ecosystem and social resilience remain high. current trends toward an increasingly large environmental footprint, further climate change, depletion of fossil fuels, and the erosion of existing ecological and social buffers are disturbing and unlikely to be sustainable (mcmichael ) . on the other hand, the increasing capacity to conceptualize, diagnose, and modify the global environment gives hope that humanity will self-organize in ways that can sustain both its social and ecological functions (crutzen ) . abrupt climate change land relations under unbearable stress: rwanda caught in the malthusian trap emerging stronger from the china crisis pan african group takes lead against the tsetse fly lyme disease and the passenger pigeon? entrapment: global ecological and/or local demographic? reflections upon reading the bmj's 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decreases aquatic ecosystem productivity of lake tanganyika world war i may have allowed the emergence of ''spanish'' influenza cholera dynamics and el niñ o-southern oscillation effects of environmental change on emerging parasitic diseases malaria's beginnings: on the heels of hoes? ecological integrity: integrating environment, conservation, and health. washington dc the population policy pendulum. needs to settle near the middle and acknowledge the importance of numbers small particles add up to big disease risk global scenarios: background review for the millennium ecosystem assessment rising life expectancy: a global history the new nature of nation-state failure development as freedom changes in life expectancy in russia in the s rapid economic growth and the four ds of disruption, deprivation, disease and death: public health lessons from th-century britain for st-century china? drug resistant tb is spreading worldwide a universal pattern of mortality decline in the g countries world population prospects: the revision. the demographic impact of hiv/aids tragedy in rwanda: the political ecology of conflict global change: the heat on lake tanganyika complexity. the emerging science at the edge of order and chaos a new conceptual base for food and agricultural policy: the emerging model of links between agriculture, food, health, environment and society what drives societal collapse? infectious disease: an ecological perspective world health organization china's forest policy for the st century we thank the millennium ecosystem assessment for providing many opportunities to discuss the main ideas expressed in this article with colleagues. we also thank professor d. crawford-brown and two anonymous reviewers for their comments and suggestions. key: cord- -z x hnu authors: baykasoglu, adil; durmusoglu, zeynep d. u. title: a classification scheme for agent based approaches to dynamic optimization date: - - journal: artif intell rev doi: . /s - - -x sha: doc_id: cord_uid: z x hnu several papers in the literature employ agent-based modeling approach for providing reasonable solutions to dynamic optimization problems (dops). however, these studies employ a variety of agent-based modeling approaches with different strategies and features for different dops. on the other hand, there is an absence in the literature of a formal representation of the existing agent-based solution strategies. this paper proposes a representation scheme indicating how the solution strategies with agent-based approach can be summarized in a concise manner. we present these in a tabular form called “agent based dynamic optimization problem solution strategy” (abdopss). abdopss distinguishes different classes of agent based algorithms (via communication type, cooperation type, dynamism domain and etc.) by specifying the fundamental ingredients of each of these approaches with respect to problem domain (problems with dynamic objective functions, constraints and etc.). this paper also analyzes generic studies in the literature employing agent-based modeling based on abdopss. on the other hand, some of the researchers studying operations research (or) have attempted to solve these problems by using the well-known traditional optimization techniques like linear programming and integer programming . however, solutions obtained for a certain time that is desirable or optimal for that time slot, often have not be preferable for another time slot. furthermore, obtaining exact solutions using mathematical methods for each time slot has been unaffordable or even sometimes has not ended-up with feasible solutions. therefore, notwithstanding its widespread use, the static optimization approach has increasingly approached its limits (borst et al. ) . this fact certainly inspired many researchers to employ agent-based modeling for the optimization of dynamic systems. as indicated by jung et al. ( ) , the agent paradigm has been shown to be a promising approach to develop intelligent, heterogeneous, and open systems, because of its features, such as autonomy, flexibility, and cooperative problem solving behavior (jung et al. ) . in this regard, reusable agents have provided many opportunities and they saved researchers from moving beyond reinventing, representing, and re-implementing the problem and thereby the cost of providing solutions has been decreased (neches et al. ) . agent-based models addressing dynamic optimization problems (dops) have also allowed several optimization mechanisms to track the moving optima in the search space. this fact certainly inspired many researchers to employ agent-based modeling for the optimization of dynamic systems. even though there have been several papers in the literature employing agent-based modeling approach to provide significant solutions for dops, each of these existing studies employs different agent-based modeling approaches having different strategies and agent features. therefore, currently, we don't have a common successful methodology for the design of agents (corchado et al. ) . with dependability and safety in mind, it is vital that the mechanisms for representing and implementing agents are clear and consistent (fisher et al. ) . in this regard, it is certain that there is a vacancy in the standard representation and classification of agent-based solution approaches employed for dops. a previously developed scheme (calégari et al. ) for evolutionary algorithms for combinatorial optimization has also inspired us to prepare a standard representation format. in this perspective, one of the objectives of this paper is to present the fundamental features/ingredients of dops solved by agent-based modeling. the features positioned in a standard scheme provide an opportunity to present dops in a standard way. it can succeed further and in future studies these representation forms can be used to study the role of these features and their importance for solution quality. thereby, this study provides a basis to analyze the best ways of implementing agent-based approaches to the dops. in this perspective, this paper presents an attempt to develop a notion for dops solved by agent-based approach. the paper shows how solution strategies with agent-based approach for dops can be summarized in a concise manner by informing about the agents employed and the key elements of dop's. a classification scheme is introduced and presented in a tabular form called agent based dynamic optimization problem solution strategy (abdopss). abdopss distinguishes between different classes of agent based algorithms (via communication type, cooperation type, dynamism domain and etc.) by enumerating the fundamental ingredients of each of these agent-based approaches. at the end, possible uses of the abdopss are illustrated and exemplified for some agent based approaches. the paper is organized as follows. sections and introduces the features listed as the ingredients of abdopss. section specifically focuses on the features of typical dops. subsequently, sect. introduces the generic features of agent-based approaches. in sect. , some typical agent-based solution approaches that are implemented for dops are described by using the abdopss. section illustrates the application of abdopss for relevant articles collected from the literature. finally, sect. presents concluding remarks with the possible benefits of the proposed classification scheme. a dynamic problem is considered to be a problem that changes some or all of its characteristics in time (lung and dumitrescu ) . thereby, optimization problems that are subject to changes, belongs to the class of dynamic optimization problems (dops) (allmendinger and knowles ) . in other words; dops are "optimization problems whose optimal solution changes over time during the optimization, which could result from change of environmental parameters, change of constraints, change of objectives and change of problem settings (representations)" (jin ) . all of the changes that are mentioned above take place in the dynamic environment and these changes create unpredictability even chaos in some situations. these changes can be opportunities improving the solutions or sometimes they can be threats decreasing level of desirability of a solution. there are different approaches in the literature that is classifying these changes in the dynamic environments. yang ( ) defines environmental change as: cyclic and random. in cyclic change of environments, with the time going, an old environment reappears exactly (yang ) . a cyclic change covers the same fixed length sequence of contraction and/or growth is repeated over time (abbas et al. ). the pattern repeats itself indefinitely (abbas et al. ). however, in random change, there is no information about the structure of change. random change is also called as acyclic and in random change there are no repeated patterns over the time frame; that is, there is an indefinite random sequence of contractions, growths, and random changes (abbas et al. ) . all of these changes in dynamic environment cause significant changes in model's structure, objective function, restrictions/constraints, parameters, and in the decision variables. bui et al. ( ) describe three of these changes through the examples as presented below. time-varying objective functions: for example enemy units arrive at a location, making some parts of the objective more difficult. the objective function is not constant over time. therefore, the objective value of a solution can be different at different times. this usually causes the occurrence of new optima (bui et al. ) . time-varying variables: an example problem of this category is dynamic machine scheduling where unexpected new jobs arrive. a time-varying variable can be used for determination of the objective value, but can be a late addition or change (bui et al. ) . time-varying constraints: for example the precedence relationship of tasks. the objective function and variables do not change for this category; however, the constraints may change over time. this category of change does not change the fitness landscape driven by the objective function, but it will affect the areas of feasibility. it is interesting to note that this category of change has not been analyzed in detail in comparison to the other two categories (bui et al. ) . cruz et al. ( ) provides a valuable survey of research done on optimization in dynamic environments over the past decade. in addition to the content of their paper, they provide a web site (cruz et al. ) and present characteristics of the dops with respect to objective function or the restrictions change with time. we have considered the study of abbas et al. ( ) to illustrate correspondence between the change types and the changes in the modeling components. abbas et al. ( ) defines change in the sample bias parameter change since processing times for machines are estimated using sampling, an unexpected sampling error can end up with the change in actual processing times six common kinds of changes for a typical system having certain data flow structure. these changes are: change in the model, change in the number of concepts, change in the number of features, change in the level of noise, change in the class distribution, and change in the sample bias. table both summarizes the change states defined by abbas et al. ( ) and their corresponding change in a typical mathematical model which is similar to the one defined by cruz et al. ( ) . examples of each change categories have also been provided through the possible changes in a dynamic production facility. according to the example case, a typical production facility that is operating in a dynamic environment faces with several expected and unexpected changes with the above given change states. change : change in the model: change in objectives of a system may lead a significant "change in the model". think of the production facility trying to minimize the cycle time and adjusting all of its production strategy with respect to cycle time minimization. if the cost of tardy job increases significantly and the managers will be obligated to change their objective as the minimization of the number of tardy jobs. in this case, the objective function, constraints and the number of decision variables along with the type of parameters may change. change : change in the number of concepts: sometimes, concept classes may not be as clear-cut as initially thought so that additional classes may arise or old classes may vanish over time (abbas et al. ) . in case of change in the number of concepts, resources used by the system can increase or decrease. new machines can arrive to the system or some machines can be removed from the system because of the oldness or in functionality. this change can affect the model by changing the variables used in objective function and constraints. this type of change is not expected to create structural change. it is a kind of reconsideration of the model with added/removed variables that is significant for decisions. change : change in the number of features: in some problems, the number of available features characterizing a problem instance may vary over time. additional information may become available or new tools may be developed that allow more accurate classification (abbas et al. ). orders received for new product types are a typical example of this change. in the face of such a change, a production facility may face with new operations required and this can lead changes in objective function, constraints and the number of decision variables. change : change in the level of noise: the noisiness of the data may change as well (abbas et al. ). this type of change is particularly common when dealing with data coming from sensors. for example, acoustic data collected in an open area can have different noise levels based on the state of the environment (abbas et al. ) . in this respect, in the production facility example, if some customers broke the deal and cancel their orders for a production facility, the plans unfortunately changes and it may cause change in objective function and constraints. change : change in the class distribution: abbas et al. ( ) exemplifies the change in the class distribution with sars virus. when the sars outbreak took place, the class distribution changed almost every day with more healthy people becoming infected effectively increasing the proportion of positive cases. similar to sars example, in case of an economic boom, arrival of some certain jobs to the system can be more frequent and this leads change in parameters. change : change in the sample bias: since system parameters like processing times for machines are estimated by the sampling of several actual processes, an unexpected sampling error can end up with the change in actual processing times and/or system arrival times. all those changes defined above also directly effects the unpredictability of the problem domain. level of unpredictability is vital to determine a proper agent-based solution strategy. although there are several efforts to match the corresponding changes with the unpredictability, they are still in their infancy. therefore, there is a serious need for measuring the level of unpredictability of the dops. the relation between the corresponding changes in constraints, objective function and the both with uncertainty and dynamism has been previously presented by (cruz et al. ) in the web site (cruz et al. ) as shown in fig. . while their framework as shown in fig. does not cover the variability in parameters and variables; it can still be a good reference for future studies. considering parameter variability and variability in the number of decision variables will certainly increase the complexity of such an analysis. it is also remarkable that dynamism is one of the components of environmental uncertainty. indeed, as presented in fig. , environmental uncertainty has three dimensions: dynamism, heterogeneity, and hostility (newkirk and lederer ) . dynamism, as being the one dimension of the environmental change, (teo and king ) found to have two dimensions. the dimensions are referred to here as changeability (i.e., concerned with the rate of obsolescence and of technology change) and unpredictability (i.e., concerned with competitors' moves and demand changes). the rigor of that study and relative recency of the findings motivated the use of those two dimensions in the current research (newkirk and lederer ) . in this regard the above defined six different changes cover different level of changeability and unpredictability. categorizing changes into these two dimensions is a topic of future research. objective function objective function different dops tried to be solved in the literature for different scopes by using various approaches. some of the existing studies on dops via agent-based modeling propose only frameworks some other studies also present applications. it is remarkable to state that a framework is not a detailed hypothesis or set of hypotheses; rather, it is a suggested point of view for an attack on a scientific problem, often suggesting testable hypotheses (aggestam and söderström ) . therefore, studies covering solely the frameworks do not provide comparable outputs. some of the applications cover real-life instances and some have hypothetical data obtained via simulation. some of the studies attempting solve dops by using agent-based modeling use test problems that has been previously presented in the literature. thereby, they compare their findings with the best known results in the literature. some other studies compare the results with themselves by providing different methodologies to solve the problems. examples of these cases will be discussed in the applications section. although some problems like scheduling, production planning and travelling salesman have been previously solved as they are static problems, indeed these problems are not totally static problems due to the nature of the real life. consider an example of scheduling and production planning problems where new machines with advanced capacities are required to be included to the systems or some machines are required to be removed from the system and/or new products are can also be introduced to the system etc. another example is for a more real life oriented travelling salesman problem (tsp) has been defined by (homayounfar et al. ) . they defined three different types of changes for tsp: * changing the distances (time) between the cities (due traffic congestion, road repair etc.) * changing the number of the cities (i.e. adding or deleting some cities) * swapping the cities a distance (e.g. the distance between the first and second cities) is changed after a specific time or specific generation, determined by dynamic frequency. the amount of change is referred to as the dynamic rate. these two parameters are set prior to the test. after each period of time, which is after a generation specified by dynamic frequency, dynamic rate is added to the specified cost value (i.e. distance between city and ). this shifts the peak (optimum cost) to another location, so a new global solution can be found (homayounfar et al. ) . numerous examples can be considered for real-life oriented versions of these well-known problems. the distinction between these conventional problems and real-life oriented ones can be performed as follows. if during the solving of an optimization problem, parameters of the system (i.e. objective and constraint functions) do not change due to nature of the problem then optimization is static, otherwise it is considered to be dynamic (homayounfar et al. ) . in such environments, the optimization process is non-terminating. in the real world, a function to be optimized may vary from time to time and the optima have to be found in time (guan et al. ) . therefore, it is very difficult to optimize such type of dynamic optimization problems with conventional methods which are developed to deal with non-stationary environments. moreover, it is also quite hard to define dependencies or correlations between the solutions obtained at a time slot and in another time slot. as stated by o'hare et al. ( ) , the real world is both unpredictable and unforgiving. decisions often need to be made where the contributory evidence is uncertain, incomplete, contradictory and highly dynamic (o'hare et al. ). therefore, performing design of experiment may not end up with significant findings. since finding a feasible solution to the static problem is np-hard, we must make certain assumptions about the input. intuitively, if the input is such that even finding a static solution is hard we cannot expect to find a good solution with respect to the dynamic objective function. thus the problem instance must be "easy enough" that a relatively straightforward agent can find a feasible solution at each time step. in practical terms, this means there must be enough resources to easily satisfy the demand if we ignore the quality of the solution in the sense of the dynamic objective function. in the worst case, we can fall back on the heuristic to find a feasible solution. in fact, agent-based algorithm will degrade gradually to this extreme, but should perform much better in the common case. this challenging dynamism was apparently a significant problem for researchers and it could only be effectively handled by using more advanced approaches like agent-based modeling. although there is no universal agreement in the literature on the precise definition of agent, their property of being autonomous has been common to all definitions (kulkarni and tai ). an agent is an encapsulated computer system that is situated in some environment and that is capable of flexible, autonomous action in that environment in order to meet its design objectives (jennings et al. ) . agents that act in an autonomous fashion to perform delegated tasks have aroused much attention over the last decade (liu et al. ) which are named as intelligent agents. an intelligent agent performs interactive tasks tailored to a user's needs without humans or other agents telling it what to do (máhr et al. ) . the intelligent agent has been proposed as a software design paradigm, which is different from the sequential, the structural and the object-oriented approaches by its autonomy in deciding when to invoke its action (parunak ) . the fundamental approach of agent based system is to simulate real-world systems with a group of interacting autonomous agents modeled as computer programs (zhou et al. ). since agent based modeling is a consideration that is developed according to system requirement, it is expected that they have several common and distinguishing characteristics. in fact, in spite of the growing interest in multi-agent systems, there is no agreement on what actually constitutes agenthood, that is, what are the fundamental characteristics of agents (garcia et al. ). on the other hand, jou and kao ( ) defined the characteristics of an agent based on agent's behaviors: fundamental, auxiliary, implicit and application oriented (role based). the first one is fundamental characteristics. as a goal is state of affairs to be achieved in the environment, an agent must perceive and process the environment changes (jou and kao ) . the second one is auxiliary characteristics. an agent also needs information from users and other agents through communication mechanisms such as user interfaces and agent communication language to complete the delegated tasks (jou and kao ) . in addition to fundamental and auxiliary characteristics, some implicit characteristics with presumptive feature exist within an agent (jou and kao ) . trustworthiness (veracity and benevolence), perceptibility, rationality, persistence, flexibility, and competence are some examples (jou and kao ) . the final characteristics are related to specific applications, e.g., the surfing behaviors of cyberspace, the facial expressions attached to entertainment, and so forth (jou and kao ) . all these features make agent technology an interesting approach for a wide set of applications (garcia-montoro et al. ). an agent based model has a set of agents defined by the model creator. number of agents can be variable or constant. their types or functions may also vary or they can be assigned for repetitive tasks. in meta-heuristics based agents, number of agents can directly be proportional (or the same) with the population/colony size. both "number of agent types and number of agents" should be considered in the design of agent-based modeling for dops since they can affect the solution space directly. in his study, tan ( ) , claims that, as the number of agents is increased, state space exponentially increases in terms of the number of agents. a large state space means more state ex-ploration for the model that tan ( ) considers, and this yields slower learning. multi-agent system, also called 'self organized system' is a computational system in which multiple interacting intelligent agents work together to solve difficult problems which may be impossible for an individual agent (yan et al. ) . multi-agent based modeling allows complex natural behavior of various interacting entities to emerge from a set of simple individual rules (razavi et al. ) . communication is the most common means of interaction among intelligent agents. any observable behavior and its consequences can be interpreted as a form of communication (mataric ) . two different approaches have been defined for communication (genesereth and ketchel ) . direct communication covers the agents handling their own coordination. designer of an agent-based system can consider direct communication with respect to problem domain. the cost of communication and availability of obtaining qualified solutions are the factors to be considered for employing direct communication mechanisms in the models. on the other hand, assisted/indirect coordination covers the systems in which agents rely on special system programs to achieve coordination (genesereth and ketchel ) . one of extensively employed indirect communication method is stigmergy. stigmergy is a class of mechanisms that mediate animal-animal interactions. it consists of indirect communication that is taking place between individuals of an insect society by local modifications induced by these insects on their environment (hadeli et al. ) . although communication is essential for agent-based modeling, it may not be perfect as expected. in most of the current research on multi-agent systems, people assume that communication of agents is guaranteed (satoh et al. ) . however communication can be broken, suspended or delayed depending on the flow of data or information. in this regard, research of problem solving under incomplete communication is very important (satoh et al. ) . since dops may have varying objectives, constraints/restrictions and parameters; agents cannot be informed on the global state of the system by themselves. therefore, in order to achieve global objectives, coordination is essential to allow the agents to adjust their local states or conditions. communication provides channels for coordination. coordination is a property of a system of agents performing some activity in a shared environment (huhns and stephens ) . the degree of coordination is the extent to which they avoid extraneous activity by reducing resource contention, avoiding livelock and deadlock, and maintaining applicable safety conditions (huhns and stephens ) . figure illustrates the taxonomy for the types of coordination defined by (huhns and stephens ) . panzarasa et al. ( ) prefer to use the generic term "interaction" instead of "coordination". they (panzarasa et al. ) define cooperation as working together to achieve a common objective and they also define negotiation as coming to a mutually acceptable agreement on some matter (panzarasa et al. ) . in other words, cooperation is coordination among nonantagonistic agents, while negotiation is coordination among competitive or simply self-interested agents (huhns and stephens ) . however, perhaps the most fundamental and powerful mechanism for managing interagent dependencies at run time is negotiation-the process by which a group of agents come to a mutually acceptable agreement on some matter (jennings et al. ) . negotiation underpins attempts to cooperate and coordinate (both between artificial and human agents) and is required both when the agents are self interested and when they are cooperative (jennings et al. ). planning distributed planning negotiation fig. taxonomy of coordination for agent-based modeling (huhns and stephens ) there are different approaches used as optimization mechanism in the agent-based systems for dops. these methods can be classified as: heuristic based approaches, exact mathematical methods, and market based approaches. heuristic based approaches cover, evolutionary based approaches and other techniques like: particle swarm optimization, immune-based algorithms ant colony optimization etc. heuristics: for large size problems it is hard to obtain the optimal solution due to the large size of the problem files. in order to reduce the computational time, heuristic approaches can be used for obtaining solutions . heuristics includes some set of procedures for obtaining acceptable solutions to the problems by decreasing the time requirements. there is a natural correspondence between autonomous entities and meta-heuristics, and problem solving with an optimization problem (pelta et al. a,b) . in the literature, agents are sometimes defined as individuals, particles etc. (wang and shixin ) which is the main actors for heuristics. if one takes a look at natural process like bird flocks or fish schools a strong similarity to multi-agent systems can be found very quickly (wagner et al. ) . in this regard, there are many studies adapting heuristic approaches to agent-based systems. mathematical methods: although it is too complex to deal with dops using classical mathematical methods, increasing power of computer technologies let some researchers to use mathematical methods. change frequency and the response time of the mathematical methods directly affect the solution's availability at the desired time. in this regard, the preferability of mathematical methods appears to be less when compared with the other methodologies. market based approaches: for an optimization approach to be considered as market oriented, it must at least fulfill the following basic requirements (karlsson et al. ): -there must be a well-defined market mechanism which includes some notion of prices (which often are expressed in terms of some monetary unit). the market mechanism regulates how negotiations and trade are performed among the participating agents and hence determines how certain commodities can be traded for certain other commodities (karlsson et al. ). -there must be some arguments for why the agent strategies are reasonably realistic, given the market model. that is, assume we have some model of information available for the different agents and a well-defined model for how they interact (the market mechanism). then the strategies must be consistent with the agents' attempt to maximize utility, given bounded rationality (karlsson et al. ). the first generic step of developing a solution strategy for any optimization problem is to define specifying class of the problem. class of the problem is an important factor that is affecting the required solution approach. therefore, the ingredients that characterize an agent-based modeling approach for a dop must somehow posses a presentation scheme to provide a solution strategy and a basis for comparison of one with another. therefore, this paper presents how solution strategies with agent-based approach can be summarized in a concise manner. in this regard, a classification scheme is designed and presented in a tabular form called abdopss (agent based dynamic optimization problem solution strategy). abdopss distinguishes different classes of agent based algorithms applied to dops (via agent-related features like: communication type, cooperation type and problem related features like: dynamism of objective functions, parameters etc.) by enumerating the fundamental ingredients of each of these algorithms with respect to problem domain. the ingredients that have been considered for abdopss have been discussed in the previous sections. the main structure of the table consists two rows (row , row as illustrated in table ), indicating the problem related features (row ) and agent related features (row ), respectively. at each corresponding column of a row, an entry, gives the necessary indication for the corresponding criteria. table shows such an empty table and corresponding values for the table. since it takes a considerable time to determine features listed by abdopss, articles are exemplified within the contents of this paper. it is remarkable to state here that, this paper does not seek to find all publications on dops and represent those using abdopss, but it is rather sought for exemplification of the proposed abdopss. in this regard typical versions of different approaches have been tried to be illustrated in table . two rows are merged into one for easiness of readability. the studies covered here are roughly classified and some unclear parts made it relatively difficult to prepare abdopss. it is certain that those papers could be better presented by their developers. pelta et al. ( a,b) presented a multi-agent decentralized cooperative strategy (mad-cos) for dynamic optimization problems. in madcos, a population of cooperative agents has moved over a grid containing solutions. in order to test and analyze both different communication schemes for the agents and real need of an explicit diversity preserving mechanism in madcos, researchers focused on cooperation and diversity mechanisms. moreover, two types of diversity mechanisms were investigated. different configurations of the moving peaks benchmark problem were employed as a test bed. a set of computational experiments have been set to evaluate how different communication strategies affect the search; and the usefulness of having explicit and implicit diversity mechanisms. last offline error (oe) ("accuracy") and relative error between the best found solutions ("error") have been recorded to measure the performance of each configuration. results showed that having an researchers indicated that regarding diversity, the implicit mechanism provided by their multi-agent model seems to be enough for the scenarios tested. however, it was stated that the explicit mechanism proposed, based on randomization, did not perform as expected. wang and liu ( ) presented an agent-based evolutionary search algorithm (aes) for solving dynamic travelling salesman problem (dtsp). in their study, the term agent indicated a candidate position in the search space. agents resided in a lattice-like environment and a collection of such agents were termed as population. they applied a recombination and local updating procedure to the fittest agent referring to a predefined neighborhood. they also combined the perturbation learning strategy to further reinforce the performance of the local updating rule and hope to seek the global optimum rapidly under changing environments. dynamic version of kroa tsp was selected as the case of implementation. offline performance which was the best of generation fitness averaged over runs and then averaged over the data gathering period. experimental result and relevant t test result indicated that the performance of aes was excellent. researchers indicated that the superiority of aes lie in its faster convergence and optimum tracking ability in dynamic environments. it is concluded that aes algorithm had a more quickly and robust convergence capability than standard genetic algorithm (sga) on dynamic problem. billiau and ghose ( ) proposed a new algorithm for solving distributed constrained optimization problems (dcops). the proposed algorithm, which has been called as support based distributed optimization (sbdo), has used agent level objectives instead of weighted or soft constraints that have been used by other dcop algorithms. in dcops, constraints/ objectives of the problem change over time by adding or removing constraints/objectives. researchers compared the performance of this algorithm with asynchronous distributed optimization (adopt) and distributed pseudotree optimization procedure (dpop) by using meeting scheduling problem. result showed that, although sbdo algorithm has not guaranteed to find the optimal solution, it reliably found solutions within two percent of the optimal solution. results also revealed that sbdo was able to find near optimal results significantly faster than adopt and in a comparable time to dpop. máhr et al. ( ) compared two structurally different planning approaches, which were agent based solution and on-line optimization approach, for drayage operations in an uncertain environment. the structurally differentiating feature of the two solution approaches was the level of control: centralized (on-line optimization) and decentralized (agent based solution). dynamic vehicle routing problem (dvrp) with two types of uncertainty, arrival time and service time, was employed in order to compare the performance of agent-based solution and on-line optimization approach. moreover, scenarios were developed under different arrival time and service time. in their study, containers and truckers were defined as agent. each container agent sold itself on an auction to the truck agents. researchers concluded that when the online optimization approach performed better, it was by capitalizing the optimal (or near optimal) balance between routing and rejection cost. when agents performed better, their flexibility provided by their distributed nature was the competitive advantage. voos ( ) focused on dynamic resource allocation problems especially in continuous systems. in this study, resource allocation was expressed as an optimization problem, which could be decomposed into single optimization problems, under certain constraints. researchers proposed to solve this optimization problem in a distributed fashion by using multiple agents. these agents have acted as local optimizer and coordinated their local solutions to an overall consistent solution. in this study, market based interaction mechanism was employed and the agents have calculated and negotiated complete supply and demand trajectories using model based predictions. in order to test the performance of the proposed approach, researcher employed this approach to three technical examples. results revealed that this approach could be used to cope with resource allocation in dynamic environments. li and li ( ) proposed a multi-agent coordination and integration method which has been called intercommunication job-sharing hybridization, for solving complex problems. these complex problems could be decomposed into smaller separate sub-problems, with communications/exchange between sub-problems identified, human decision-makers' roles clearly defined and managerial judgment incorporated. with the proposed method, the overall problem was divided into distinct jobs which were then assigned to relatively independent and distributed agents. these agents have shared data, information knowledge and carried out different tasks synchronously or asynchronously in the context of internet/intranets/extranets to produce solutions. in addition, these agents have linked human participants' judgments and intuition together for joint problem-solving. the architecture of the internet-enabled multi-agent-based hybrid support system for international marketing planning was exemplified. researchers constructed a prototype, which has been called agent international, of the proposed multi-agent hybrid framework. this prototype covered some key features of the conceptual framework and its architecture. the potential and value of the prototype was evaluated by a questionnaire which was prepared and delivered to the corresponding firms in london. according to the responses, researchers concluded that the prototype system was rated somewhat moderately in helping understand pertinent marketing decision making factors, exploring various alternatives, performing analysis, coping with uncertainty, incorporating managers' judgment an improving the confidence and quality of international marketing decision-making. tang et al. ( ) presented an auction based dynamic optimization decision support system. the presented decision support system was applied in solving an automobile load makeup planning problem. the proposed system included three types of agents. these were the yard agents, representing the shipping yard, the truck scheduler agent, representing the transportation company who sold trucks and executed transportation and load agent representing a truck during the planning state. each type of agent had its own behaviors. in order to solve the static load make up problem, researchers implemented heuristics which were empirical (em), minimum spanning tree (mst), vehicle routing optimization (vro). in addition to these heuristics, a virtual market enabled by auction mechanism was employed to solve dynamic optimization problem. researchers developed the mixture of static optimization with mst algorithm and dynamic optimization with the auction mechanism, mst dyn, at the beginning of the day, they apply static optimization on the guaranteed information, and then they apply the dynamic optimization until the loads are fixed at the beginning of lining up. in order to test the performance of the algorithms, same scenario was used and each algorithm has run for days. transportation cost and dwell time were selected as the performance measures. results showed that, em algorithm produced the worst performance and mst dyn the best. the mst algorithm produced the worst performance and em algorithm the best based on the transportation cost. researchers indicated that mst dyn algorithm produced a little bit more transportation cost than em algorithm. therefore, it was concluded that mst dyn algorithm produced the best comprehensive performance. berro and duthen ( ) presented an optimization method in dynamic environment. the proposed method, using software agents, tried to provide accurate solutions and react quickly to changes in the state of the problem. in this method, software agents were randomly created and they try to colonize an optimum of the function. the system composed of two parts namely control system and agent. an agent has not communicated but it perceived the presence of other agents. when the functions to optimize and the dimensions of the search space have been defined, the user must specify two parameters namely force which would influence the searching speed and epsilon which calculated precision of the optimal points. in order to test the proposed method, cases of optimization problems, multimodal function and multi-objective function, were used. the test results were compared with the genetic algorithm based approaches. researchers concluded that the first tests result of the proposed method were satisfactory in particular for the computing speed and precision. jiang and han ( ) presented a simulated annealing (sa) based algorithm to solve real time multi-agent decision making problem. in this study, the sa algorithm was implemented in a centralized version and performed by the agents in parallel, without assuming the availability of communications. since there was no standard benchmark to evaluate multi-agent decision algorithm, a random generator was used to generate all test sets. the sa algorithm was tested by comparing it, with other algorithms, especially with variable elimination (ve) algorithm with respect to the scalability and relative payoff. results revealed that, the proposed method was almost optimal with a small fraction of the time that ve took to compute the policy of the same coordination problem. in addition, researchers indicated two main benefits of this approach as follows: the time taken by the algorithm has grown polynomial with the number of agents and the algorithm could report a near-optimal answer at any time. researchers concluded that, sa was a feasible approach for action selection in large complex cooperative autonomous systems. zhou et al. ( ) proposed a model combining discrete event systems and multi agent systems (mas) to simulate a real time job shop. all entities of the generic job shop were modeled as autonomous agents namely job agent, machine agent, work-center agent, shop floor agent, controller agent and job releaser agent. all agents pursued their own interest with unique functions. all communications in the mas were realized through message passing. proportion machines busy (work-center), average number in queue (work-center), maximum number in queue (work-center), average daily throughput (shop floor), average time in system (shop floor), average total time in queues (shop floor), maximal size of work in process (shop floor) were selected as performance indicators. results of the case study demonstrated the advantage of distributed data collection and analysis. it was indicated that, the case study also validated the proposed system by statistical analysis and comparison to existing simulation results in similar test case. gonzález et al. ( ) presented a new centralized cooperative strategy based on trajectory methods (tabu/taboo search) for solving dops. the proposed strategy was compared with two methods namely a multi-qpso and agents. the multi-qpso is a particle swarm optimization (pso) variant with multiple swarms and different types of particles where there exists an implicit cooperation within each swarm and competition among different swarms. on the other hand, agents are an explicit decentralized cooperation scheme where multiple agents cooperate to improve a grid of solutions. researchers tried to assess the possibilities of trajectory methods in the context of dops and to draw attention on explicitly including cooperation schemes in methods. a set of solver	hreads were consisted in the proposed strategy. each solver could implement the same of a different resolution algorithm for the problem at hand. the coordinator was responsible for processing the information received from the solver and producing subsequent adjustments of their behavior by sending "orders". exchange of data was achieved by using a blackboard model, with two blackboards. one of them was written by the solvers that wrote the reports of their performances and read by the coordinator; and another was written by the coordinator that wrote the orders and read by the solvers. the information flow in the proposed strategy was achieved by using the following three steps: firstly, performance information was sent from the solvers to the coordinator, and then this information was processed and stored by the coordinator and finally, the coordinator sent directives to the solvers. a set of rule, based on reactive search ideas, was employed to control the solvers. in order to test compare the performance of the proposed strategy, moving peaks benchmark problem and three commonly used multimodal real test functions were selected. to emphasize the importance of dynamism and optimal tracking, and to reduce the number of variables to control in the experiments, only the position of the peaks was altered. proposed strategy and the other two methods were compared according to offline error. just before a change, offline error of each algorithm was recorded and this value was averaged over all changes, for all independent runs. therefore, mean fitness error was calculated. researchers indicated that the proposed strategy could consistently outperform the results of the two other methods. they concluded that the cooperation included in agents provided some benefits over multi-qpso on the easier problems, but since the optimization done in agents relied only on using simple random perturbations of solutions it may be enough to cope with more difficult problems, even with the help of the cooperation. lepagnot et al. ( ) presented a new method called multi agent dynamic optimization (mado) to solve dynamic optimization problems. in mado, a population of agent has explored the search space. three modules namely, memory module, agent manager and coordinator were employed. the number of agents in the system may have varied temporarily, but the number of agents along the whole search process tended to be equal to the predetermined value. this could be achieved by the coordination who would send a delete instruction if the number of agents was higher than a predetermined value. to test the performance of the mado, moving peaks benchmark problem was employed. offline error and standard deviation (sd) were selected as performance measure. different variants of mado were compared with the proposed one. results indicated that mado was better than all the simpler variants. researchers concluded that the proposed mado was a promising method for solving dynamic optimization problems. yan et al. ( ) proposed an agent based evolutionary search (aes) search method. in aes, a population of agents has represented potential solutions. similar to eas, aes gradually converged in the search space during the run, especially when the environment has been stationary for some time. in order to improve the performance of aes for dops, two diversity maintaining mechanisms, random immigrants and adaptive dual mapping were employed. dynamic - optimization problems which were generated from static optimization problems by using xor generator were investigated. nine different dynamic test problems were constructed. the environment was periodically changed every predetermined number of generations. different change severities were employed to test the performance of aes. the performance of aes was compared with traditional sga, the primal dual ga and the ga with random immigrant, where the worst % individuals were replaced with random individuals every generation. mean best of generation fitness was selected as performance measurements. according to the mean best of generation fitness, researchers indicated that aes could always outperform other eas on almost all problems. researchers stated that the competitive and learning behaviors of agents could always help aes to obtain a better performance than the peer gas could do. they concluded that some dynamic characteristics of the environments may affect the performance of the algorithm. hanna and cagan ( ) presented an evolutionary multi-agent system (emas) for adaptive optimization. emas which has employed the evolution of design strategies within a cooperative virtual team has evolved as conditions change and as new solution states were discovered during the optimization process. a set of strategies for creating solutions were represented by population of agents. in emas, the strategies for generating solutions have been recombined, altered, and removed by applying genetic operators that are in typical genetic and evolutionary algorithms. however, researchers have made emas different from genetic and evolutionary programming techniques by adding cooperation dimension. cooperation was employed to combat the problem of not knowing which strategy to use in an unknown but static design space. it has been provided by embodying each strategy in an autonomous agent and allowing the population of agents to communicate. researchers used the well known combinatorial optimization problem of travelling salesman. researchers tried to illustrate that the proposed framework was capable of increasing the effectiveness of individual solution strategies by evolving and coordination them a decentralized manner. three simple heuristic construction algorithms called nearest insertion, farthest insertion and arbitrary insertion were employed. beside these, three heuristics based algorithms, -opt, -opt and simple mutation were also used. two basic reduction algorithms, random reduction and best partial reduction were employed. different from the typical genetic algorithms, gene strings represented agent strategies for generating solutions, not the solutions themselves. the design architecture used in this study was similar to asynchronous team architecture. in this study fitness was based on the ability of the agent to make positive changes in its destination memory. first of all researchers selected a -city problem to compare the resulting tours generated by emas with those generated by both individual algorithms on their own as well as priori determined hybrid algorithms. when the results of emas were compared with the results from running construction algorithms from each starting city and then running improvement algorithms on the resultant tour, researchers found that trials that correlated more strongly with the average patterns had better final values in terms of distance from the optimal value of the average solution quality. in addition to the -city problem and -city problem, researchers applied these patterns to a team solving a euclidean -city problem modeled on a very large scale integration layout problem. researchers has seen that the cooperative teams of individual strategies evolved to generate better solutions than both individual strategies alone and a priori set hybrid strategies. it was indicated that the strength of the emas algorithm lies in its ability to evolve the best team of agent dynamically. it was also stated that, one of the strength of the emas algorithm was as a predictive or learning guide for which set of algorithms or strategies should or should not employed and when. researchers concluded that, utilizing emas in the proposed way has been shown to lower computation time while maintaining or even improving solution quality. pelta et al. ( a,b) investigated if the type of rules employed previously in cooperative systems for static optimization problems have had sense when applied to dops. the proposed strategy was based on the joint use of a population of solutions and optimizers (agents). researchers analyzed the roles that diversity and decentralized cooperation mechanisms in the performance of the methods. researchers aimed to propose and compare two kinds of control rules to update the solution's set. these rules were a simple frequency based re-sampling (probabilistic) rule and a fuzzy-set based rule. researchers also tried to understand the behavior of both rules in order to develop more efficient cooperative strategies for dops. in this study, cooperation was understood as a mechanism for information sharing. the population of solutions had two purposes which were to serve as an implicit memory that has evolved by means of the action of agents and to be a communication channel for them. an explicit cooperation mechanism was proposed. this mechanism, which was not always triggered, was based on a simple idea. in order to test the performance of the proposed strategy, a set of experiments were developed by using moving peaks benchmark problem. the main aim of the experiments was to evaluate the behavior of both rules which may trigger the explicit cooperation mechanisms. offline error was used as performance measurement. the performance of the cooperative system for different setting of each rule and the system's dynamic behavior were also investigated. the results of the proposed strategy were compared with some published results. researchers stated that the fuzzy-based rule has been better than the frequency rule. as a conclusion, researchers indicated that both rules have been competitive when compared with a state-of-the art of the algorithm. xiang and lee ( ) proposed an agent-based dynamic scheduling approach which employs ant colony intelligence (aci) with local agent coordination. the goal of their study was to represent a dynamic manufacturing system through an mas. they also used aci to improve the global performance of the system. in the proposed system, entities were modeled as intelligent agents with related knowledge of their own functions and goals. mas was used to provide parallel execution of commands. beside this, mas had the intelligence of negotiation to enhance system performance. the agent coordination mechanism used in the study was inspired by both foraging and division of labor of ant colony in mas. there were five types of agents in the proposed mas. they were order agents, job agents, machine agents, work center agents and shop floor agents. researchers indicated that, different from the previous studies, a more generic manufacturing model with less unrealistic assumptions was considered. furthermore, aci was integrated with both machine agents and job agents to solve both task allocation and task scheduling problems. two types of disturbances were introduced. one of them was resource related disturbance including machine break down and machine recovery. the impact of integrating aci in agent coordination was investigated by simulating a realistic shop as a multi-agent manufacturing system. in this disturbance, unreality was expressed in terms of mean time between failure and the mean time to repair. another was source related disturbance including new order\job arrival and existing order\job cancellation. to types of agent coordination, namely coordination based on aci (mas + aci) and coordination using fifo dispatching rule (mas + fifo) were compared. mean flow time, mean tardiness, throughput, buffer size and machine utilization was employed to measure the performance of the proposed approach. results showed that a mas + aci reduced buffer size, max queue number, mean flow time and tardiness. researchers concluded that a mas + aci were outperformed mas + fifo. wang and usher ( ) presented an agent-based job shop model which employed the contract-net protocol as an agent's negotiation mechanism. in this study, two types of agents, named as job agents and machine cell agents, were used with pure hierarchical control structure. researchers considered routing flexibility to provide more options for job agents. average flow time and average queue time were selected as performance measurement. in order to measure the impact of the collaborative factor that was incorporated into the contract-net protocol on the performance, a job shop with five different loading levels were simulated. according to the findings, the collaborative factor did not have much effect on mean flow time when the system load is light, but a significant decrease was observed when the system was heavily loaded. when they examined the average queue time for jobs at each machine cell, they observed that the negotiation scheme with the proposed factor reduced the wip levels of the bottleneck machine cells when the system was under heavy load. based on the simulation results, researchers concluded that the collaborative factor could improve the performance of the contract net-based negotiation scheme in agent-based scheduling problems. wang et al. ( ) proposed a multi-agent approach to study the dynamic scheduling problem in a flexible manufacturing system (fms) shop floor. the proposed approach was combined with a filtered beam search (fbs). researchers aimed to show the feasibility of the proposed approach and to evaluate the approach via computational experiments. dynamisms in this system were provided by new job arrivals. minimization of a weighted quadratic tardiness function was selected as the objective function. there were two types of agents, a system optimal agent (soa) and several cell coordinated agents (ccas). cooperation and coordination among distributed ccas and soa were employed to realize the scheduling goal. five modules which were called as communication, cooperation and coordination, fbs-based algorithm for decision making, execution and monitoring, human interface, were used. in addition to these modules, one local knowledge base and one capacity database were also included. fipa cnip-based negotiation protocol was selected. fbs was performed by filtering phase and beam selection phase. a prototype system was built to show the practicability of the proposed approach. the performance of the proposed scheduling scheme on the prototype system was compared to two dispatching rule combinations. two dispatching rules were used for cell selection, named as dispatching by objective function value and dispatching by make-span, one dispatching rule for routing assignment, named as modified shortest processing time in the selected cell and one dispatching rule for determination of starting time of an operation on the selected machine. when the results on the performance of the average number of jobs tardy were investigated, researchers indicated that the quality of the global schedules generated with the proposed scheduling scheme was better than those of two dispatching-rue combinations. in addition, researchers sought the time required for a complete process of scheduling negotiation. they concluded that the proposed scheduling scheme was promising for real world implementation in multiple manufacturing cells of size. this paper proposes a classification scheme (abdopps: agent based dynamic optimization problem solution strategy) for agent-based approaches which are employed for solving dynamic optimization problems (dops). in this paper typical articles providing agent based solutions to the dops are scanned through the literature and represented using the abdopps. the abdopps is expected to be beneficial to researchers in many ways. similarities of the features located in abdopps can be used to define classes of solution strategies by their descriptions. in this regard, classes of the problems may orient researchers to focus on certain strategies. using the dynamism related features of the corresponding dops presented in abdopps, unpredictability levels of certain problems can be determined and be used to reclassify problems. these representation forms can also be used to discover the role of presented features and their importance for solution quality. online adaptation in learning classifier systems: stream data mining. illinois genetic algorithms laboratory managing critical success factors in a b b setting evolutionary optimization on problems subject to changes of variables solving vehicle deployment planning problem by using agent based simulation modeling dynamic optimization in a dynamic and unpredictable world search for optimum in dynamic environment: an efficient agent-based method robust, flexible multi-agent optimization using sbdo dynamic optimization in future cellular networks adaptation in dynamic environments: a case study in mission planning a taxonomy of evolutionary algorithms in combinatorial optimization replanning mechanism for deliberative agents in dynamic changing environments optimization in dynamic environments: a survey on problems, methods and measures models of decision and optimization (modo) research group web 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for dynamic optimization problems simple control rules in a cooperative system for dynamic optimization problems multi-agent based simulations using fast multipole method: application to large scale simulations of flocking dynamical systems speculative computation by abduction under incomplete communication environments multi-agent reinforcement learning: independent vs. cooperative agents wireless-based dynamic optimization for load makeup using auction mechanism integration between business planning and information systems planning: an evolutionary-contingency perspective agent-based distributed resource allocation in continuous dynamic systems, intechopen. multi-agent systems agent-based problem solving: the ant colonies metaphor an agent-based evolutionary search for dynamic travelling salesman problem an agent-based evolutionary search for dynamic travelling salesman problem fbs-enhanced agent-based dynamic scheduling in fms an agent-based approach for flexible routing in dynamic job shop scheduling ant colony intelligence in multi-agent dynamic manufacturing scheduling agent based evolutionary dynamic optimization explicit memory schemes for evolutionary algorithms in dynamic environments simulating the generic job shop as a multi-agent system agent-based simulation of electricity markets: a survey of tools key: cord- -jxymy e authors: cuttini, marina; forcella, emanuela; rodrigues, carina; draper, elizabeth s.; martins, ana f.; lainé, agnés; willars, janet; hasselager, asbjørn; maier, rolf f.; croci, ileana; bonet, mercedes; zeitlin, jennifer title: what drives change in neonatal intensive care units? a qualitative study with physicians and nurses in six european countries date: - - journal: pediatr res doi: . /s - - - sha: doc_id: cord_uid: jxymy e background: innovation is important to improve patient care, but few studies have explored the factors that initiate change in healthcare organizations. methods: as part of the european project epice on evidence-based perinatal care, we carried out semi-structured interviews (n = ) with medical and nursing staff from randomly selected neonatal intensive care units in countries. the interviews focused on the most recent clinical or organizational change in the unit relevant to the care of very preterm infants. thematic analysis was performed using verbatim transcripts of recorded interviews. results: reported changes concerned ventilation, feeding and nutrition, neonatal sepsis, infant care, pain management and care of parents. six categories of drivers to change were identified: availability of new knowledge or technology; guidelines or regulations from outside the unit; need to standardize practices; participation in research; occurrence of adverse events; and wish to improve care. innovations originating within the unit, linked to the availability of new technology and seen to provide clear benefit for patients were more likely to achieve consensus and rapid implementation. conclusions: innovation can be initiated by several drivers that can impact on the success and sustainability of change. innovation was defined as "the intentional introduction and application within a role, group or organisation, of ideas, processes, products or procedures new to the relevant unit of adoption", regardless of the time elapsed since development or availability in the marketplace. in the healthcare field, innovations typically include new services, ways of working or technologies directed at improving the health outcomes, cost-effectiveness, administrative efficiency, or user's experience. , according to greenhalgh et al., an important element of healthcare innovation is implementation "by planned and coordinated actions". implementing innovation, including the abandoning of ineffective or obsolete practices, is crucial to improve the effectiveness and safety of health care. however, "invention is hard but dissemination is even harder", and the slow or uneven adoption of innovation has been frequently reported. , theoretical models of change have identified different primary catalysts for the adoption of innovation including the recognition of the need to change, as indicated by "unfreezing the target behaviour" in lewin's change theory or "establishing a sense of urgency" in kotter's eight-step process; the innovation characteristics in rogers' diffusion theory; and the balance between benefits and costs of adoption according to the risk-based framework. focussing on implementation, the consolidated framework for implementation research (cfir) has provided a comprehensive and pragmatic structure to promote theory development and effectiveness verification across multiple contexts. however, studies providing empirical data about the factors that initiate change in healthcare organizations or the relation between the origin of innovation and the success of implementation are few. we carried out a qualitative study with physicians and nurses to explore how clinical or organizational innovations are introduced and implemented in neonatal intensive care units (nicus), a setting characterized by extreme patient fragility, complex technological environment, highly specialized multidisciplinary personnel. this paper focusses on the initial motive for change, broadly defined as the reasons, goals or events that started the process leading to actual clinical or organizational innovation. this study is part of the "effective perinatal intensive care in europe" project (epice, https://epiceproject.eu) on the use of evidence-based interventions in perinatal care in regions of european countries. for this study, we involved one region from each country with resources to carry out qualitative interviews and analysis: the eastern region in denmark, ile-de-france in france, hesse in germany, lazio in italy, northern region in portugal and east midlands in the uk. in each region, we stratified the nicus according to academic status (yes/no) to ensure variability in terms of organization and policies of care, resources, involvement in research and training. we then randomly selected two nicus per region within these strata. in each nicu, we interviewed two physicians and two nurses with experience of at least years clinical work in that unit. priority was given to staff with involvement or interest in evidence-based medicine. we focused on physicians and nurses because the presence of other professionals (e.g. physiotherapists, psychologists) in nicu teams may vary between countries. additionally, these professionals are often attached to different hospital units and provide only consulting or part-time work in nicu. we had no explicit refusals. however, in denmark, only one unit was able to schedule the interviews within the study period. the study was approved by the ethics committees in all regions. signed informed consent by informants was obtained except in denmark and france, where the committees waived the requirement. we carried out individual semi-structured interviews in local language, focussing on the most recent clinical or organizational innovation introduced in the unit regarding care of very preterm infants (vpt, < weeks of gestation). we broadly referred to the theory of organizational change outlined by pettigrew, emphasizing the categories of content ("what has changed"), context (inner and outer) and process of change, including the actions, reactions and interactions of the various interested parties. we developed an interview guide to ensure standardization across units and interviewers. the guide followed a chronological order starting with the content of the change and the main initial motive, or driver, for introducing it; the planning, dissemination and implementation phases; compliance and perceived benefits of the innovation. pilot interviews carried out in italy, france, portugal and uk and discussions within the study team were used to finalize the guide. a -day training workshop was held for interviewers to standardize the conduct of the interviews and methods of transcribing and analysing the data. interviews were carried out by appointment at the informant's hospital, and were recorded and fully transcribed anonymously. we used thematic analysis to identify the patterns across the dataset ("themes") relevant to the description and motives for change. the analysis was performed in two stages to address the challenges of our multilingual sample. firstly, at regional level each interviewer carried out an initial coding of the raw data using a common scheme based on the interview guide and supplemented with additional codes emerging from the data and shared within the coding team. the coded text was then translated into english for the second stage of analysis, which was carried out at the coordinating centre in italy by e.f. and m.c. e-mail discussions between the interviewers and the italian team were used to check the interpretation of results. similar themes continued to emerge in all nicus after the first half of the interviews, indicating saturation. the analyses were performed using the nvivo software (qsr international, australia). the characteristics of nicus and informants are presented in tables and , respectively. the selected nicus were diverse with respect to structural and functional organization, including number of cots and admissions, provision of specialized services, and use of protocols and guidelines. most informants (n = ) were females. the mean interview duration differed by profession ( min for physicians and for nurses) and country, with longer interviews in the uk and italy. table shows the reported innovations. for physicians, the most frequently reported changes concerned ventilation, followed use of probiotics (n, %) ( ) by feeding and nutrition, and by infant care and pain management. this latter was the most frequently reported by nurses, who also discussed the care of parents. six "themes" emerged from the data in relation to the initial main motive for change ( fig. ) . again, nurses were more likely to report care-related motives, while physicians appeared more sensitive to the availability of new knowledge and technology. findings in scientific literature, attendance at conferences and informal opportunities to exchange information with colleagues were reported as main triggers for change by seven physicians and four nurses. the idea has emerged from the literature. since some years we have managed respiratory distress in very premature infants in a fairly systematic way by intubation and surfactant administration. and then new randomised trials appeared in the literature showing that stabilization of these infants by non-invasive ventilation could avoid intubation and unnecessary administration of surfactant, because these children did not need it. […] so it's a real change of practice. (physician/ france/ ) new staff arriving from other hospitals were also a source of innovative ideas, and often took the lead in promoting implementation: …we've been aware that other units had been using a different mode of infant ventilation and also this consultant came from a unit where that was being used. so we were sort of fairly happy that it's not going to do any harm and might have the potential to do some good. […] this consultant i mentioned actually took ownership of it, did produce a guideline. (physician/uk/ ) the availability of innovative technology, such as new equipment, was a very strong driver for change: a very recent change that, i really believe, has modified quite a bit our management, both for physicians and nurses, are the ram cannulas for nasal ventilation. they allow us to use a high flow and apply pressure both during inspiration and expiration. […] the previous system was more powerful, but more aggressive. (physician/france/ ) while most of the new technology mentioned concerned ventilation, other types of equipment were also reported: …as now there is an easy-to-use machine that allows us to analyse every time the lipid, protein and carbohydrate content [of mother's milk], well we do it, everything is analysed and we will know exactly what we are doing. because we know that maternal milk is perfect for preemies, but it does not exactly meet the needs of the premature baby. thus we give what is called a fortifier. (physician/france/ ) considering the advantages of a technology and obtaining the resources to acquire it may take time, but its adoption seems to raise little resistance, and benefit from easy implementation: we had attended a conference as a team and looked at the equipment, and thought that it would be something really good. […] we spoke to the representatives there and had a quote, and decided that this was something we were going to buy. (physician/uk/ ) thus if one finds something easier at the technical level, which seems as effective, one wants to use it. that's it. (physician/ france/ ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) nurses ( ) ( ) guidelines and regulations from outside the nicu change may be motivated by factors external to the unit, such as a new guideline or regulation issued by official agencies: so with nice, they get a national launch and when they come out, you are told by commissioners that you need… you need to find a way to meet this guideline. and then we would usually adapt the national guideline into a local policy. because there is a regulation on the use of breast milk that is very clear and very strict and does not allow the use of raw frozen-thawed milk within a hospital service. it is possible at home but not in the hospital because of obvious bacteriological reasons and so, there you go, it was imperative that we follow the rules and not be outside of bounds. (physician/ france/ ). the guidelines produced by scientific or professional groups seem to be more acceptable that those issued by official agencies, particularly when some of the staff had been involved in the process: …a [hospital] pediatric pain group was created, including the various sub-specialties at the level of paediatrics. i am the coordinator of the group, myself and nurse x, the nurses are obviously involved, in fact they are the ones who are with the children and carry out the pain assessment and administer the medications, basically. (physician/portugal/ ) for the nicu, this was … an opportunity to bring some things up to date, things which were being done but there was no proper protocol. (physician/portugal/ ) in contrast, guidelines issued by official agencies were viewed as more bureaucratic, and possibly less appropriate to the unit patients and care. they appeared to encounter more resistance, particularly when there was disagreement about the strength of the evidence: so one of the difficulties with nice guidelines is that they come out with a government stamp on them saying you must do it and you must audit them. but actually sometimes the evidence on which the guideline is based is quite weak. and ultimately it comes down to expert opinion. (physician/uk/ ) the decision to standardize practices generally arose within the unit team, aiming at more consistent and homogeneous care: there was a need for a common framework. (nurse/italy/ ) even issues considered common knowledge could benefit from standardization, as was the case with actions in response to vancomycin blood level monitoring: we did know, that you must react. everyone knew that, but it wasn't standardized. now it's rather precise, regardless of who is working, whether it's weekend or night or whatever, the reaction is always exactly the same. (physician/germany/ ) the need for a common approach was felt especially in case of rapid staff turnover, or because of new professionals joining the unit: now every morning we have the physiotherapists […]. therefore also this part, which was not included in the old protocol, had to have relevance, and we all had to standardize our approach to the children. (nurse/italy/ ) modification of the characteristics of the patients admitted to nicu was mentioned as a motive to revise policies: also the type of patients change, they are becoming smaller and smaller… the weekers were only in the mind of god, now they survive, therefore clearly you have to update progressively, as both research and type of patients lead you to adapt some parts. (physician/italy/ ) the experience of participating in a study requiring modification of usual routines was the main driver leading to the establishment of a structured protocol for kangaroo skin-to-skin maternal care: there were many reasons that motivated it, one of them-and this was very important-was that we are part of a project which is called care. so we took advantage of this situation to say that we would like our unit to be part of this change and we went ahead with it. (physician/portugal/ ) in italy, participation in an intervention study to increase parental presence in nicu led to the establishment of a new routine for the first communication with the parents: before, in case of a new neonatal admission, we were busy only with the baby. now we go to talk to the parents, we reassure them and tell them that as soon as the baby has stabilized, they will be able to enter [the nicu]. (nurse/italy/ ) in germany, study participation led to the development of an educational protocol identifying the situations when endotracheal intubation could be performed by trainees: well, we had taken part in a study, a study on intubation, and in that framework we thought, good now, we'll thoroughly reorganise it, so that there are guidelines. (physician/germany/ ) in one case only the reported research, a randomized clinical trial to identify the lowest baby's weight for safe transition from incubator to open cot, was designed by the informant and carried out within the unit itself: nevertheless, when the change involves, as in this case, organizational modifications requiring compliance by the whole team and the parents, implementation can still be challenging: it's a process which is a bit different, and [initially] the medical staff weren't really motivated. i mean one thing is a new ventilator, a new ventilation strategy, and then you have all the scientific enthusiasm inherent to intensivists. as long as there is the money to buy the ventilators, there's a meeting, and if there is agreement they move forward and the process is established and everyone follows along, the parents have no word in it, nor the nurses, they just have to adapt to it. whereas here, there is involvement of all staff, it is different and very interesting, at least from my point of view. (portugal/physician/ ) occurrence of an adverse event adverse events, and the urgency to prevent similar cases in the future, were reported as rare but highly emotionally charged triggers leading to the change of unit practices: this change occurred because we diagnosed late, late a baby who was in hospital with aortic coarctation…in other words, there was an error in our work. […] i think that it was because of this urgent case that [the cardiac screening] was implemented from one day to the next. it had to be done. (nurse/ portugal/ ) while describing the newly introduced daily mandatory inspection of the resuscitation equipment at the labour ward, documented and signed by the nurse in charge, the informant reported about: …a suction device that actually didn't work, the device was faulty, it just didn't build up any suction and you would have noticed that if you had checked it beforehand. so that happened in a situation with an emergency caesarean section where we really came in at the same time as the patient. […] we discussed, how do you do that, how do others do that, how can we match that? and then we said, okay, it's got to be a daily check. (nurse/germany/ ) adverse events appeared to prompt immediate change even when unrelated to identified clinical errors: …the one that springs to mind that is probably the most recent is a guideline on difficult intubation. what prompted it initially was… we actually had an incident where a baby was very difficult to reintubate. and when we looked at the incident in detail, we identified that we hadn't got a guideline on the difficult to manage airway. […] what was interesting when we wrote the guideline was that what had been done during the incident was what the guideline said we should do. (nurse/uk/ ) interestingly, the nurse commented on the value of guidelines precisely for infrequent events: it isn't a circumstance that is going to happen often, but then i feel that those are particularly the occasions you need guidelines for. obviously for things you do day-in day-out, it's perhaps less important, because you know what you're doing all the time. whereas something that happens less often, it's always useful to have a guideline to refer to. (nurse/uk/ ) improvement of care improvement of care was explicitly identified as the main motive for change by three doctors and eight nurses. two physicians from the same nicu mentioned the importance of starting personalized parenteral nutrition since the first hours after birth: …this nutrition prevents the babies from entering a catabolic state, which is what happens when they are born and so, it is fundamental that we stop the catabolic state, right, and try to nourish them from the first day. (physician/portugal/ ) this aim conflicted with the opening hours of the hospital pharmacy, that would not prepare parenteral nutrition bags unless ordered before pm: …and so, all newborns who were born outside of those hours had to receive [only] basic fluid therapy until the next day. (physician/portugal/ ) additionally, parenteral bags could only be ordered with reference to a specific, legally identified individual: and here the problem is that legally the baby only exists after it is born. (physician/portugal/ ) the change that was implemented involved ordering a parenteral bag "in anticipation": so we ended up by speeding things a little and planning ahead and making requests for babies, who aren't yet identified with a file number, by using only the mother's identity and the baby's predicted weight. (physician/portugal/ ) and then that bag is adjusted in terms of the rhythm of perfusion for the infant who is born in the meantime. there, it's something new. (physician/portugal/ ) the nurses focussed on the importance of care, and on the appropriate handling of the fragile very preterm babies: at the beginning of last year we discussed that we would like to introduce kinaesthetics into our care practices. […] it's not that anyone ever felt that we weren't handling the children correctly, rather we just wanted to improve because the children stay in our wards for a very, very long time. we wanted to do something good for the children, and we noticed that we treat them differently from our medical colleagues. so, we have a medical colleague who always positions the children semi-upright, because he says that's good for their breathing. then you come to one of these little cots and you see that the infant is in this position but is sliding down and being held up by the air tubes. breathing improves, no question about that, but you need to give the child a support, some security, so the baby doesn't have the feeling, oh god, what do i do now? (nurse/ germany/ ) improvement of patient care may also be achieved by increasing parents' involvement and empowerment: we chose our focal points that we want to communicate to the parents. we really pushed the advice for breastfeeding, reinforced skills in feeding and handling the infants. our experience was, the more skilled the parents are, the more they can contribute to the care. (nurse/germany/ ) this study provides, from the perspective of the healthcare staff, an overview of the type of clinical and organizational innovations introduced in the nicus and of the main factors that motivated the decision to change. consistently with our initial question, some of the innovations described referred to the same topic. however, the relative frequency of selection varied by professional role, with ventilation at the top of the physicians' list while issues of infant and parental care were more prominent among nurses. we found that the introduction of innovative policies or treatments in nicus can be triggered by several factors. some were external to the unit, such as the publication of a new law or official guideline in need of implementation. others were internal, arising from a reflection on own clinical experience promoted by reading the literature, attending conferences, participation in research, or as reaction to an adverse event. while external triggers were generally backed-up by formal assessment of scientific evidence, this was not always the case for changes that originated from staff personal experience and observations. this dichotomy seems to mirror two views of evidence-based medicine: the rigorous identification of scientific evidence with results of randomized clinical trials and meta-analyses only, versus the broader view including other sources of evidence such as different study designs, clinical experience, interactions with peers and patients, and "pragmatic science" methods of learning. [ ] [ ] [ ] in our study, pragmatic methods and clinical experience were used to promote organizational changes especially in areas where formal scientific evidence was lacking or controversial. they were more often used by nurses, because of their special interest in the patient perspective and, possibly, the relative paucity of experimental evidence in nursing. , however, clinical experience is effective only when accompanied by the other three elements of the kolb's experiential learning cycle, namely reflective observation, conceptualization and experimentation. the development, reported by one of our informants, of a randomized trial based on the initial observation of a declining trend of babies' weight at transfer to open cots, with its final finding of safe shortening of hospital stay, was a perfect example of the application of the cycle as well as demonstration of the complementarity between ebm and quality improvement methods. we found that the type of motivation to change influenced the speed of implementation and staff compliance. innovations arising within the nicu team, out of personal experiences and exchanges with colleagues, new information from the literature or participation in research, appeared to lead to smoother and possibly quicker implementation. an extreme example of internal trigger was the occurrence of an adverse event, where the emotional burden led to immediate action and widely shared consensus about the necessary change. this finding confirms the learning potential for staff after an adverse event, which is at the root of audit initiatives such as incidence reporting, case analyses and mortality statistics conferences for the nicu or all hospital staff. , the nature of the innovation was also relevant. technological innovations, such as new drugs or equipment, offer the promise of improving care within limited resources and without need for complex reorganization of unit routines. in contrast, changes requiring policy reorganization, coordination across disciplines and agreement by all components of the team remain challenging, and may take more time to gain consensus. however, glamour and underlying commercial interests may foster the adoption of new technologies even before evidence of benefit and cost-effectiveness is established. preliminary testing to the same standards of other innovations is absolutely necessary. , the active participation of the unit staff in the development of the new policy, or at least in the decision to adopt it, appeared crucial to increase compliance. changes imposed completely from outside, such as mandatory regulations or official guidelines issued by ad hoc agencies, raised more frequent criticism and resistance, particularly when the strength of the evidence was considered controversial. an additional important issue was the opportunity to test the innovation before adoption, as was the case for participating in a research project on kangaroo care, highlighting the importance of clinical research not only in generating new knowledge but also as learning experience for participants. finally, the presence of a staff member with personal interest and expertise in the specific field of change was the third element that emerged as important for successful implementation. this study has strengths and limitations. to our knowledge, this is the first study providing an overview of the factors that trigger change and promote the introduction of clinical and organizational innovation in nicus. the study benefited from the epice mixed-method design as we based our research hypothesis on quantitative data showing variable uptake of a range of evidencebased interventions by region. however, our decision to focus on the process of change allowed any unit to participate, as the interview contents were not contingent on use of a specific intervention. we did not provide a pre-set definition of innovation, allowing a better understanding of what constitutes innovation for the medical and nursing professionals. the choice of discussing a real experience, identified as the most recent, aimed to avoid theoretical discussions and prevent the selection of the most successful event. the inclusion of a maximum of eight interviews per country inhibited a full analysis of country-level specificities. nonetheless, many of the themes emerged in multiple contexts, suggesting broader application. the findings of our study can contribute to inform strategies aimed at fostering successful implementation of the intended change and staff compliance. whether the change originates within the nicu or from outside, it is advisable to involve all the staff concerned in its uptake, including listening to objections and criticisms and proposals for local adaptation. in case of innovation promoted by official agencies, early partnering with professional associations may be important. clinical audits represent a powerful opportunity for change, particularly when carried out with a nonjudgemental, pragmatic attitude. regular literature review meetings can promote the transfer of scientific findings into clinical practice. actual participation in research studies can be an effective way to test innovations before adoption and monitor the outcomes. overall, these strategies may represent interesting research hypotheses to be tested in future studies. along with the established role of evidence-based methods to assess the value of innovative interventions, qualitative research can contribute to a better understanding of the circumstances and conditions that foster the development, dissemination and implementation of clinical innovations. the social psychology of innovation in groups innovation in healthcare: a systematic review of recent research diffusion of innovations in service organizations: systematic review and recommendations evidence-based de-implementation for contradicted, unproven, and aspiring healthcare practices road map for diffusion of innovation in health care disseminating innovation in health care outcomes of adoption: measuring evidence uptake by individuals and organizations field theory in social science leading change. why transformation efforts fail diffusion of innovation st edn the decision to adopt evidence-based and other innovative mental health practices: risky business? fostering implementation of health services research findings into practice: a consolidated framework for advancing implementation science canadian neonatal network epic study group. a qualitative examination of changing practice in canadian neonatal intensive care units use of evidence based practices to improve survival without severe morbidity for very preterm infants: results from the epice population based cohort context and action in the transformation of the firm applied thematic analysis broadening the view of evidence-based medicine strengthening the contribution of quality improvement research to evidence based health care knowledge 'translation' as social learning: negotiating the uptake of research-based knowledge in practice clinical nurse specialists' use of evidence in practice: a pilot study a descriptive study of research published in scientific nursing journals from to experiential learning experience as a source of learning and development specialty-based, voluntary incident reporting in neonatal intensive care: description of incident reports a comprehensive overview of medical error in hospitals using incidentreporting systems, patient complaints and chart review of inpatient deaths translating research into clinical practice: making change happen unproven technologies in maternal-fetal medicine and the high cost of us health care putting innovation to the test we are grateful to all the physicians and nurses who participated in the interviews and shared their experiences with us. we thank nikola jeck for carrying out the interviews in germany. the m.c. designed the study, drafted the study protocol, coordinated data analyses and prepared the manuscript. e.f. coordinated data acquisition and analyses across the participating regions, carried out and transcribed the interviews in italy and performed the overall second-stage data analyses. c.r., e.s.d., r.f.m. and m.b. participated in the finalization of the study protocol and materials, supervised data acquisition in their region and contributed to the manuscript for important aspects related to the interpretation of results and the discussion. a.f.m., a.l., j.w., a.h. carried out and transcribed the interviews in their region and performed first-stage data analyses; they contributed to the paper as regards the validity of data reporting and interpretation. i.c. prepared and maintained the quantitative database of the study, prepared the tables and figure, and performed the literature review. j.z. initiated and coordinated the epice project, participated in the definition of this study aims, protocol and instrument, and substantially contributed to the manuscript for important aspects. all authors have read and approved the final version of the manuscript. competing interests: the authors declare no competing interests.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- -pv doe authors: novossiolova, tatyana title: twenty-first century governance challenges in the life sciences date: - - journal: governance of biotechnology in post-soviet russia doi: . / - - - - _ sha: doc_id: cord_uid: pv doe the chapter explores the rapid advancement of biotechnology over the past few decades, outlining an array of factors that drive innovation and, at the same time, raise concerns about the extent to which the scope and pace of novel life science developments can be adequately governed. from ‘dual-use life science research of concern’ through the rise of amateur biology to the advent of personalised medicine, the chapter exposes the limitations of the existing governance mechanisms in accommodating the multifaceted ethical, social, security, and legal concerns arising from cutting-edge scientific and technological developments. so far as to suggest that the 'life sciences knowledge, materials and technologies are advancing worldwide with moore's law-like speed.' and whilst some commentators have questioned the extent to which the ongoing progress of biotechnology has translated into practical applications and novel products, there is some consensus that the biotechnology landscape has been fundamentally transformed over the recent decades with the possibilities now unlocked holding revolutionary potential. indeed, rapid advances in the field have produced a knowledge base and set of tools and techniques that enable biological processes to be understood, manipulated and controlled to an extent never possible before ; they have found various applications in numerous spheres of life, generating enormous benefits and offering bright prospects for human betterment; and they have come to be regarded as a key driver of economic development with potential to close the gap between resource-rich and resource-poor countries. the progress of biotechnology has been largely driven by three sets of forces, namely social, political and economic. the social dynamics at work in this context are understood as the efforts to improve public health and overall wellbeing of individuals both in the global north and global south, boost agricultural yields and encourage environment-friendly practices to mitigate the adverse effects of climate change. several factors account for the significant value attached to the life sciences in the context of intense globalisation and continuous change. surging population numbers and extended life expectancy are augmenting the demand for developing effective and affordable medications, novel approaches for the treatment of chronic diseases and additional cost-effective sources of energy and food production. at the same time, rising global trade and travel, coupled with increased urbanisation, and an uneven distribution of wealth are creating optimal conditions for disease outbreaks, pandemics and environmental degradation. against this backdrop, biotechnology appears full of promise and critical to tackling social and natural concerns; enhancing disease prevention, preparedness and surveillance; promoting development; and alleviating human suffering. economic dynamics include national expenditure on research and development, purchasing power, trends in consumerism and market pressures and fluctuations. besides public funding for r&d which remains a key factor in the growth and flourishing of bioindustry in developed and emerging economies alike, private investment from venture capital firms, start-up companies and transnational corporations (tncs) have also played an indispensable role in capturing new markets and further facilitating the extension of bioeconomy on a global scale. dupont's significant footprint in india is indicative in this regard, not least because of the depth and diversity of the activity that the company has undertaken via its offshore r&d centres ranging from crop science to biofuels. likewise, merck has outlined a . billion dollar commitment to expand r&d in china, as part of which it intends to establish an asia headquarters for innovative drug discovery in beijing. political dynamics are triggered by states' increasing commitment to support the progress of biotechnology as a way of maximising their power and boosting their status in the international arena. in the aftermath of / and the 'anthrax letters' attack of october , substantial effort has been given to harnessing life science research for the purposes of national security. biodefence and bioterrorism preparedness are thus considered high-priority areas for national investment by government agencies and the military alike. an illustrative example of this two-tiered approach is the funding policy in the usa, where biodefence research is financed by the nih, department of homeland security (dhs) and defense advanced research projects agency (darpa), to name a few. under the synergistic influence of these three sets of forcessocial, economic and politicalbiotechnology has been transformed into a truly global fast-evolving enterprise encompassing a multitude of stakeholders, delivering considerable benefits and holding out still greater promise, with profound and far-reaching implications for virtually every aspect of human well-being and social life. the pharmaceutical industry is a case in point, for its steady expansion would hardly be possible were it not for the vast array of techniques and methods enabled by the progress of the life sciences. worth roughly billion dollars, the global pharmaceutical market dominates the life sciences industry and arguably determines the trajectory of life sciencesrelated technological development and global spread. gene cloning, dna sequencing and recombinant construction of cell lines, to name a few, are all deemed indispensable for the development of novel medicines and therapeutics. it suffices to mention that more than half of the top selling commercially available drugs in the usa would not exist without those methods. agriculture, too, has been heavily influenced by the ongoing biotechnology revolution, as evidenced in the rapid growth and dispersion of commercialised transgenic crops (biotech crops) and the efforts to use gmos (both animals and plants) for the production of vaccine antigens and other biologically active proteins ('biopharming'). indeed, the increase in the area of farmland planted with transgenic crops rose dramatically from . hectares in to about million hectares in and is still growing. in addition, technological convergence between biotechnology, nanotechnology, information technologies and cognitive science has unlocked a broad scope of opportunities for maximising public (and private) welfare, offering substantial benefits in wideranging areas such as medicine, pharmacy, crime investigation and national security by ensuring precision and reliability, while at the same time, reducing the amount of time previously required for the performance of certain tasks. four key features of biotechnology make it so appealing to the majority of stakeholders involved. first, biotechnology innovation is characterised by duality, whereby research yields results that simultaneously lead to advances in basic knowledge and stimulate product development. second, the output that the life sciences generate in the form of new medicines, improved nutrition products, enhanced yields and novel materials, is 'strongly positive'. the increasing utility of tools and strategies for human enhancement, whether in professional sport, for cosmetic and aesthetic purposes, or on the battlefield, vividly reflects the firm conviction that the transformative capacity of biotechnology, even at the most fundamental level, is something to be welcomed and vigorously embraced. what is more, biotechnology possesses proven economic viability, as illustrated in the burgeoning industries and new markets it has spurred. against this backdrop, the high rate of biotechnology expansion is anything but surprising, since every increment in biological capability pays back the researcher and the researcher's sponsors in short order. payback comes in the esteem of peers, in promotions, and in increases in the academic or corporate salaries of the researchers whose work generates knowledge and new therapies. payback comes in the form of profits for the manufacturers of kits to perform the manipulations, royalties for the writers of the methods manuals profits for the drug industry. payback comes for the public in the form of new drugs and therapies. fourth, besides being cost-effective, many of the benefits that biotechnology offers are easy to obtain and disseminate. in other words, many of the various prospects for public (and private) betterment are not situated at some distant moment in the future but can be realised immediately, as a result of which pressing problems can be alleviated, if not fully resolved, and substantial revenue can be generated in the short term. last but not least, while there are some risks and concerns associated with the advancement of biotechnology, few of those are deemed urgent or significant enough to impact on the pace of innovation. as the actual manifestation of such risks is often contingent upon the interplay of a variety of factors, this renders the likelihood of a major crisis unfolding as a result of the progress of biotechnology low. moreover, there is a genuine belief that any challenges that may arise from the proliferation of novel technologies can either be foreseen or dealt with on a case-by-case basis. given the enormous potential of biotechnology for addressing societal, economic and environmental challenges, it is unsurprising that most states have readily endorsed scientific and technological innovation and embarked on largescale generously-funded r&d programmes in the life sciences. given the powerful multifaceted impetus for biotechnology advancement, it is possible to identify at least five key trends in the governance of biotechnology that are common for highly industrialised and developing countries alike. those include: high-level coordination, facilitation and funding; synergies within and between both the public and private sector; emphasis on strategic and competitive interests at the expense of precaution; regulations that seek to promote rather than restrict scientific and technological progress; and overreliance on technical solutions. at international level, the on-going expansion of biotechnology has been hailed not only as an inherently positive development but also as an essential prerequisite for enhancing human welfare and addressing various socio-economic, environmental and health concerns. in its world health report, the who called for: increased international and national investment and support in [life science] research aimed specifically at improving coverage of health services within and between countries. the who has also strived to promote research on specific diseases, such as hiv/aids, cancer, pandemic influenza, tuberculosis and malaria, with the goal to improve methods for prevention and diagnostics and facilitate the development of effective therapeutics and vaccines. in a similar fashion, the un food and agriculture organisation (fao) has highlighted the positive impact that biotechnology could have on the development of agriculture: . . . biotechnology could be a major tool in the fight against hunger and poverty, especially in developing countries. because it may deliver solutions where conventional breeding approaches have failed, it could greatly assist the development of crop varieties able to thrive in the difficult environments where many of the world's poor live and farm. it is not difficult to see how those assertions have been translated into national policies and practical steps across the globe. the us nih that provide the bulk financial support for medical and health-oriented r&d in the us spent over . billion dollars during the fiscal year , about a third of which was allocated for funding biotechnology and bioengineering projects. within its sixth framework programme for research and technological development spanning the period - the european union (eu) distributed more than . billion euro for projects under the theme 'life sciences, genomics and biotechnology for health'. developing countries, too, are increasingly investing in 'red' biotechnology as part of their efforts to address public health concerns. according to a recent who report, support for biotechnology and particularly, for cancer research, in cuba has soared over the past years, amounting to over one billion dollars. as a result, the cuban biotechnology industry is burgeoning, holding around international patents and exporting vaccines and pharmaceuticals to more than countries. the prospect of climate change coupled with rising population numbers has compelled governments in the global north and south alike to explore 'green' biotechnology as a means of ensuring food security. the usa remains by far the largest commercial producer of gm crops. several eu member states (france, germany, spain, poland, romania, czech republic, portugal and slovakia), canada and australia further feature in the list of industrialised nations that have embarked on growing gm plant breeds. more and more emerging economies are striving to expand their agrobiotechnology sector, most notably brazil, india, argentina, south africa, mexico, burkina faso, myanmar and chile. in , the chinese government launched a major r&d initiative worth billion dollars to develop new plant varieties by that will enhance yields, have improved nutritional value and be resistant to pests. public-private partnerships underpinned by access to early-stage risk capital and strong linkages between business, universities and entrepreneurial support networks constitute an important vehicle for promoting innovation and fostering technology transfer and product development. for instance, the chinese government has launched a major initiative mobilising . billion dollars in venture capital to support start-ups in the immense zhangjiang science park outside shanghai ; russia's rusnano has entered a million dollar partnership with the us venture capital firm domain associates to fund 'emerging life science technology companies and establish manufacturing facilities in russia for production of advanced therapeutic products'; and cleveland's university hospital has allocated million dollars for setting up a 'non-profit entity to fund and advise physician-scientists on transitional research and a related for-profit accelerator that will develop selected compounds to proof of concept.' the kauffman foundation in the usa, a wealthy philanthropic establishment dedicated exclusively to the goal of entrepreneurship has been particularly zealous in its quest for promoting university-based entrepreneurial activities nationwide. its kauffman campuses initiative launched in early enjoyed so much popularity among universities that following the initial round of grants totalling million dollars, the foundation announced its resolve to leverage a million dollar investment for the creation of new interdisciplinary education programmes. university-industry partnerships, while not a novel phenomenon in the area of biotechnology, have considerably intensified over the past several decades, thus facilitating the widespread commercialisation of life science research. indeed, per cent of the companies in the us surveyed by blumenthal et al. in had relationships with an academic institution in that year and in more than half of those cases industry provided financial support for research in such institutions. according to another study, the total industry investment in academic life science research in the usa tripled between and reaching almost billion dollars and has been growing ever since. against this backdrop, some commentators have put forward the 'triple helix' model, which serves both as a conceptual tool and a policy blueprint. in the former case, it is used to elucidate the academic-industry-government relationships that underpin the institutional arrangements and changing practices in the processes of production, transfer and application of knowledge in post-industrial societies; in the latter, it is promoted as a framework for economic development through state investment and knowledge sharing between academia and industry. others, however, have remained sceptical of the close integration of universities and the private sector voicing concerns about the possible deleterious effects arising therefrom: as in other activities, when big money flows fast, temptations and opportunities arise for risky behaviour and stealthy or even brazen wrongdoing in pursuit of personal or institutional advantage. the new world of academiccommercial dealings is characterised by some grey areas and evolving rules for permissible and impermissible conduct. the people who manage and conduct research in scientific organisations are not immune to the weaknesses and foibles so plentiful elsewhere, despite the accolades for probity that science bestows upon itself. with more and more universities joining the biotechnology 'gold rush' and corporate values and goals steadily penetrating the professional academic cultures, scholarship turns into a result-oriented activity subject to the priorities and interests of business partners and industrial sponsors. strategy and careful planning deemed essential to the pursuit of for-profit knowledge can have a restraining effect on the spontaneous vigour characteristic of academic research, limiting the range of problems that could be studied to those defined by the market. at the same time, scientists often find themselves under tremendous pressure striving to satisfy the demands of their industrial clients without utterly neglecting their academic duties ranging from mentorship through filing grant applications to publishing. the extensive workload coupled with the bright prospects for securing long-term research funding and achieving some individual gain and prominence provide a favourable environment in which instances of dubious, sometimes fraudulent, behaviour, conflicts of interest and lack of transparency, unless too severe, are unlikely to encounter widespread opprobrium and may even go unnoticed. in the race for patents and venture capital, the business mentality dulls scientific rigour and the ethics threshold appears not too difficult to cross. interests at the expense of precaution given the tremendous benefits that biotechnology is expected to generate in virtually any sphere of human activity, it is not difficult to understand why its progress is predominantly viewed through an explicitly positive lens by policy-makers. since the opportunities for achieving public betterment and enhancing state prestige and international standing are too tempting and too abundant, there is a powerful urge to dedicate both will and resources to promoting the large-scale expansion of the life sciences. for one thing, the prospect of conquering disease and maximising human wellbeing provides solid justification for a deliberate and sustained investment in fostering scientific and technological prowess. lack of commitment and reluctance to support r&d in the life sciences then becomes an unfavourable option in the political calculations of states regardless of their level of economic development and international status. within the context of political calculus pervaded by realist fears, competition and power, the perceived risks of inaction with regard to scientific and technological development justify vast expenditure, lower regulatory barriers to innovation and product development. political choices concerning biotechnology support are therefore frequently made at the expense of calls for caution and potential social, environmental and ethical concerns. the regulation of genetic engineering is a case in point. as discussed in the previous chapter, from the outset, the attempts of governments to impose strict controls on research involving rdna faced a severe backlash from academic scientists and business executives alike. by the s, the various legislative initiatives put forward in the usa were abandoned in favour of the regime established by the nih guidelines, which virtually exempted the biotechnology industry from formal regulation. while the leading us-based companies pledged to 'voluntarily comply' with the guidelines, behind the scenes they craftily continued to push for a system that would insulate them from governmental and public scrutiny. indeed, during the s when the states parties to the btwc strived to strengthen the treaty by negotiating a binding verification mechanism corporate interests proved too big and too important to be ignored. both the pharmaceutical research and manufacturers of america (phrma) which represented the country's major research-based pharmaceutical and biotechnology companies, and the biotechnology industry organisation (bio) which at that time represented some biotechnology firms, became vocal opponents of any measures designed to promote international arms control which seemed to hinder in any way the protection of proprietary information and intellectual property. in the period between and the associations invested considerable effort, time and ingenuity in lobbying the us government and influencing the diplomatic talks in geneva to secure an outcome that was in line with the demands of their constituencies. of course, it would be naive to ascribe the us resolve to reject in both the text of the protocol and its utility in general for providing adequate verification and enhancing confidence among states parties solely to the activity of the biotechnology industry; nevertheless, it would be equally naive to suppose that corporate interests played no significant role in the process. besides economic priorities, national security and military calculations can also provide a compelling rationale for downplaying the potential risks associated with biotechnology expansion. following the 'anthrax letters' attack in october , the us government embarked on a massive financial investment to boost its bioterrorism preparedness and enable the prevention, early detection, monitoring and emergency response to biological threats. as outlined in biodefense for the st century, a presidential directive that set out a comprehensive framework for national biodefence policy, between and the federal government provided roughly billion dollars 'to state and local health systems to bolster their ability to respond to bioterrorism and major public health crises'. along with the highly controversial vaccination programme that the government envisaged, another important development designed to enhance america's biodefence preparedness and capability was the drastic increase both in the number of high-containment labs (bsl- and bsl- ) and the number of researchers with access to some of the most dangerous pathogens known to mankind, including the causative agents of ebola, plague and q fever. some commentators have questioned the logic behind this policy highlighting the heightened risk of accidental or deliberate release of pathogens. far from being ill-founded or hypothetical, such fears stemmed from a range of high-profile cases that occurred after across the us in which the lack of proper training and professional negligence resulted in scientists being exposed to or infected with deadly microbes. real-life horror stories about vials of plague being transported in the hand-luggage of researchers on passenger aircraft without the required authorisation, and deadly cultures gone missing from what appeared to be secure laboratories further fuelled the criticism toward the us biodefence policy raising difficult questions about its appropriateness and actual goals even before the 'anthrax letter' investigation revealed that the attack was 'insider's business'. life science research, just as any other sphere of professional activity, is subject to a range of institutional, national and international regulations. along with the more general rules such as those related to occupational health and safety, fair pay and job competition, conflict of interests, labour rights, and professional liability, there are also specific ones addressing particular aspects of the research process including project clearing (e.g. review by local biosafety committees), safe laboratory practice and transport of pathogens (e.g. international health regulations), exchange of viral strains (e.g. pandemic influenza preparedness framework, ), handling of dangerous pathogens (e.g. us select agent programme) and ethical treatment of human subjects and samples obtained therefrom (e.g. the human tissue act in the uk). while hardly exhaustive, this list suffices to convey the idea that the regulatory regime governing the practice of life science research is dense and comprehensive. with more than international organisations overseeing biotechnology from various perspectives, there is a prima facie reason to assume that the regime in its current form is sufficiently flexible to accommodate novel advances and hold any potential risks, which they may pose, at bay. yet in reality over the past decade the opposite trend has prevailed, that is, the existing governance mechanisms have struggled to respond adequately to the proliferation of new scientific developments with multiple adaptive uses and the multiplicity of cutting-edge developments posing profound ethical quandaries. how to account for this discrepancy? part of the problem stems from the fact that since at least the late s the regulation of biotechnology has been streamlined so as to become compatible with and not a restriction on continued technological change and economic growth. as such, it rests upon the barely questioned assumption that the progress of biotechnology is inherently good and needs to be harnessed and vigorously promoted. needless to say, any measures that seem to slow down or restrain its advancement are deemed undesirable and even detrimental to socio-economic development. hence, when developing regulations, policy-makers have generally pursued a twofold objective: first, to promote the safe practice of life science research by reducing any risks arising therefrom both to scientists and the general public; and second, to ensure that any issues that may hinder the expansion of biotechnology are not subject to restrictive legislation. a vivid manifestation of this approach is the way in which the ongoing debate on 'dual use research of concern'benignly-intended research that seeks to maximise human welfare by responding to health, societal and environmental ills but could also facilitate to the development of more sophisticated and potent biological weapons and enable bioterrorism has been handled. for more than a decade, researchers, journal editors, security experts and policy-makers have strived to devise oversight mechanisms and governance initiatives that could adequately tackle the challenge of dual use without stifling innovation. unfortunately, to date their efforts have met with little success, as a result of which virtually each experiment of dual use concern is dealt with separately on a case-by-case basis. this is not to say that there are no similarities across the studies of this kind. on the contrary, a few of the most notable examples follow a similar paradigm, including the creation of a vaccine-resistant strain of the mousepox virus, the artificial synthesis of the polio virus, the recreation of the spanish influenza virus and, most recently, the production of a mammalian-transmissible h n avian influenza virus (see fig. . ). all four of them were performed in strict compliance with the rules and procedures in place for laboratory biosafety, biosecurity and biorisk management and under appropriate physical containment conditions; all had passed thorough review by the respective local biosafety and bioethics committees; and all of them were deemed essential in terms of public health benefits. above all, the ethical and security concerns that the studies have raised go far beyond the laboratory door, posing fundamental questions about how life science research is reviewed, conducted and communicated. yet none of the high-profile experiments of concern has proved critical enough to provoke a radical change in the way dual-use research is governed. three points merit consideration in this regard. the first pertains to the manner in which the dominant discourse on dual use is framed, that is, in purely ethical terms as a dilemma. while bioethics undoubtedly has a role to play in the discussions on dual use, the language in early , the journal of virology published a report of the creation of a highly virulent strain of the ectromelia virus, the causative agent of mousepox. the work described in the report was carried out by a group of australian scientists based in canberra. its original goal was the development of an infectious immunecontraceptive that could be used against wild mice for the purpose of pest control. to achieve this, the group drew upon previously published work. during the course of the experiment, the researchers unexpectedly discovered that the newly engineered strain of the mousepox virus, which they created, killed % more mice than the parent virus, including mice that had been vaccinated or that had natural immunity. when the research was published, concerns were raised that it could potentially be misapplied for hostile purposes, or even that the same technique could be utilised for creating a more virulent strain of the variola virus, which causes smallpox in humans. in , a team of scientists led by dr eckard wimmer from the university of new york at stony brook announced that they had successfully created a polio virus 'from scratch'. to carry out the research, the scientists 'followed a recipe they downloaded from the internet and used gene sequences from a mail-order supplier'. a once the virus created, it was tested on mice, as a result of which the infected animals became paralysed and died. the study spurred a wide-ranging debate, not least because it drew attention to the possibility of using synthetic biology for constructing de novo viruses for the purposes of bioterrorism. in , it was announced that cdc scientists, together with colleagues from several research institutions across the usa, had successfully recreated the influenza virus, that was responsible for the pandemic, which killed between and million people worldwide. using dna from a tissue of a flu victim buried in the permafrost in alaska, the researchers managed to reconstruct the influenza virus and thus study its pathogenesis and properties that contributed to its virulence. despite the scientific justification that was put forward, critics have argued that the study is 'a recipe for disaster', not least because the availability of the virus' full-genome sequence and detailed method for its reconstruction on the internet may facilitate its synthesis by a of 'dual-use dilemmas' is too abstract to offer appropriate analytical tools for dealing with the issues at play. as discussed above, the questions that dual-use research poses such as data sharing, research funding and project planning are far from hypothetical but they feature explicitly in everyday professional practice. however, the 'dilemma framework' automatically strips them of the complex socio-technical arenas in which they have actually presented themselves by laying an emphasis on what action should ideally be taken, rather than what is practically feasible given the circumstances. moreover, such issues are typically structural in nature for they constitute fundamental elements of the life sciences professional culture, and as such, could hardly be adequately addressed solely at the level of individual researchers. yet framing social, legal and security concerns in terms of moral dilemmas allows for structural issues to be omitted from the discussion, rendering life scientists the chief, if not the only, moral agents expected to reach what is deemed to be the 'right' answer. assigning abstract duties then comes to be regarded as an appropriate 'solution', even if those are virtually impossible to fulfil given the complexities of the working environment within which researchers operate. the second point is related to the reductionist view that dominates the discourse of what counts as a risk in life science research. perhaps one of the most significant legacies of the asilomar conference on rdna (see chapter ) is the emphasis on laboratory risk that could be effectively managed by dint of physical containment and rules and procedures for safe laboratory practice. it suffices to mention that the bulk of guidelines and formal regulations published by the who focus exclusively on promoting and refining measures that aim to maximise laboratory biosafety and prevent the accidental release of pathogens. hence, it is hardly surprising that the concept of dual use and the idea of risks beyond the laboratory door implicit in it seem alien to the majority of practising researchers. striking as it may appear, even though dual use research has been debated for more than a decade now, the level of awareness among life scientists of the broader social, security and legal implications of their work remains low. the third point deals with the way in which risks in life science research are assessed and mitigated. given the narrow definition of risk encompassing technical particulars, physical containment and biosafety, risk assessment is considered an appropriate and reliable tool for ensuring research safety. the heavy reliance upon risk assessing tools is underpinned by two underlying assumptions. one is that it is possible to foresee and calculate most, if not all, things that could potentially go wrong both during the development phase of the project and after its completion. the other is that it is possible then to use the produced data as a basis for devising measures and strategies for eradicating, or at least, mitigating the risks likely to occur. attractive as it may seem, this 'new alchemy where body counting replaces social and cultural values' presupposes a clear distinction between the risk assessment 'experts' and the general public, whereby the former are granted a licence to make decisions about the risks that the latter cannot do without. likewise, costbenefit analysis on the basis of which research proposals are screened for potentials risks and security concerns has attracted some serious criticism. in the view of some commentators, besides being sometimes deeply inaccurate, the cost-benefit analysis is 'ethically wrong' since 'applying narrow quantitative criteria to human health and human life' is unacceptable. but there are other problems, too. as pointed out by dickson, the cost-benefit analysis distorts political decision-making by omitting any factors that cannot be quantified, thus obscuring questions of equity, justice, power, and social welfare behind a technocratic haze of numbers. as a result, complex and politically charged decisions are reduced to a form that fits neatly into the technocratic ways of making regulatory decisions, whereby calculations and approximations made by the few substitute for the judgements of many. the wide-ranging controversy that unraveled in late when two teams of scientists working independently in the netherlands and the usa managed to produce an air-borne strain of the h n avian influenza virus, a highly pathogenic and lethal microbe with over per cent mortality rate in humans arguably constituted the pinnacle of the deliberation on dual use research. both studies set alarm bells ringing for the security community who almost immediately jumped in the debate voicing concerns over the possibility of biological proliferation and bioterrorism. some commentators even argued that the experiments ran counter to the spirit if not to the letter of the btwc. against this backdrop, the resultant controversy was deemed at least initially to offer a timely opportunity to evaluate the existing governance mechanisms, determine their gaps and weaknesses, and broaden the scope of deliberation inviting participation of a wide range of stakeholders. unfortunately, the outcome of the debate proved far more moderate, signalling preference for preserving the status quo without disrupting the established systems for governance and oversight. despite the extensive mass media coverage of the controversy, only few public consultations were held and none of those was designed as a platform for making policy proposals or developing action plans. moreover, the denselypacked agenda prepared duly in advance left very limited scope for posing 'tricky' questions which the participating 'experts' might have struggled to answer. needless to say, all consequential decisions were made behind closed doors away from public scrutiny and on some occasions the people with the greatest vested interest in the publication of the studies were also the ones with the greatest say in the process. there were no significant changes in terms of governance initiatives, either. far from being ground-breaking developments, the us government policy for oversight of life sciences dual use research of concern and the decision of the dutch government to invoke export control legislation before allowing the publication of the study conducted within its jurisdiction were little more than desperate moves that aimed to obscure the inadequacy and shortcomings of the measures already in place. overall, the manner in which the h n debate was handled could be treated as a missed opportunity, whereby those in charge of the decision-making process did little to address or even acknowledge the broader issues underpinning dual-use research of concern but simply 'kicked the can down the road to the next manuscript' waiting for the next controversy to erupt. technology seems to play a significant role in the governance of life science research. high-containment laboratories, well-equipped biosafety cabinets, sophisticated waste management systems, enhanced personal protective equipment and secure containers for the safe storage and transportation of biohazard materials are just a few of the tools and systems in place that allow the safe handling of dangerous pathogens and toxins and, at the same time, protect both laboratory personnel and the general public from exposure to deadly microbes. that said, the effectiveness of technical solutions should not be overstated if only for the fact that 'problems' of governance are barely technical matters per se but rather constitute complex issues of human relatedness. nevertheless, the attractiveness of technological fixes as offering reliable risk mitigation and reassurance in the safety of biotechnology is ever growing. it suffices to mention that the h n controversy discussed above was in part resolved after the lead researchers in the netherlands and the usa respectively agreed to add a detailed section on the technical specificities and laboratory biosafety and biosecurity measures taken during the experiments. the strategy has proven effective in diverting attention from the rather inconvenient questions regarding the utility and significant potential for hostile misuse of the so called 'gain-of-function' (gof) research and concentrating it on more mundane issues dealing with in-house precautions and safety procedures. once the latter were deemed adequately resolved, the former were effectively forgotten. still, the value of technical means in ensuring reliable risk management should not be taken for granted. for one thing, laboratory biosafety precautions, however sophisticated, are far from perfect and accidents do occur. such is the case with the pirbright site in the uk which was at the centre of a major outbreak of foot-and-mouth disease in , as a result of which over animals were slaughtered. in the bioterrorism bsl- laboratory at the us cdc in atlanta suffered repeated problems with airflow systems designed to help prevent the release of infectious agents. the faulty system could perhaps be regarded as an exception had it not been for the authoritative investigation report of the us government accountability office (gao) released in march . according to the report, the cost of building and maintaining high-containment laboratories, coupled with the absence of national standards for their design, construction, operation, and maintenance 'exposes the nation to risk'. far more critical is the situation in the developing world and emerging economies where lax regulations and technical failures have significantly heightened the risk of accidental release of pathogens, as demonstrated by the numerous 'escapes' of the severe acute respiratory syndrome (sars). but even if technology functions impeccably, this hardly reduces the likelihood for a human error or inappropriate behaviour. unlocked doors in high-containment facilities hosting deadly pathogens, eating and drinking in laboratories and poor waste disposal practices are just a small part of the otherwise long list of mundane mishaps that may result in severe consequences. it is worth mentioning that the us cdc came under the spotlight after internal e-mail correspondence revealed that doors in the bsl- block where experiments involving the causative agenets of anthrax, sars and influenza were performed were left unlocked on numerous occasions, thus increasing the risk of unauthorised access or theft. given the chance of technical flaw and the potential for human error, some life scientists have begun to question the reliability of existing laboratory precautions and demand thorough review and evaluation. in a recent letter to the european commission the foundation for vaccine research has asked for 'a rigorous, comprehensive risk-benefit assessment' of gof research that 'could help determine whether the unique risks posed by these sorts of experiments are balanced by unique public health benefits which could not be achieved by alternative, safe scientific approaches'. engines that drive biotechnology momentum by and large, the ongoing progress of biotechnology is largely viewed and assessed through an explicitly positive lens which allows focusing almost exclusively on the benefits likely to be accrued notwithstanding the risks, actual and potential. the resultant distorted image is problematic, not least because it precludes any comprehensive discussion on the potential side effects and negative implications of novel life science advances. above all, it sustains the barely questioned assumption that the existing governance mechanisms are adequate and sufficient to cope with the stresses and strains of the rapidly evolving biotechnology landscape. yet given the complex and multifaceted dynamics shaping the life science enterprise, the rapid pace of innovation, and the limits to predicting the synergistic and cumulative effects of the proliferation of new technologies, the uncritical acceptance of such assumptions is at best naïve and at worst dangerous. arguably the advancement of the life sciences has greatly benefited from the fascinating breakthroughs made in other areas of study, such as chemistry, engineering, computing, informatics, robotics, mathematics and physics. some commentators even talk about a third revolution in biotechnology underpinned by scientific and technological convergence: convergence does not simply involve a transfer of tools sets from one science to another; fundamentally different conceptual approaches from physical science and engineering are imported into biological research, while life science's understanding of complex evolutionary systems is reciprocally influencing physical science and engineering. convergence is the result of true intellectual cross-pollination. the resultant 'new biology' has opened up a range of marvellous possibilities enabling the manipulation of living matter at the full range of scales, as well as the application of biological systems principles for the development of novel materials, processes and devices. as such, it has been largely hailed as possessing the 'capacity to tackle a broad range of scientific and societal problems.' this is not an exaggeration. as noted by a recent report of the us nas, the precipitous decline in the cost of genome sequencing would not have been possible without a combination of engineering of equipment, robotics for automation, and chemistry and biochemistry to make the sequencing accurate. likewise, it is the combination of expertise from fields as diverse as evolutionary biology, computer science, mathematics, and statistics that has allowed both the analysis of raw genomic data and the subsequent use of these data to other fields. at the same time, advances in nanoscience and nanotechnology have considerably enhanced drug delivery making it more accurate by targeting specific parts of the body. yet the transformative potential of scientific and technological convergence comes at a price, not least because parallel to the benefits it offers, there are risks the effects of which could be truly devastating. take drug delivery, for instance. thanks to the technological breakthroughs over the past decade, doctors have gained unprecedented access to the human body which, in turn, has facilitated the treatment of previously incurable disease and conditions (e.g. some forms of cancer). nanoparticles and aerosols are now utilised for delivering a precise dose of therapeutics to tissues and cells via novel pathways circumventing body's natural defences and evading immune response. it is not difficult to imagine how such knowledge could be misapplied for malicious ends, including incapacitating and killing. research on bioregulators is a case in point. bioregulators are natural chemicals in the human body that play a vital role in the maintenance of the homeostasis but when administered in large quantities or in healthy individuals could be toxic and lead to serious disorders, even death. given their properties, bioregulators constitute the perfect bioweapon: efficient and virtually impossible to detect. and if in the past, security analysts discounted the risk of their weaponisation due to the instability of the compounds when released in the atmosphere, the emergence of novel drug delivery techniques has significantly altered the security calculus. this is just but one example of the challenges that the increasing convergence between biology and chemistry poses to the integrity of the international biological and chemical non-proliferation regimes. even though some effort has been made over the recent years to address those and other areas of concern and strengthen the international prohibition against biological and chemical warfare, in practical terms little has been achieved, as a result of which the risk of the hostile exploitation of novel scientific developments remains far from hypothetical. along with the risk of misuse of new knowledge, there is the risk posed by the lack of sufficient scientific knowledge. cross-disciplinary convergence opens a multitude of opportunities for manipulation and modification of living matter but, at the same time, it precludes almost any sensible assessment of the potential interactions likely to occur in the process. nano-based medicine is but one area that has attracted criticism in this regard. since some elements behave differently at nano-scale, it becomes extremely difficult to assess their level of toxicity or other negative side effects that they may exert. such is the case with long carbon nanotubes, which having been initially praised for their potential to improve implant development were later blamed for exhibiting asbestos-like behaviour that could lead to cancer. another area of converging science with far-reaching implications is synthetic biology, a cross-disciplinary field that draws upon strategies and techniques from molecular biology, chemistry, engineering, genomics, and nanotechnology and thus enables the design and modification of biological systems at a fundamental level. empowered by the tools of synthetic biology, in scientists managed to assemble a polio virus 'from scratch' in the absence of a natural template. and in craig venter and his team announced the construction of the first self-replicating synthetic cell which, in their view, was 'a proof of the principle that genomes can be designed in the computer, chemically made in the laboratory and transplanted into a recipient cell to produce a new self-replicating cell controlled only by the synthetic genome.' the controversial work has attracted criticism on several grounds, including the potential negative effects of the accidental or deliberate release of the novel organism in the environment and the arrogance of scientists to 'play god'. more broadly, both the polio and synthetic cell studies have exposed the obstacles to the regulation of synthetic biology. while some commentators dismiss the risk of bioterrorism, underscoring the key role of tacit skills and knowledge and the difficulties that the lack thereof poses to the replication of the experiments, other issues still merit attention. consider the question of access to commercially available genomic sequences. even though the oversight system for screening base pair orders has improved since the guardian report that exposed the lax regulations under which virtually anyone could order gene sequences, gaps still remain leaving scope for abuse by those with malign intent. for example, schmidt and giersch have outlined at least three areas of emerging challenges that the existing governance regimes would struggle to accommodate, including 'split orders', 'outsourcing', and the potential for non-natural biological systems. the human genome project completed in lasted over ten years and cost close to billion dollars; by contrast, about a decade later, wholegenome sequencing can be performed within hours at a price of roughly dollars or less. while still in its infancy, personalised medicine and individual genetic testing are steadily gaining popularity. indeed, 'up to people in england are expected to have their entire genetic makeup mapped in the first stage of an ambitious public health programme' launched by the national health service in that aims to 'revolutionise the treatment and prevention of cancer and other disease.' according to its proponents, genomic testing offers numerous advantages vis-à-vis traditional evidence-based medicine, including the possibility of early diagnostics of disease, of individually-tailored treatment and, perhaps most importantly, of disease prevention, as illustrated in the resolve of the hollywood actress angelina jolie to undergo double mastectomy after discovering she has an inherited genetic mutation that puts her at high risk of breast and ovarian cancer. but this is just the beginning. in scientists managed to sequence a foetus's entire genome using a blood sample from the mother and a saliva specimen from the father, a development that could potentially allow for a range of genetic disease conditions to be detected prenatally. and laboratory experiments have already demonstrated the efficacy of genetic therapy to cure mitochondrial disease by creating an embryo with genetic material from both parents and a third person acting as a donor. while truly breathtaking, the advances outlined above raise a host of thorny issues of ethical, social, and legal concern that merit public scrutiny and extensive deliberation before decisions regarding their widespread application are made. at a very basic level, there is the question of whether and to what extent we as individuals are capable of assimilating the information that our own genetic makeup may reveal. are we sufficiently resilient to cope with the emotional distress, anxiety, shame, stigma and guilt that the awareness of severe medical conditions that we or our closed ones are suffering or likely to develop? far from hypothetical, this question has prompted the establishment of a novel profession, that of the genetics counsellor whose task is to help patients overcome any negative effects, stress, or psychological trauma that the disclosure of their genomic map may create. this is just a partial solution though, for the crux of the matter lies in finding a way to deal effectively with risk and probabilities and we as humans are yet to demonstrate a capacity for understanding or relating them to our own lives. individual emotional turmoil, however significant, constitutes only the tip of the iceberg. according to daniel kevles, the torrent of new genetic information has already begun to fundamentally reconfigure social practices and inter-personal relations: it has been rightly emphasised that employers and medical or life insurers may seek to learn the genetic profiles of, respectively, prospective employees or clients. employers might wish to identify workers likely to contract disorders that allegedly affect job performance while both employers and insurers might wish to identify people likely to fall victim to diseases that result in costly medical or disability payouts. whatever the purpose, such genetic identification would brand people with what an american union official has called a life-long 'genetic scarlet letter' or what some europeans term a 'genetic passport'. linking genetic makeup with human identity would ultimately set the scene for the proliferation of technologies aimed at human enhancement: after all, if a gene therapy could allow one to stand a chance in a job competition, boosting one's capabilities would potentially make them a more desirable candidate. other issues of more immediate concern are also likely to arise. one is privacy. gene-sequencing companies usually hold the genetic data of their clients in digital format on online platforms, which automatically creates a risk that personal information may be leaked, hacked or stolen. further, there is the question of ownership. consider, for instance, the controversial issue of human gene patenting, whereby patented genes are treated as research tools and, as such, are controlled by the patent holder who may restrict and charge for their use. thus created, the system often operates to the detriment of patients by hindering research practice, elevating diagnostics prices and denying access to second and independent medical opinion. gene identification alone has a potential 'dark side' too, for it could enable the development of weapons targeted at groupspecific gene markers (e.g. ethnicity). pre-natal genetic testing is yet another significant bone of contention, not least because it evokes notions of state-mandated eugenic programmes and assaults on human rights and dignity. while a nazi-like campaign for a superior race seems improbable in the twentieth-first century, this is not to say that other forms of eugenics may not be encouraged. indeed, some commentators have highlighted the rise of 'homemade eugenics', whereby individual families can make decisions on the attributes of their progeny: the lure of biologically improving the human race, having tantalised brilliant scientists in the past, could equally seduce them in the future, even though the expression of the imperatives may differ in language and sophistication. objective, socially unprejudiced knowledge is not ipso facto inconsistent with eugenic goals of some type. such knowledge may, indeed, assist in seeking them, especially in the consumer-oriented, commercially driven enterprise of contemporary biomedicine. it is plausible to assume that when presented with the opportunity of having their future child tested for genetic disorders, many parents would barely hesitate to accept. such a resolve could have far-reaching implications though. for instance, some genetic therapies entail the use of donor dna different from that of the parents, whereby any genetic modifications in the embryo will pass down to future generations. despite the government support for the 'three-parent babies' in the uk, local religious organisations have protested vociferously against the legalisation of the technique. at the same time, there are certain genetic disorders that can be diagnosed at an early stage but, as of yet, cannot be cured, which inevitably poses the tough choice between raising an unhealthy child and abortion. to be sure, such questions constitute more than individual parents' dilemmas, for they touch upon established social and cultural values, something evident in the profound differences across national reproductive policies. more broadly, there are concerns that reproductive genomics may remain a prerogative of those affluent enough to afford it, thus further exacerbating the divide between the global rich and the global poor. the growth of life science capacity over the past few decades across the globe has been truly astonishing, leading to the emergence of a vibrant research community that brings together researchers from various parts of the world. indeed, a nas report highlights the extension of both north-south and south-south partnerships, which has played a key role in synergising strengths and maximising competitiveness by improving the quality and effectiveness of research and facilitating data sharing. at the same time, increasing collaboration in the realm of biotechnology industry has offered companies situated in emerging economies access to the global market, thus contributing to economic development and growth. recent advances in technology and laboratory and experimental equipment have further impacted on the practice of life science research in profound ways. improvements in dna sequencing technology have significantly shortened the time required for the preparation of nucleic base-lines, thus relieving scientists of the burden of completing the task themselves and allowing them to focus on their actual project instead. studies and experiments once performed by senior researchers with extensive experience are now carried out by masters students. aided by specially designed genetic engineering toolkits, children as young as the age of ten start exploring the realm of biology in an interactive and engaging manner. needless to say, their notion of science and the world in general would differ significantly from that of their parents whose primary sources of knowledge used to be textbooks and encyclopaedias. indeed, the increasing commercialisation of synthetic biology offers anyone curious enough to fiddle with biological systems the chance of doing so in the comfort of their own home. such modern gene hackers often lack formal background in biology and come from various walks of life. driven by an insatiable appetite for knowledge and the vision of a ground-breaking discovery that could be turned into a multimillion dollar profit, they take up the rather unusual hobby of biohacking which entails the redesign of existing and the creation of novel biological systems. for just few hundred dollars bio enthusiasts set up laboratories easily obtaining all essential requisites and equipment from online sales. and if to some biohacking equates to little more than an unusual hobby, others highlight its potential to generate substantial revenue and fuel economic development. contrary to popular expectation, biohackers are not just eccentric individuals who work in solitude away from public attention. rather, they are members of a wide global movement dedicated to the ideal of do-it-yourself biology (diy), which has branches in locations on four continents. the movement has been partially institutionalised through the establishment of the biobricks and international genetically engineered machine (igem) foundations, which seek to promote the open and ethical conduct of biological engineering and stimulate innovation and creativity. to this end, igem holds an annual competition open to high school students, university undergraduates and entrepreneurs from all over the world. with more than participating teams, the competition constitutes the premiere forum at which biohackers can showcase their skills through project presentation. exciting as it may seem, the ongoing diffusion of life science expertise poses an array of governance conundrums. at the level of professional practice, the proliferation of research facilities around the world has exposed the urgent need for laboratory biosafety and biosecurity training, especially in developing states where a tradition of handling dangerous pathogens is lacking. the issue is further complicated, for such countries often lack the required legal and institutional infrastructure to ensure that professional practice is in compliance with relevant international regulations. foreign aid has gone some way in helping overcome those deficiencies but it has given rise to new problems, too. for instance, it is far from unlikely for a donor state to provide material support for the construction of a state-of-theart laboratory eventually leaving its maintenance to the local government, which can hardly afford the subsequent costs. a similar trend is observed in the area of capacity building and human resource development. most projects that aim to promote biorisk management and a biological security culture tend to be severely constrained in terms of time and funding and overly ambitious in terms of agenda and expected outcomes. lack of adequate mechanisms for quality assessment hinders progress evaluation and sometimes leads to duplication of effort and resources. the emergence of the diy biologists in the life science arena has further added to the challenge of ensuring that novel scientific and technological developments are utilised in a safe and ethical manner. even at the level of everyday practice, difficulties still persist. for instance, many amateur scientists have complained of the lack of manuals and guidelines regarding the safe operation and maintenance of home laboratories. issues such as waste disposal, safe handling and storage of biological material and prevention of contamination pervade the work of biohackers who unlike professional researchers conduct experiments in a much more volatile environment. potential security concerns are also present. with more and more individuals gaining access to biological engineering technologies, ensuring appropriate oversight of what goes on in garage laboratories becomes increasingly difficult. the experience of the us fbi is a case in point. back in the fbi arrested steven kurtz, a professor at the university of buffalo under the suspicion of plotting a bioterrorist attack. the subsequent investigation revealed that all laboratory and dna extraction equipment found in kurtz's house was legitimately obtained and used in his artwork. in an attempt to avoid mistakes of this kind, the fbi has drastically changed its approach to dealing with the diy movement launching a series of outreach activities that seek to raise awareness of the potential security implications of biohacking. while undoubtedly necessary, such initiatives may well be seen as too little, too late in light of the wide spread of materials, tools and devices that could facilitate the malign misuse of the life sciences. indeed, it is worth noting that as early as the late s the us defence threat reduction agency (dtra) managed to build a research facility that simulated the manufacture of weaponised anthrax using only commercially available materials and equipment. the role of states: both a poacher and gamekeeper structural factors have an important bearing on the development and growth of biotechnology. economic considerations, power interests and realist fears generate potent dynamics that shape and influence and sometimes direct the life science trajectory. within this context, states assume a dual role. on the one hand, they are expected to act as gamekeepers and regulate, monitor and control the process of life science research and the dissemination of novel technologies. on the other hand, though, they also have powerful incentives to act as 'poachers', not least because of the fascinating opportunities for enhancing their prosperity, prestige and security that scientific and technological development open up. the following passage effectively outlines states' dual function: government has an important role in setting long-term priorities and in making sure a national environment exists in which beneficial innovations will be developed. there must be a free and rational debate about the ethical and social aspects of potential uses of technology, and government must provide an arena for these debates that is most conducive to results that benefit humans. at the same time, government must ensure economic conditions that facilitate the rapid invention and deployment of beneficial technologies, thereby encouraging entrepreneurs and venture capitalists to promote innovation. given that the agent (i.e. state governments) in charge of initiating ethical debates on the progress of biotechnology is also the one expected to provide the conditions that would allow this progress to generate outcomes likely to contribute to economic growth and political superiority, it is hardly surprising that any issues likely to slow down or otherwise hinder the enormous momentum of the life sciences are omitted from public discussion. this duality further informs how risks are perceived, framed and addressed. for instance, even though most of the developing countries lack capacity to manage dual use research of concern, they do not see this as an immediate priority and prefer to invest effort and resources in improving their laboratory biosafety and laboratory biosecurity infrastructure and capacity. in the view of their governments, the dangers of naturally occurring and circulating diseases constitute a far greater worry than the potential for misuse of cutting-edge research. by contrast, some developed countries, most notably the usa, have embarked on building their biological defence systems highlighting the grave threat posed by the potential use of bioweapons by non-state actors. their activities have encountered severe opprobrium as some analysts see them as a contravention of the norms embedded in the btwc. the evolution of the chemical and biological non-proliferation regime epitomises the attempts of states to avert the hostile exploitation of the life sciences whilst promoting their use for 'peaceful, prophylactic and protective purposes'. the entry into force of the btwc and the chemical weapons convention (cwc) in and , respectively, is indicative both of states' renunciation of chemical, biological, and toxin weapons and of their commitment to the goals of arms control and disarmament. that said, the imperfections and shortcomings of these treaties signify the influence of realist fears and political calculations that pervade international negotiations. in the case of the btwc, two points merit attention. the first pertains to the lack of verification mechanism when the treaty was first agreed back in the early s. subsequent revelations of secret state-led offensive biological programmes in the former soviet union, south africa and iraq up until the early s have significantly undermined the convention. second, the failure to negotiate a binding protocol in has further dimmed the prospects for strengthening the regime and thus ensuring universal compliance with its prescriptions. less acute but just as worrying is the situation regarding the cwc. even though the convention is exemplary in many respects, not least because of its verification system, almost universal membership and implementing body -organisation for the prohibition of chemical weapons (opcw)it still faces serious challenges that need to be considered. for instance, while the treaty bans the development, production, acquisition, and retention of chemical weapons, the definition of 'purposes not prohibited under th[e] convention' entails 'law enforcement including domestic riot control purposes' (article ii. d). some commentators have argued that given the lack of a universally agreed definition what kind of activities count as 'law enforcement', this text opens a major loophole in the convention. several states parties of the convention have voiced concerns in this regard. australia has noted that: the weaponisation of [central nervous system] acting chemicals for law enforcement purposes is of concern to australia due to the health and safety risks and the possibility of their deliberate misuse, both of which have the potential to undermine the global norm against the use of toxic chemicals for purposes prohibited by the convention. [ . . . ] australia's position is that it is not possible for a state party to disseminate anaesthetics, sedatives or analgesics by aerial dispersion in an effective and safe manner for law enforcement purposes. critics highlight the possibility for the deployment of novel chemical weapons for the purposes of countering terrorism, something evident in the moscow theatre siege (dubrovka) when the russian security forces used a fentanyl-derivative agent, as a result of which about a sixth of the hostages and all of the terrorists involved died. in the european court of human rights ruled with regard to the dubrovka operation that: there had been no violation of article (right to life) of the european convention on human rights concerning the decision to resolve the hostage crisis by force and use of gas. the court, nonetheless, noted that: even if the gas had not been a 'lethal force' but rather a 'non-lethal incapacitating weapon', it had been dangerous and even potentially fatal for a weakened person [ . . . ]. the court further confirmed some of the earlier criticisms that were levelled against the government, particularly in terms of preparedness and provision of medical assistance. according to the ruling, russia had to pay damages to all the applicantsrepresentatives of siege victims. to date, russian officials have withheld information concerning the exact formula of the gas, which was used during the dubrovka operation, on security grounds. given the lack of an internationally agreed definition of what constitutes 'terrorism' on the one hand, and the rise of irregular/asymmetric warfare and sporadic conflicts, on the other, some commentators have warned against the possibility of a 'grey area' which may enable states to utilise non-traditional methods of war to gain advantage. deliberative systems encompass a vast array of practices, processes and mechanisms, both formal and informal, whereby a polity considers the 'acceptability, appropriateness and control of novel developments in or impacting on, shared social and physical arenas'. by design, they reflect and are informed by the values, beliefs and standards shared among the group, or in other words, by the prevalent culture. as such, deliberative systems vary across societies with their intensity, inclusiveness and structure depending on the established political and social norms. yet their chief purpose and function remain virtually the same, namely to help societies adapt to the changing circumstance of their milieu in a way that ensures stability, sustainability and safety. public deliberation requires time; and wide-ranging life science advances, current and planned, offer profound challenges to shared ideas and ideals about the foundations of human relatedness and of social coherence, justice, human dignity and many other norms, both formal and informal. yet given the ruminative nature of deliberative processes, on the one hand, and the fast speed at which biotechnology innovation is evolving on the other, the danger of the former being steadily outpaced and overburdened by the latter is far from hypothetical. consider the following passage sketching the scale of social changes likely to arise from the increasing convergence between nanotechnology, biotechnology, cognitive neuroscience and information technology: in the foreseeable future, we will be inundated with new inventions, new discoveries, new start-ups, and new entrepreneurs. these will create new goods and new services. [ . . . ] as expectations change, the process of politics and government will change. people's lives will be more complex and inevitably overwhelming. keeping up with the changes that affect them and their loved ones exhausts most people. they focus most of their time and energy on tasks of everyday life. in the future, when they achieve success in their daily tasks, people will turn to the goods and services, the new job and investment opportunities, and the new ideas inherent in the entrepreneurial creativity of the age of transitions. no individual and no country will fully understand all of the changes as they occur or be able to adapt to them flawlessly during this time. this vision of a 'brave new world' merits attention on two important grounds. first, it implies that the changes likely to occur in the not too distant future as a result of the rapid progress of science and technology are imminent and unavoidable in the sense that their advent hardly depends on or even requires extensive public deliberation. second, given that our capacity for adaptation to and grasp of those changes will be considerably impaired, the age of transitions leaves little space for public deliberation. to add to this gloomy picture, there is already some evidence that the progress in the life sciences is overwhelming the existing deliberative mechanisms. for instance, kelle et al. argue that the rapidity of biotechnology advancement coupled with the immensity and complexity of the knowledge accumulated therefrom complicates efforts to deal with potential risks, something evident in the regulatory gap that the convergence of chemistry and biology has created in the area of arms control. this is problematic, for the reduced resilience of deliberative systems provides favourable conditions in which scientific and technological innovation can continue unabated. a vicious circle is thus created in which the inability of deliberative systems to cope with the strain exerted by biotechnology advancement fuels the latter turning it into a self-propelling force. the proliferation of contentious 'gain-of-function' research is a case in point. even though the h n controversy discussed in the preceding sections exposed the limitations of existing governance mechanisms for addressing the potential security, ethical, and legal implications arising from such studies, it hardly precluded scientists from conducting similar experiments. indeed, less than four months after the moratorium on research involving contagious h n virus was lifted, a team of chinese researchers announced the creation of a hybrid of the h n strain and the h n virus that caused the flu pandemic. and it was not long until the newly-emerged h n influenza virus became airborne, as well. if anything, those examples indicate that in light of the rapid pace of life science progress, addressing governance concerns on a caseby-case basis is not only self-defeating but given the number and variety of conundrums, it is likely to become unsustainable in the long run. given the significant potential of biotechnology to bring about multifaceted changes in different spheres of life and generate considerable benefits in the form of new products, enhancement of public and private capital and alleviation of social ills, there is a powerful urge to allow the ongoing expansion of the life sciences to proceed largely unfettered. risks are carefully calculated and, where possible, downplayed as hypothetical at the expense of comprehensive deliberation. and even when proposals for risk mitigation measures are entertained, preference is usually given to those unlikely to hinder the progress of life sciences. by and large, there is a genuine belief that the existing governance mechanisms in the area of biotechnology can accommodate and cope with the wide-ranging pressures exerted by scientific innovation and the rapid diffusion of technologies with multiple uses, by offering 'solutions' and handling concerns on a case-by-case basis. in particular, the technology of safety is still 'celebrated as an unadulterated improvement for society as a whole'. yet there are reasons for scepticism toward the adequacy and effectiveness of the governance approaches currently in place. much of the discussion in the preceding sections has focused on the ways in which the increasing pace, growth and global diffusion of biotechnology advances are beginning to expose the limits of the existing measures for control and risk management by challenging accepted values and beliefs and redefining established norms of practice. as the multifaceted dynamics driving the biotechnology momentum continue to intensify and multiply, it becomes more and more difficult to comprehend, let alone foresee, the various impacts that the large-scale deployment and proliferation of novel scientific and technological advances have both on our social systems and the environment. given the tight coupling between human-made and natural systems and their complex, often unanticipated interactions with catastrophic potential, the existing narrow definitions of risk are rendered inadequate. at the same time, the advent of new technologies with multiple adaptive applications opens up an array of possibilities for hostile exploitation thus compelling governments to make tough decisions in an attempt to reconcile the benefits of biotechnology with the potential security concerns arising therefrom. while the advancement of biotechnology promises tremendous public health benefits, it also holds a considerable catastrophic potential, as the case of 'gain-of-function' experiments illustrate. as scientific capabilities and work involving dangerous pathogens proliferate globally, so do risks and the prospects of failures, whether technical or arising from human error. indeed, assessing the rapidly evolving life science landscape some security commentators argue that 'current genetic engineering technology and the practices of the community that sustains it have definitively displaced the potential threat of biological warfare beyond the risks posed by naturally occurring epidemics' . laboratories, however well equipped, do not exist in isolation but are an integral part of a larger ecological system. as such, they constitute a 'buffer zone' between the activities carried out inside and the wider environment. and despite being technically advanced and designed to ensure safety, this 'buffer zone', just as other safety systems is far from infallible. for one thing, mechanical controls leave room for human error and personal judgement, both of which are factors that could be highly consequential but which could hardly be modelled or predicted with exact certainty. the speed at which the transformation of the life sciences is taking place is yet another factor that adds to the complexity of life science governance. stability is a fundamental condition for the development and preservation of human and natural systems alike. in social systems, culture is the primary source of stability, for it determines what values, beliefs, practices and modes of behaviour are deemed acceptable and, as such, lays the foundations of order. all forms of governance therefore are cultural artefacts and manifestations of culture. culture also provides the tacit standards whereby change is assessed and treated as acceptable or unacceptable. hence, any state of affairs in which the rate of change precludes regulation disrupts the ordinary functioning of the system and jeopardises its preservation: the breakdown of human regulation does not extinguish regulation of a simpler sort. [ . . . ] the system formed by men and the rest of the natural world will continue to regulate itself after a fashion, even if human regulation wholly fails at all levels above the primary group. but the resulting 'order' would exclude all those levels of human order which man-made stability makes possible. to be sure, a world characterised by a runaway biotechnology would be far different from the one we know. the main challenge to averting this prospect lies in ensuring that the systems of governance are in sync with the progress of life sciences. history has shown that even highly developed, long-standing systems of governance can fail for reasons as diverse as disasters; loss of authority/legitimacy of governing bodies; and pervasive corruption. one further source of failure includes the inability of a society to adapt to its changing milieu: men are adaptable; they can learn to live even in harsh and hostile environmentsso long as the environment remains constant enough to give them time to learn. [ . . . ] if they form the habit of adapting by constantly changing that to which they are trying to adapt, they build uncertainty into the very structure of their lives. they institutionalise cluelessness. the process of adaptation is closely connected to cultural patterns and any serious disruptions in the latter could have detrimental effects and impair it severely. the extent to which change is taking place within the framework of the prevalent culture defines the borderline between system evolution and system disintegration. the governance mechanisms currently in place, both formal and informal, are all a function of historical, cultural, and sociopolitical contingencies. as such, their capacity for adaptation largely depends on our ability to comprehend and assimilate the complex changes that the progress of biotechnology brings about. they can only evolve as fast as our shared standards, values, routines and perceptions allow them to. and that is why governance can hardly be reduced to a technocratic exercise; on the contrary, to be effective, it requires extensive deliberation and full appreciation of the far-reaching implications of novel life science advances. trends and drivers of change in the biomedical healthcare sector in europe: mapping report (dublin: european foundation for the improvement of living and working conditions chemical synthesis of poliovirus cdna: generation of infectious virus in the absence of natural template the test-tube synthesis of a chemical called poliovirus: the simple synthesis of a virus has far-reaching societal implications craig venter creates synthetic life form', the guardian moore's law pertains to the rapid rate of technological development and advances in the semiconductor industry, specifically the doubling of the number of transistors on integrated circuits that occurs approximately every months. although advances in the life sciences occur at more random intervals and are driven by new conceptual breakthroughs in understanding of biological processes, it is a useful metaphor for the exponential growth of knowledge related to biology. see committee on the advances in technology and the prevention of their application to next generation bioterrorism and biological warfare threats, an international perspective on advancing technologies and strategies for managing dual-use risks the myth of the biotech revolution: an assessment of technological, clinical and organisational change globalization, biosecurity and the future of the life sciences innovation in global industries: u. s. firms competing in a new world biotechnology and the un's millennium development goals top ten biotechnologies for improving health in developing countries rising to the challenge: u.s. innovation policy for global economy innovating in the new austerity, burrill & co's th annual report on the life sciences industry globalization and the future of the life sciences, op cit an international perspective on advancing strategies for managing dual-use risks the valley of the shadow of death', dspace@mit globalization and the future of the life sciences the biotechnology promise: capacity-building for participation of developing countries in the bioeconomy converging technologies for enhancing human performance: science and business perspectives converging technologies -shaping the future of european societies taking care of the symbolic order: how converging technologies challenge our concepts enhancement technologies and the modern self the ethics of nbic convergence bringing converging technologies closer to civil society: the role of precautionary principle', innovation: the innovation in global industries, op cit in the valley of the shadow of death see the 'infectious diseases' section of the who website for an overview of the nih budget for , see david malakoff and jeffrey mervis, 'first look: us spending deal a mixed bag for science within nih's flat budget, a few favourites', scienceinsider for information on the eu sixth framework programme and the activity area of life sciences cuba -battling cancer with biotechnology global status of commercialised biotech/gm crops plant genetic engineering: china hesitates on the brink', gmo safety rising to the challenge, op cit government academic, and venture firms come together in march to fund translational and early-stage development', fiercebiotech science for sale: the perils, rewards and delusions of campus capitalism relationships between academic institutions and industry in the life sciences -an industry survey the expanding role of university patenting in the life sciences: assessing the importance of experience and connectivity university-industry relationships and the design of biotechnology research entrepreneurial science in the academy: a case of the transformation of norms the triple helix of university-industry-government relations the triple helix: university-industry-government innovation in action the dynamics of innovation: from national systems and "mode " to a triple helix of university-industry-government relations commercialisation of the university and the problem choice by academic biological scientists see also dina biscotti et al. 'the "independent investigator": how academic researchers construct their professional identity in university-industry agricultural biotechnology research collaborations toward more secrecy in science: comments on some structural changes in science -and on their implications for an ethics of science varieties of secrets and secret varieties: the case of biotechnology for a detailed analysis on the us decision to reject the draft btwc protocol, see malcolm dando the white house the pitfalls of bioterrorism preparedness: the anthrax and smallpox experiences bioterrorism and smallpox planning: information and voluntary vaccination taking biodefence too far new labs, more terror a plague of researchers mixing bugs and bombs lab loses trio of plague mice', nature news anthrax letters' attack and the controversy of the us biodefence programme see jeanne guillemin army suspends germ research at maryland lab back to bioweapons? international health regulations (who, ), specifically section 'laboratory pandemic influenza preparedness framework for the sharing of influenza viruses and access to vaccines and other benefits (who for further discussion on the implementation of biotechnology regulations, see bo sundqvist et al. 'harmonisation of european laboratory response networks by implementing cwa : use of gap analysis and an "insider" exercise as tools on the development of health and safety regulations on the use of nanotechnology, see eileen kuempel et al. 'risk assessment and risk management of nanoparticles in the workplace: translating research into practice there is a debate on whether the 'american model' of science-policy making underpinned by neoliberal ideology is fully embraced in europe. see, for example, gabriele abels, 'the long and winding road from asilomar to brussels: science, policy and the public in biotechnology regulation further, a policy paper issued by a business taskforce appointed by the uk government issued a policy paper in late demanding the liberalization of the existing eu legislation which, in their view, 'places restrictions on products and technologies without adequate evidence of risk'. see department for business, innovation & skills and the prime minister's office, cut the eu red tape: report form the business task force proposed framework for the oversight of dual use life science research: strategies for minimising the potential misuse of research information biosecurity reconsidered: calibrating biological threats and responses some commentators have expressed scepticism toward the claim that scientific and technological advancement poses serious threats underscoring the importance of other factors, such as socio-economic and socio-technic contexts. see, for example, kathleen vogel, 'intelligent assessment: putting emerging biotechnology threats in context others, however, argue that advances in modern biology and medicine have implications for the evolution of biological weapon programmes. see malcolm dando, 'the impact of the development of modern biology and medicine on the evolution of offensive biological warfare programmes in the twentieth century expression of mouse interleukin- by a recombinant ectromelia virus suppresses cytolytic lymphocyte responses and overcomes genetic resistance to mousepox the efficacy of cidofovir treatment of mice infected with ectromelia (mousepox) virus encoding interleukin- disaster in the making creation of killer poxvirus could have been predicted chemical synthesis of poliovirus cdna: generation of infectious virus in the absence of natural template a tale of two studies: ethics, bioterrorism and the censorship of science first synthetic virus created', bbc news university of bradford, available at www.brad.ac.uk/ acad/sbtwc (accessed / / ). see also german ethics council, biosecurity -freedom and responsbility of research on the framing of risk in biotechnology, see geert van calster, 'risk regulation, eu law and emerging technologies: smother or smooth? ethics of risk analysis and regulatory review: from bio-to nanotechnology european biotechnology regulation: framing the risk assessment of a herbicide tolerant crop framing the uncertainty of risk: models of governance for genetically modified crops denmark's regulation of agri-biotechnology: co-existence bypassing risk issues a distorted regulatory landscape: genetically modified wheat and the influence of non-safety issues in canada on the shortcomings of the existing models for public deliberation on the risks of biotechnology, see les levidow, 'european public participation as risk governance: enhancing democratic accountability for agrobiotech policy for critique of cost-benefit analysis, see also brian rappert the creation of contagious h n avian influenza virus: implications for the education of life scientists all meetings of the us national science advisory board for biosecurity (nsabb) convened to discuss the manuscripts were restricted to selected individuals and full proceedings were never published. moreover, the consequential consultation meeting organised by the world health organisation (who) in february which rejected the nsabb recommendation for a redacted publication of the manuscripts featured the lead scientists who conducted the experiments and representatives of the us national institutes of health flu experts -and one ethicist -debate controversial h n papers', scienceinsider fight over dutch h n paper enters endgame', scienceinsider fight over dutch h n paper enters endgame', scienceinsider dutch appeals court dodges decision on hotly debated h n papers', scienceinsider bias accusation rattles us biosecurity board experimental adaptation of an influenza h ha confers respiratory droplet transmission to a reassortant h ha/h n virus in ferrets airborne transmission of influenza a/h n virus between ferrets: materials/methods, supporting text, tables, figures, and/or refences safety incidents" at animal lab', bbc news notes airflow problems plague cdc bioterror lab high-containment laboratories: assessment of the nation's need is missing the scientist lab infection blamed for singapore sars case between and , several high-containment facilities in the us have experienced serious biosafety lapses. accidents involving dangerous pathogens such as the causative agents of anthrax and bird flu were reported biosafety in the balance' anthrax? that's not the real worry cdc closes anthrax and flu labs after accidents about the same time, there were also reports about smallpox vials being retrived after having been left unaccounted for over years. see ap, 'forgotten vials of smallpox found in storage room gain-of-function' influenza research and proposal to organise a scientific briefing for the european commission and conduct comprehensive risk-benefit assessment the third revolution: the convergence of the life sciences see committee on biomocular materials and processes, national research council, inspired by biology: from molecules to materials to machines a new biology for the st century nanotechnology: what it can do for drug delivery facing the truth about nanotechnology in drug delivery carbon nanotubes -bullets in the fight against cancer on the governance challenges brought about by the convergence between biology and other fields of science, see francis fukuyama and caroline wagner, information and biological revolutions: global governance challenges -summary of a study group report of the first workshop the body's own bioweapons innovation, dual use, and security: managing the risks of emerging biological and chemical technologies life sciences and related fields: trends relevant to the biological weapons convention building better implants some nanotubes could cause cancer and breeding: panacea or pandora's box?', news release on the security implications of synthetic biology, see international council for the life sciences synthetic biology and biosecurity awareness in europe on the social and ethical aspects of synthetic biology, see presidential commission for the study of bioethical issues, new directions: the ethics of synthetic biology and emerging technologies synthetic biology: social and ethical challenges framing biosecurity: an alternative to the biotech revolution model vogel's view are contested in jonathan tucker 'could terrorists exploit synthetic biology lax laws, virus dna and potential for terror on the issue of commercial order screenings, see stephen maurer et al. making commercial biology safer: what the gene synthesis industry has learned about screening customers and orders on the governance of synthetic biology, see catherine lyall dna synthesis and biological security industry self-governance: a new way to manage dangerous technologies unpacking synthetic biology: identification of oversight policy problems and options synthetic biology, security and governance'¸biosocieties dna synthesis and security science enters $ , genome era', bbc news happened when i had my genome sequenced', the observer for information about commercial companies offering full-genome sequencing genome-based therapeutics: targeted drug discovery and development: workshop summary dna of , people to be mapped for nhs', the guardian systems cancer medicine: towards realisation of predictive, preventive, personalised and participatory (p ) medicine' grateful and moved" by reaction to her mastectomy decision', the guardian dna blueprint for fetus built using tests of parents cure for illness raises ethical fear', the guardian what happened when i had my genome sequenced another point that cadwalladr raises is the danger of a negative placebo effect whereby doubts about certain genetic disorder may lead to psychosomatic symptoms from eugenics to patents: genetics, law, and human rights unhidden traits: genomic data privacy debates heat up from eugenics to patents see also harriet washington, deadly monopolies: the shocking corporate takeover of life itself -and the consequences for your health and our medical future taking life: private rights in public nature benefits and threats of developments in biotechnology and genetic engineering' in sipri yearbook, armaments ethnic-affiliation estimation by use of population-specific dna markers racial differences in the response to drugs -pointers to genetic differences the achilles' helix from eugenics to patents three-person" embryos to combat genetic disease', the guardian innovative genetic treatment to prevent mitochondrial disease, press release three-parent embryos for mitochondrial disease? twelve reasons for caution february , the uk passed legislation allowing the use of the technique. see james gallagher, 'uk approves three-person babies', bbc news building baby from the genes up one commentator distinguishes between 'big science' which was 'todown, hierarchical, vertical' and 'networked science' characterised by 'open systems, open software, open participation'. see diane rhoten biopunk: solving biotech's biggest problems in kitchens and garages life hackers biology is technology: the promise, peril, and new business of engineering life diffusion of synthetic biology: a challenge to biosafety do i understand what i can create: biosafety issues in synthetic biology charge dropped against artist in a terror case strategies to educate amateur biologists and scientists in non-life science disciplines about dual use research in the life sciences governing amateur biology: extending responsible research and innovation in synthetic biology to new actors, research report for the wellcome trust project 'building sustainable capacity in dual use bioethics secret project manufactured mock anthrax', the washington times biological weapons and america's secret war global governance and the twenty-first century technology converging technologies for improving human performance: nanotechnology, biotechnology, information technology and cognitive science report of the who informal consultation on dual use research of concern biological threat assessment: is the cure worse than the disease? dangerous ambiguities: regulation of riot control agents and incapacitants under the chemical weapons convention weaponisation of central nervous system acting chemicals for law enforcement purposes, xix session of the conference of the states parties moscow theatre siege: questions remain unanswered', bbc news press release: use of gas against terrorists during the moscow theatre siege was justified, but the rescue operation afterwards was poorly planned and implemented spasenie zalozhnikov ili unichtozhenie terroristov?', novaya gazeta the dubrovka and beslan hostage crises: a critique of russian counter-terrorism sekretov bol'she net', rossiskaya gazeta see also national research council, avoiding surprise in an era of global technology advances the challenge to deliberative systems of technological systems convergence converging technologies for improving human performance, op cit preventing a biochemical arms race, op cit bringing the bwc conventions closer together', the cbw conventions bulletin h n hybrid viruses bearing /h n virus genes transmit in guinea pigs by respiratory droplet infectivity, transmission, and pathology of human-isolated h n influenza virus in ferrets and pigs limited airborne transmission of h n influenza a virus between ferrets shifting the blame: literature, law, and the theory of accidents in nineteenth-century america normal accidents, op cit building a bio world shifting the blame, op cit freedom in a rocking boat: changing values in an unstable society some commentators have critiqued the work of diamond on the grounds of simplicity. for a summary of some of the criticisms levelled at his work, see eric powell key: cord- - uu dh authors: ford, lea berrang title: climate change and health in canada date: - - journal: mcgill j med doi: nan sha: doc_id: cord_uid: uu dh nan national governments from around the world met in poznan, poland in december at the th conference of the parties to the united nations framework convention on climate change ( ). this conference came at a time of increasing political and scientific confidence in the role of human-induced greenhouse gas emissions in changing global temperatures. the most recent ( ) report of the intergovernmental panel on climate change ( ) states that climate change is now "unequivocal", based on increasing evidence from global average air and ocean temperatures, melting of snow and ice, and rising global average sea level" ( ) . while there remains uncertainty regarding the specific nature and rate of climatic changes and their impacts, there is negligible scientific doubt that the global climate is changing and that these changes will have significant and potentially profound impacts on a wide range of ecological and human systems across the planet ( ) ( ) ( ) ( ) . that climate change predictions are both scientifically and politically daunting lessens neither their verity nor implied imperative ( , ) . climate change will involve an average increase in global temperatures of approximately . - . °c by the end of the century ( ); this range reflects both uncertainty in climate modeling, as well as a range of possible scenarios for how we will respond to climate changes, including mitigation, technology development, economic development and population growth. this temperature shift will be manifest in average global climatic changes, including higher maximum temperature, more very hot days, increased occurrence and severity of heat waves, fewer cold and frost days, fewer cold spells, more intense precipitation events, increased risk of drought in continental areas, increased cyclone intensities, and intensified enso events. these effects will, however, vary significantly by region and act within existing climate conditions. for example, while parts of latin america will see minimal changes in temperature, arctic regions are expected to experience an average temperature shift of - °c by the end of the century ( ) and recent research suggests that even these projections may be conservative ( , ) . changes in our global and regional climate systems will have important implications for health and health systems ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . the world health organization estimated that in the year , climate change caused approximately , excess deaths worldwide, as well as million disability-adjusted life years ( ) . temperature and weather have direct effects on mortality and morbidity through the occurrence of extreme weather events, including heatwaves, cold periods, storms, floods, and droughts ( , ( ) ( ) ( ) . such events can, in turn, affect the incidence of food-borne and water-borne disease ( , ) . the habitat and survival of insect species capable of transmitting many vector-borne diseases are affected by temperature and water regimes ( , ) . many pathogen replication cycles are also determined by temperature conditions. indirect impacts of climate change on livelihoods, such as increased economic vulnerability, reduced availability of food resources, and reduced allocation of government funding for health systems, may also have important, though unquantified, impacts on global health systems ( , , , , , ) . health impacts due to climate change have already been documented, including changes in the range of some vector-borne diseases ( , , ( ) ( ) ( ) and an increase in heatwave-related deaths ( , , ) . indirect effects will also include increases in regional food insecurity, migration resulting from environmental degradation, and loss of environmental-dependent livelihoods resulting from ecological shifts in weather or species distributions ( ) . table summarizes the reported and projected health impacts of climate change documented by the intergovernmental panel on climate change (ipcc). the ipcc, an intergovernmental body open to all united nations member countries, comprehensively assesses existing literature related to climate change science, potential impacts, and options for adaptation and mitigation. the published reports represent the consensus of thousands of scientists around the world contributing as authors and reviewers, as well as political consensus required by member countries for the acceptance, adoption and approval of the final document. these projections and measures of confidence are therefore believed to be conservative ( ) ( ) ( ) . current climate change effects on global health are small but increasing in most countries ( ) . this is due to the lag effect between greenhouse-gas emissions, climate system warming, and the weight of evidence documenting health impacts. while the burden of negative health impacts will be disproportionately high in poorer countries, even high-income countries will be vulnerable to morbidity and mortality related to increases in the number and severity of extreme weather events such as storms, heatwaves, and floods ( ) . vulnerable populations in all countries include the urban poor, the elderly and children, traditional communities, subsistence farming communities, and coastal populations ( , , ( ) ( ) ( ) . evidence does not support the potential for economic development to combat the health impacts of climate change ( ) . canada will not be immune to the health impacts of climatic change ( ) ( ) ( ) ( ) . canada has observed approximately °c rise in temperature since the beginning of the century, and we can expect this to continue by about . degree per decade, up to °c over the next century ( ) . this temperature change is not insignificant; the earth's temperature was, for example, only - degrees cooler during the last ice age ( ) . the effects of climate change in canada will differ between regions ( ) . while the prairies are expected to experience warmer, drier summers and more sever summer droughts, ontario and quebec can expect decreased snow, increased rainfall, and an increase in the incidence of severe summer storms ( ) . the greatest changes will occur in the canadian north, where temperature changes are projected to be among the highest in the world, and where traditional, resource-dependent communities are considered highly vulnerable ( , , , ) . indeed, for northern inuit communities, climate change poses a significant threat to traditional livelihoods in the short to medium term ( , ( ) ( ) ( ) . canada will experience a number of significant direct and indirect impacts of climate change ( ) ( ) ( ) ( ) . extreme heat events are expected to become more frequent, longer in duration, and more intense ( ) . extreme heat events can exacerbate health conditions, such as asthma, as well as lead to an increased number of deaths ( , , , ) . heat waves were responsible for over five hundred deaths in chicago during a -day level of uncertainty * alter the distribution of some infectious disease vectors medium confidence alter the seasonal distribution of some allergenic pollen species high confidence increase heatwave-related deaths medium confidence increase in malnutrition and consequent disorders, including child growth and development high confidence increase in morbidity and mortality related to heatwaves, floods, storms, fires, and droughts high confidence continued changes in the ranges of some infectious disease vectors high confidence mixed effects on malaria incidence and distribution (expansion in some areas, contraction in others) very high confidence increase in the burden of diarrhoeal diseases medium confidence increase in cardio-respiratory morbidity and morality associated with ground-level ozone high confidence increase in the number of people at risk of dengue low confidence health benefits include fewer deaths due to cold high confidence ( ) . children and the elderly are particularly vulnerable during heat wave events ( ) . the increasing number of summer days in urban areas of quebec and ontario declared unsafe for outdoor activity due to smog and heat can be expected to negatively impact public health through reduced outdoor and exercise activities ( ) . poor air quality, resulting from smog and air pollution, is associated with asthma, chronic respiratory disease and cardiovascular disease, and is a serious public health issue in canada. smog and air pollution are expected to continue to increase with climatic change ( , ) . toronto public health recently predicted that climate change would cause a % increase in air-pollution related deaths in the city by ( ). many regions are expected to see an increase in summer storms. this may affect risks associated with flooding, and will have implications for water quality and contamination ( , ( ) ( ) ( ) . a prolonged drought followed by a high rainfall event -such as a summer storm -can pick up surface contaminants and flush them into local waterways, causing a 'pulse' in the contaminant load of local water treatment facilities ( , ) . this scenario was determined to be one of the factors contributing to the e. coli outbreak in walkerton, ontario in ( ) . in addition, higher than normal rainfall events may exceed expected norms for sewage treatment facilities, overwhelming treatment systems. in the inuit community of arctic bay, increased rainfall has been observed to overflow the local sewage ponds, contaminating the bay and roads ( ) . these scenarios are consistent with climate change predictions and can be expected to occur more frequently. the distribution of vector-borne diseases will change ( , , ( ) ( ) ( ) . warmer and wetter summers will affect the distribution and survival of pathogens and some disease vectors such as mosquitoes and ticks ( , ( ) ( ) ( ) . research has already documented possible shifts in the distribution of the vector of lyme disease ( , ( ) ( ) ( ) , and possible expansion of the potential range of west nile virus (wnv) ( , , ) . mosquito vectors of wnv will be affected by longer summers ( , ) . increased incidence of the virus coincides with periods of prolonged hot weather and increased mosquito activity ( ) . recent research also indicates the potential for the re-emergence or emergence of exotic pathogens to canada, including locally-transmitted malaria ( ) . emergent disease risks are by nature difficult to predict. despite this, it is sensible to anticipate the spread of known diseases into new areas and the emergence of new diseases. there will also be a number of indirect effects on canadian health and health care. in many cases, these indirect pathways are difficult to identify, predict, and quantify, but may nonetheless be important for changing health systems in canada. for example, changing sea temperatures are likely to impact the distribution and availability of fish and tree species through impacts on local weather, affecting the viability or focus of fisheries and forestry industries, and by extension the community health and well-being of resource-dependent communities. in this case, the concern is not the loss of traditional species per senew species are likely to emerge to fill changing niche conditions -but the rate of change in ecological systems, and the ability of industries and communities to adapt to these changes. similarly, the dramatic spread of the pine beetle has been facilitated -and many suggest triggered -by increasingly favourable weather conditions ( ) ( ) ( ) ( ) . as in the case of the pine beetle, many of the impacts of climate change on health in canada are likely to be the result of indirect causal processes, and in some cases unforeseen events. the implications of climate change for health are not limited to global and national impacts. they will also be manifest at the municipal level (table ) and within canada's health sector ( , , ) . for canada's health system, this will result in changing risks. for example, the increased emergence of new, re-emergent and exotic diseases will mean that conventional expectations of likely diagnoses by family physicians and primary health care providers will be insufficient. the potential for malaria infection in patients with no history of travel is one such example ( ) . the increase in extreme weather events such as heatwaves, floods and storms will increase pressure on disaster preparedness and emergency health services and programming ( ) . program planning for emergency health provision will need to consider future rather than historical experiences or trends in demand and frequency of health crises ( , ( ) ( ) ( ) . increased health surveillance will be required to document baseline health measures and monitor changing health outcomes. this is particularly important in canada's northern communities where health provision and surveillance have faced significant challenges and where climate impacts will develop earlier and more rapidly than in the south ( , ) . the good news is that opportunities for avoidance of, and adaptation to, climate impacts on health are available, feasible, and in many cases of benefit to improved health in canada more broadly ( ) . given the unpredictable nature of many impacts, adaptation, prevention, and preparedness measures that increase overall health system capacity are most sensible and cost-effective -so called 'win win' or 'no regrets' responses ( , ( ) ( ) ( ) ( ) ( ) . these include: increased surveillance, particularly of disease vectors, water quality, and air pollutants; integration of climate projections into emergency planning and disaster preparedness ( ) ; improved access to preventive care and primary physician care to promote early detection of new disease emergence or shifting disease incidence; integration of climate change considerations into education programming for medical students and primary health care workers; integration of climate projection parameters into urban planning to increase protection against extreme weather events ( , ( ) ( ) ( ) ; increased monitoring and evaluation of food production systems and water monitoring safety given climate projections ( , , , ) ; development of heat wave alerts and responses, and mitigation of urban heat islands ( , , , ) , and; increased multi-national support for improved health capacity in low and middle income countries. the risk of health impacts resulting from climate change are not restricted to within our national borders. the impact of climate change elsewhere, particularly in low and middle income nations, will influence the potential for imported infection to canada. for example, the introduction and outbreak of sars in toronto in resulted in cases, deaths and significant economic losses ( , ) . international travel may have been responsible for promoting the introduction of west nile virus in north america ( ) ( ) ( ) . similarly, a canadian outbreak of imported malaria in - may have been brought to canada via travelers arriving from the punjab in india, where a large epidemic had occurred ( , ( ) ( ) ( ) . more recently, concern regarding the potential spread or proliferation of avian influenza has highlighted the interconnected nature of national health to health conditions around the world. as such, the health impacts of climate change in canada will be influenced by the health and response capacity of other nations from or to which canadians, visitors or trade products travel. in this context, adaptation, preparedness and response resources may in some cases be most effectively and efficiently allocated through supporting health capacity in other countries. increased interest and prioritization of health collaborations in asia following the sars outbreak provides a germane example of a developing awareness of such risk priorities. despite the magnitude and scope of climate change, the recent global financial crisis has overshadowed concern for, and prioritization of, climate change science, policy, and action. while the implications of economic crisis at the international and national levels are undoubtedly of legitimate priority and concern, placing climate issues on the back-burner is misguided for two reasons. first, while the financial crisis may be acute and possibly prolonged in the short term -years, but not decades -the climate change crisis will last well into the next century and beyond. investments in health system capacity and surveillance need to be implemented in advance of emerging impacts to avoid and/or mitigate morbidity and mortality. additionally, observed climate impacts will begin to rapidly accelerate over the next decade. in the absence of genuine and dramatic intervention, climate change impacts have the potential to be severe and acute on a scale greatly exceeding the current financial crisis ( , , , ) . the lack of action on climate change -including both mitigation of emissions and adaptation to current and future impacts -is generally rationalized based on the costs of interventions. the costs of a no-action approach, however, will be significant. the stern review, an independent assessment commissioned in the united kingdom, estimated that a - °c warming over the next century could result in losses of up to % of global gdp ( ); the report estimates the cost of mitigating climate emissions and severe impacts at approximately % of global gdp. the health sector, which makes up % of canada's gross national product (gnp) can make a 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province of pakistan stern review on the economics of climate change joint cna/cma position statement on environmentally responsible activity in the health sector.: canadian nurses association (cna) and the canadian medical association (cma) world bank. health, nutrition and population (hnp) stats key: cord- - mfiie authors: aykut, stefan c.; morena, edouard; foyer, jean title: ‘incantatory’ governance: global climate politics’ performative turn and its wider significance for global politics date: - - journal: int polit doi: . /s - - - sha: doc_id: cord_uid: mfiie the paris agreement represents a deep-rooted change in global climate governance. while existing scholarly assessments highlight central institutional features of the paris shift, they tend to overlook its symbolic and discursive dimensions. our analysis shows that the paris architecture combines two core elements: an iterative pledge and review process to stimulate global climate action, and a ‘performative’ narrative aimed at aligning actors’ expectations on the prospect of a low-carbon future. we therefore suggest calling it an incantatory system of governance. we then examine the origins of the new approach and find that the rise of ‘soft law’ approaches and communicative techniques in global climate governance are both indicative of a broader process: the entry of management culture in international organisations. against this backdrop, we examine the prospects, limitations and caveats of the new approach and discuss its wider implications for global politics. the paris agreement adopted in december is widely considered as a major breakthrough in global climate governance, with the potential of becoming a blueprint for other governance arenas (jordan et al. ) . and yet, just years after its adoption, it was already in jeopardy when us president trump announced on june his intention to withdraw from the treaty. the decision completely paralysed negotiations at the un climate summit cop in bonn in november of that same year. interestingly, however, the atmosphere was very different at the 'bonn zone', an area dedicated to non-state and sub-state climate efforts and just a few hundred metres away from the official conference space. a highlight of the 'bonn zone' was the launch of the #wearestillin coalition. under the leadership of billionaire philanthropist, former new york city mayor and un special envoy for climate action michael bloomberg, as well as california governor jerry brown, the coalition brought together american cities, states and businesses committed to fulfilling the us's national emission reduction commitments through bottom-up action. the mood was similarly upbeat at the one planet summit in paris a month later. convened by french president emmanuel macron to mark the cop 's second anniversary, the summit provided business and ngo leaders, representatives from international organisations and national and multilateral development banks, heads of state and government, philanthropists and mayors with an opportunity to both reassert their commitment to the paris agreement and to announce new measures for its implementation. the 'bonn zone' and one planet summit are revealing of the current state of global climate governance. they are symptomatic of more deep-rooted shifts in its organisation, in the levels of engagement, in the actors involved, and the mechanisms through which it operates and produces effects. global climate policy is now understood as a process that transcends the united nations framework convention on climate change (unfccc), and of which transnational initiatives and private governance schemes constitute an integral part (moncel and van asselt ) . furthermore, it is no longer aimed at the production and enforcement of binding reduction targets for states, but builds on a flexible 'pledge and review' system combining voluntary pledges by public and private actors alike, and binding reporting and transparency rules for states (keohane and oppenheimer ) . taken together, these changes have been described as a shift away from a 'regulatory' and towards a 'catalytic and facilitative model' of global governance (hale ) . while such assessments highlight central aspects of the paris shift, they also contain significant blind spots. the bulk of stand-alone articles and special issues on post-paris climate governance focusses on negotiation dynamics and outcomes, the interpretation of the agreement's legal dispositions, or institutional innovations in the post-paris process (jordan et al. ) . in doing so, such analyses tend to overlook an important feature of the new governance regime: its symbolic and discursive dimensions. as illustrated by the examples above, the post-paris process conveys a central role to the emission of 'signals' and the creation of 'momentum' for climate action, through carefully orchestrated global moments such as the one planet summit and climate action summits and highly publicised private initiatives like #werestillin. in other words, in this new governance, performances, symbols and narratives appear to be just as important as the production of rules, institutions and instruments. we therefore suggest calling the new approach an incantatory system of governance. on a general level, the notion of 'incantation' points to the ritualised and repetitive dimensions of global climate governance, with its annual meetings and recurring calls to urgency and action (little ) , as well as to the theatrical dramaturgy of climate summits and their filiation to the 'society of spectacle' (death ) . more specifically, it permits to capture what we believe constitutes a distinctive feature of the new approach: the fact that communicative and symbolic devices are explicitly recognised, by its architects and promoters, as core instruments in the agreement's implementation. a central element in this context is the grand narrative of an ongoing 'planetary transition' to a decarbonised world economy, which is crafted and circulated by key governance actors. by using the notion of incantation, we also wish to engage a discussion on the origins and wider significance of this governance shift. in an increasingly fragmented (biermann et al. ), marketised (newell and privatised (park et al. ) global governance landscape, 'soft law' approaches resting on voluntary commitments (abbott and snidal ) , indicators and best practices (merry ) have been on the rise over the last decades. the paris shift fits within this broader set of transformations, inspired by the adoption of new public management (npm) methods in international organisations. we suggest that these two dynamics-the weakening of legal and regulatory frameworks, and the inflationary use of communicative devices-can be understood as two sides of the same phenomenon: the importation of a business culture in global governance. finally, the notion of incantation points to the need to renew the methods with which we study global climate governance. our aim is not to present the new approach as ineffective per se, but to understand how it plays out in practice, and better appreciate its prospects, risks and caveats. this requires examining the role of rituals, symbols and discourses in global governance, analyse how they produce effects and study how they relate to, or combine with, more traditional governance methods-such as the negotiation of legal documents and the action of international organisations. in line with collaborative event ethnography (campbell et al. ), our analysis is therefore based on repeated collective observations of different spaces of global climate governance, particularly during the paris cop. there, we studied the circulation of people and documents, practices of text production and editing, the role of diplomatic rituals and political performances, as well as civil society mobilisations, scientific events and business happenings . we also analysed how philanthropic foundations and think tanks shaped the 'road to paris' and the discursive context surrounding cop (morena ) . in this paper, we connect the findings of these different lines of research. drawing on discourse analysis (bäckstrand and lövbrand ) , we also reconstruct the narratives circulated by promoters of the new governance approach. that being said, the article's primary goal is to advance a broader conceptual argument. the empirical material serves to shed light on our argument rather than provide a comprehensive, rigorous analysis of one conference or one discourse. the paris approach introduces a series of institutional innovations. it marks a transition from a 'regulatory' approach to global climate governance, with detailed rules and obligations that apply to developed states, to a 'hybrid' system that both combines voluntary submissions and binding review cycles for all states and associates a wider range of stakeholders. however, in the eyes of its architects and main proponents, the new approach does not only rest on new institutions, it also centrally relies on new discursive and symbolic elements. instead of legally binding reduction targets and sanctions for non-compliance, the governance framework laid out in the paris agreement is based on the submission and review of freely determined policy pledges, or nationally determined contributions (ndcs). however, the approach also differs significantly from purely voluntary systems. on the substantive side, it sets two long-term temperature goals: keeping global warming 'well below' °c and 'pursuing efforts' to stay below . °c. the cop decision also sets out the figure of billion usd per year towards developing countries' adaptation and emissions reduction efforts. apart from the . °c target, these figures had already been laid out in the contested copenhagen accord in . accordingly, the paris agreement's main innovations are procedural, rather than substantive (oberthür and bodle ). an 'enhanced transparency framework' is set up to ensure the publicity and comparability of ndcs; a 'global stocktake' is scheduled every years to collectively evaluate the adequacy of national efforts; based on this assessment, countries are expected to 'ratchet up' their pledges in line with the agreement's long-term goals. in sum, the paris framework establishes legally binding obligations of conduct, but no obligations of result (bodansky ) . its implementation has been described as a 'two level game' in which the capacity of domestic civil societies to exert pressure on their governments plays a decisive role (keohane and oppenheimer ) . the paris architecture is therefore understood by its proponents as an iterative process, in which 'the many interdependent parts […] interact in mutually facilitative ways' (hale and roger : ) . the agreement also broadens the scope of stakeholders that participate in global climate governance. in addition to developed countries, developing countries as well as private and subnational actors are encouraged to submit emission reduction pledges. in this respect, paris not only marks a historic break with the north-south divide in global climate politics; it also confirms the rise of 'private authority' and corporate self-regulation in global governance (pattberg ; andonova ) . from centrepiece of a unified and centralised climate regime, the unfccc is now considered as only one of many elements that collectively make up a broad landscape of 'transnational climate governance' (betsill et al. ; bulkeley et al. ). in the lead-up to the paris conference, climate governance scholars reassessed the unfccc's role. they encouraged it to take on an 'orchestrating' function for climate action by states, as well as businesses, cities, regions and federated states (abbott and snidal ; moncel and van asselt ; hale and roger ) . orchestration is thereby defined as an 'indirect mode of governance that relies on soft inducements', as the orchestrator 'works through like-minded intermediaries, catalysing their formation, encouraging and assisting them and steering their activities through support and other incentives' (abbott : ). an oft-cited example is the unfccc's non-state actor zone for climate action (nazca), an online platform launched in 'where actors from around the globe-countries, regions, cities, companies, investors and other organisations-can display their commitments to act on climate change.' to further encourage transnational climate action and link it to the un process, the unfccc also promoted 'high-level champions' for climate action. the 'champions'-usually personalities from the business, political and cultural spheres-put their professional networks and celebrity in the service of climate action. in return, the un arena provides social prestige and symbolic recognition to these individuals. initiatives such as nazca portal or the high-level champions are envisioned as more than mere appendages to national efforts. they are a constitutive 'fourth pillar' of global climate governance alongside mitigation, adaptation and climate finance, intended to 'galvanize' and 'catalyse' global climate action (hale ) . the underlying image is that of a virtuous cycle, in which experiences of past cooperation create trust and confidence among actors and alter their future preferences (bang et al. ) . the concept of 'catalytic cooperation' (hale ) neatly captures this idea. it rests on the claim that global mitigation efforts have wrongly been portrayed as a classic case of a prisoner's dilemma. instead, it is argued that climate action entails first mover benefits for pioneers and increasing returns as the number of followers increases. this may lead to normative change through 'norm cascades' and 'tipping points' that transform the incentive structure and hence the nature of the problem. hence, 'the entire purpose of a catalytic regime' like the paris agreement 'is to shift actors' preferences over time in favour of cooperation' (hale : ) . given the importance of norms, trust and preferences in this governance setup, however, surprisingly little attention have been paid in the literature to global climate governance's symbolic and discursive dimensions. indeed, the defining feature of contemporary climate governance is that signals, narratives and performative moments are at its core. this is explicitly recognised by key proponents of the new approach. laurence tubiana, special envoy of the french presidency to the cop negotiations and one of the architects of the paris agreement, presents the treaty as a 'selffulfilling prophecy', whereby positive narratives 'by producing a convergence of rational anticipations […] contribute as much to change as the agreement itself.' the main objective of post-paris climate governance is no longer the production of new legal norms, but the alignment of state and non-state actors' expectations on the prospect of a low-carbon future. the 'signals' and 'momentum' generated by the governance process underpin the voluntary architecture of the agreement. while the origins of the bottom-up approach in global climate governance are often traced back to the copenhagen climate conference (cop ), fully capturing how and why it came about, and what constitutes its specificities, requires us to go further back in time. indeed, voluntary approaches have been part of the discussions since the beginning of climate talks in the s. we also need to expand our horizons to other areas of global politics, as the approach adopted in paris echoes a wider 'managerial turn' in global politics. prevailing accounts of the paris shift tend to focus on dynamics within the climate regime. and indeed, the idea of a voluntary framework to coordinate the global mitigation effort historically emerged in the run-up to the rio conference. at that time, the eu favoured a 'targets and timetables' approach based on binding reduction commitments for industrialised countries. the us administration criticised the proposal as overtly 'top-down' and 'rigid', arguing that climate governance should involve a more flexible 'bottom-up' approach (bodansky : ) . as a compromise solution, japan suggested in july a pledge and review system combining voluntary country submissions and an international review process to track implementation. however, the targets and timetables approach ultimately won over in kyoto in (damian ) . the voluntary approach resurfaced in the run-up to the copenhagen conference, where countries negotiated on a successor treaty to the kyoto protocol. two years earlier, the so-called 'bali action plan' had introduced the concept of 'nationally appropriate mitigation actions' as a means of getting developing countries to contribute to the mitigation effort. the idea was to encourage emerging economies to make voluntary pledges that would be subject to measurement, reporting and verification (mrv). this, it was hoped, would trigger an incremental process, whereby pledges would progressively be strengthened and ultimately be converted into binding commitments. in the midst of the copenhagen collapse, however, the voluntary approach was ultimately extended to the global north as well. intended nationally determined contributions (indcs) were introduced as a compromise solution between 'nationally appropriate mitigation actions' and the quantified emissions reduction objectives that applied to developed states under the kyoto protocol. the origins of the voluntary approach can therefore be traced back to the early years of the climate regime. there are, however, important differences between the initial proposals and the paris approach. these relate not only to the specific ways in which the paris agreement combines binding and non-binding elements, but also to the broader global setting in which the new climate governance is embedded. this setting differs significantly from the early s. in the post-cold war context of the rio conference, the widely held view was that global governance unfolds mainly through global institution building and the gradual strengthening of international law (levy et al. ; zangl and zürn ) . advocates of pledge and review in the early climate negotiations could therefore frame such a system as a first, incremental step towards more substantial commitments later on (bodansky : ) . this argument appears less plausible today, as the voluntary turn in climate governance coincides with major transformations in global governance. the global diffusion of 'regulatory capitalism' (lévi-faur ) and the rise of 'private authority' (hall and biersteker ; pattberg ) challenge the long-standing supremacy of states and international organisations in global affairs. in a multi-actor world (kaul et al. ) , global governance no longer unfolds through state-led multilateralism alone, but also through forms of 'transnational regulation', 'hybrid governance arrangements' (graz ; andonova ) and networks of corporate self-regulation (müller and cloiseau ; short ) . these transformations had as a corollary the introduction of new governance methods, which originated in the business sector. in the s, new management techniques such as total quality management aimed to provide firms with 'remote control' over their increasingly transnational production chains, through a circular procedural sequence of goal-setting, reporting and auditing (power ) . these techniques inspired a range of national administration reforms during the 'managerial moment' of the s and s (kroeze and keulen ; pollitt and bouckaert ) , before spreading to the global level. corporate social and environmental responsibility (cser) schemes, which rely on a similar circular process of pledging, reporting and review, contributed to this dissemination (zumbansen ; crane et al. ) . through partnerships in such schemes, members of ngos think tanks and international organisations were progressively 'acculturated' to business methods, practices and vocabulary (conley and williams : , ). the spread of cser is also associated with a process of private 're-regulation' (logsdon and wood ; conley and williams ) , whereby businesses became recognised sources of policy proposals at the international level (müller ) . international organisations followed suit over the next decades and increasingly adopted 'soft' and 'experimental' governance methods (sabel and zeitlin ; eckert and börzel ) . the millennium development goals (mdgs), the un global compact and the eu's open method of coordination-three processes launched at the turn of the millennium-constitute paradigmatic examples for this trend. all three combine the definition of common goals, decentralised implementation methods and collective review and benchmarking mechanisms. in addition to coinciding with a broader 'managerial turn', international organisations' adoption of more flexible governance modes also signals their increasing difficulty to develop and enforce binding rules on states (hale et al. ) . hence, the open method of coordination was launched in response to critiques of the eu's overly centralised power structure (regent ; schout et al. ) , while the mdgs came on the back of more than a decade of structural adjustment programmes that spurred growing resistance among developing countries (mcarthur ; shawki ) . the direct consequence of these evolutions is a shift in the normative horizon of global governance. if international relations scholars could still claim in the s that the 'main purpose' of international regimes was 'to harmonize national legislation or to establish rules that can be applied by and to states' (zartman : ) , this no longer pertains to this new type of governance arrangements. from a system organised around the production of legal documents to be transposed into national law, global governance shifted towards a system grounded on the definition of shared goals, voluntary commitments by state and non-state actors, and global review and monitoring processes. while un climate governance was somewhat of a latecomer in adopting the new governance modes, it had been affected by these broader trends well before the copenhagen and paris conferences. since the turn of the millennium, a new 'transnational climate governance landscape' (bulkeley et al. ) progressively took root through the emergence of corporate social responsibility schemes (bulkeley and newell : ) , transnational city networks (betsill and bulkeley ) and corporate carbon trading systems (bernstein et al. ). in the climate diplomacy space, this evolution was closely scrutinised and promoted by a well-experienced and well-connected group of diplomats, ngo, foundation and business representatives, climate policy and communications experts in close contact with the unfccc secretariat and key parties to the convention (morena ) . bringing together individuals with a history of involvement in the international climate diplomacy space-through initiatives like the global call for climate action (gcca) or project catalyst, or informal networks such as the croissant conspiracy or the lionesses -, the international policies and politics initiative (ippi) provides a telling example of how non-state actors strategically mobilised to orientate the international climate debate. participants in the initiative's mid- 'lake tornow' meeting close to berlin include representatives from foundations (ecf, ciff, vasuda), development ngos (oxfam, care international), environmental ngos (greenpeace, wwf), campaign networks (can international, .org, avaaz, gcca), business networks (the climate group), think tanks (e g, wri, ucs, ecofys, track , iddri, germanwatch, grantham lse) and strategic communications (climate nexus) (morena : for participants in ippi, the failure to reach an agreement in was a direct consequence of stakeholders' disregard for wider political and non-state actor dynamics and their influence. experts from the think tank third generation environmentalism (e g, founded in ), for instance, suggested that the copenhagen collapse had shown that 'climate diplomacy has shifted from a relatively narrow focus on the unfccc process, to a more complex and wider discipline that now engages new constituencies and embraces broader geopolitical discussions' (mabey et al. : ) . as johannes meier, ceo of the european climate foundation (ecf, founded in ) explains, experts and activists had failed to recognise that change happens 'in rather oblique and non-linear ways' and that there is a 'need to pay more attention to politics and even to the polity' (meier ) . in its strategy document, ecf further argues that 'the radical policy change that will be required' entails moving not only policy-makers, but 'society as a whole, from the progressive to the conservative, right to left, engaged and disinterested' (european climate foundation : ). the new priority in the lead-up to paris was therefore to stimulate actions at multiple levels and locations, both within and beyond the unfccc, and involve a wide range of stakeholders, to create the conditions for a new type of global climate agreement. the idea was to deliver an agreement that combined a long-term goal that sends 'a clear signal to policy makers, businesses, investors and the public that the low-carbon climate-resilient economy is inevitable' (morgan et al. : ) , with 'bottom-up' commitments that are regularly updated and subject to robust transparency and accountability provisions. this, it was suggested, would enable climate diplomacy to use the 'groundswell' of 'nonstate action' to 'reinvigorate' global climate governance (chan et al. ) . through these and similar proposals in the run-up to paris, climate policy experts and representatives from think tanks, philanthropic foundations and environmental ngos successfully positioned the pledge and review approach as a credible and pragmatic alternative to the legally binding, top-down system that had prevailed up to copenhagen. critical governance scholars and ethnographers of global institutions have long argued that discourses, narratives and symbols constitute key elements in the making of global orders and pointed to the importance of rituals and performances in global mega-events like un climate summits. and indeed, beyond the -page treaty and accompanying -page cop decision, the paris cop also gave birth to the mobilising narrative of an ongoing 'planetary transition' to a low-carbon economy. the making of this narrative can be traced back to the aftermath of cop in copenhagen, when the production and dissemination of discursive frames became a central concern for climate governance actors. in the process, communication practices became a key strategic tool for the architects of the paris approach. making sense of the contemporary transformations of global climate governance requires an analytical vocabulary that adequately captures its discursive and symbolic dimensions. this points to at least two existing lines of research. first, research on discourses and norms highlights the constitutive power of language, knowledge and ideas in global environmental governance (bernstein ; oels ; pettenger ; hughes and paterson ) . lövbrand ( , ) , for instance, show how three broad discursive formations-'ecological modernization', 'green governmentality' and 'civic environmentalism'-distinctly shaped global climate politics in the post-kyoto and post-copenhagen eras. global climate discourses also extend beyond the realm of un climate diplomacy. they have disciplining effects on the everyday and participate in the creation of subjectivities (paterson and stripple ) . they provide 'discursive hooks' to actors seeking entry into the climate arena (allan ) and enable strategies of 'climate bandwagoning' (jinnah ) . moreover, their circulation contributes to a 'climatisation of global debates', whereby issues formerly unrelated to climate policy are increasingly scrutinised through a 'climatic lens' oels ) . un summits, which attract new actors and issues into the climate arena, play an important role in this progressive extension of the thematic scope and symbolic reach of climate governance. this resonates with a second line of research which focuses on the symbolic and performative dimensions of global environmental summits (blühdorn ; campbell et al. ) . ethnographer paul little ( ) provides a fascinating account of the role of performances and rituals at the rio conference. analysing the endless litany of speeches by heads of state and government during the opening ceremony, he shows how these conveyed to the respective home audiences the idea that 'world leaders' were best suited to address global problems. death ( ) makes a similar argument in a foucauldian study of 'theatrical techniques' at the he argues, has come to constitute a 'distinct technology of government'. despite being unsuccessful in terms of negotiations, the two summits constituted attempts 'to inspire and conduct the self-optimisation of the watching global audience'. for these authors, global mega-conferences cannot be reduced to formal negotiation outcomes; they are also important loci for the production of meaning, through the emission of signals, frames and narratives. such perspectives permit to shed new light on the discursive context of the pre-paris process. indeed, copenhagen also marks the start of a new 'positive' narrative around climate change, which would come to form a core feature of the new climate governance. for the group of stakeholders mentioned above, copenhagen had not only been a diplomatic fiasco, but also a failure in terms of communication. it had effectively failed to shape the overall narrative on climate change in a positive way (morena : , ) . too little attention had been paid to the symbolic and discursive dimensions of climate diplomacy. to succeed, the paris conference therefore had to send 'unambiguous signals that the world will shift its economic and social activity toward more climate-friendly and sustainable pathways' (oberthür et al. : ) . to do this, a range of individuals were mobilised and tools were created to ensure that stakeholders in the climate debate sent the right message to the right audience at the right time (morena ) . communications efforts were orchestrated by discreet 'unbranded' initiatives such as the global strategic communications council (gscc) or climate briefing service (cbs) whose communications experts '[coordinated stakeholder] voices at the national and international levels to help shape the national offers as they are being drafted and the thinking around the international agreement'. they focused their communications efforts on global and national climate-relevant 'moments' leading-up to the paris conference; from g and g summits, to the rio + conference ( ) and associated green growth/green economy agenda, to china's adoption of its new -year plan, to the launching of climate-related reports (ipcc reports, new climate economy report, unep emissions gap reports…). these communications efforts mobilised a wide range of stakeholders, from climate 'outsiders' active on the margins of the official negotiation process to climate 'insiders' working closely with parties to draft a new treaty (de moor et al. ; newell ) . christiana figueres, unfccc executive secretary at the time, played a key role in these efforts. she provides a fascinating account of her intense lobbying work for a climate agreement in a recent nature commentary. her primary task, she contends, consisted in spreading optimism: i immediately realized that, before we could consider the political, technical and legal parameters of an eventual agreement, i had to dedicate myself to changing the mood: there could be no victory without optimism. i decided to set a clear intention: even if we did not know precisely how, a global deal would emerge, simply because it was necessary. it was that contagious frame of mind that led to effective decision-making, despite the enormous complexities under which we were operating. when the paris agreement was achieved, the optimism that people felt about the future was palpable -but, in fact, optimism had been the primary input. (figueres ) among the groups that actively promoted a new climate narrative were also progressive business interests like the we mean business coalition launched at the nyc climate week (benabou et al. ) . in its first report the climate has changed, the coalition argues that 'the transition to a low-carbon economy is already happening' (we mean business : xiv) and attempts to demonstrate that 'ambitious climate action makes business sense' (ibid.: viii). the transition is depicted as a dynamic, polycentric process where 'bold business action' and ambitious policy-making are mutually reinforcing (ibid.: vii). a follow-up publication shaping a catalytic paris agreement contains a detailed proposal for a new climate treaty (we mean business ) . according to the authors, such an agreement should combine voluntary and binding elements to '[create] an inclusive enabling environment for all stakeholders-including business' and fix an ambitious temperature target to 'send a political signal that long-term decarbonisation is inevitable' (ibid.: ). in other words, its purpose would be largely symbolic. by further substantiating the narrative of an ongoing and unavoidable low-carbon transition, the successful adoption of an 'ambitious' agreement would encourage low-carbon efforts by businesses, investors and citizens. this would in turn generate momentum for more ambitious national policies, thereby setting in motion a self-reinforcing process towards decarbonisation. as former us secretary of state john kerry explains in the rolling stone: if nations are taking it seriously and setting targets, even if they don't make them, that will generate massive investment and a huge amount of private-sector activity […] and then you have to hope that somebody comes up with clean-energy technology, which makes it competitive with fossil fuel, and then, boom, you get your low-carbon economy. in the lead-up, during and on the back of cop , the agreement's core architects set up an elaborate communications campaign whose purpose was to shape a new climate narrative centred on three elements: the low-carbon transition is already underway; it presents unprecedented economic opportunities, and its successful implementation rests on the cooperation of actors from all sections of society. this, it was believed, would generate 'momentum' around the 'paris moment', and more generally the benefits of decisive climate action. for the architects of the paris approach, narratives and signals were not only key to achieving a positive outcome at cop ; they are equally important in the implementation of the paris agreement. for laurence tubiana, the post-paris process '[is] all about momentum.' christiana figueres ( ) urges all stakeholders 'to move firmly into a state of stubborn optimism' and to 'conceive of success and take immediate steps towards it'. following the adoption of the paris agreement and its subsequent ratification and entry into force, a priority for its main proponents was therefore to keep the 'paris prophecy' alive in the hope that this would lead stakeholders to ramp up their levels of ambition in the lead-up to the next global stocktake in . forging the right narrative and controlling the discursive context of global climate governance thereby become key concerns. in the final chapter, 'a new story' from their book, the future we choose, c. figueres and tom rivett-carnac describe this task as follows: right now, the predominant stories we are telling ourselves about the climate crisis are not very inspiring. but a new story can reinvigorate our efforts. when the story changes, everything changes (figueres and rivett-carnac : ) . the purpose of climate summits changes accordingly. in the post-paris period, negotiations increasingly lose their pivotal role. instead of focusing on the arduous and conflict-ridden process of political bargaining, rituals and performances occupy centre stage. 'the ideal cop would send a positive signal(s) to the international community, including investors, regarding the parties' and other stakeholders' direction of travel' writes susan biniaz ( : ) , lead climate lawyer for the u.s. state department from to and another key actor in paris. in a growing number of high-level and highly mediatised climate action summits, the unfccc now takes on the role of 'travelling salesman' for ambitious climate action. cops or climate action summits are essentially about communicating on the urgency of the climate crisis, highlighting the economic and social benefits of climate action and showcasing existing efforts-especially corporate climate action-to address the crisis (aykut et al. ) . hence, while rituals, discourses, theatrical techniques and political performances have always played an important role in global politics more generally, the post-paris climate governance stands out. whereas in other governance arenas their role tends to be understated or played down, in the climate arena, communicative and symbolic elements are explicitly recognised as core instruments in the implementation of the paris agreement. we suggest the term 'incantatory governance' to characterise this new approach. in so doing, we aim to highlight both the iterative, cyclical process created by the paris agreement's review mechanism and the central role of performative narratives and signals in the post-paris setup. as pointed out earlier, our intention is not to dismiss the approach as 'merely' symbolic and therefore ineffective. ethnographic research shows that incantatory rituals can produce real-world effects and fulfil important social functions. claude lévi-strauss, for instance, famously investigated the 'pragmatic effectiveness of symbols' in shamanistic cure (muniesa : ) . the repetitive utterance of words and mobilisation of symbols, he writes, 'provoke[s] an experience', which can produce therapeutic effects (lévi-strauss : ) . an increasing body of research shows that modern institutions also heavily rely on symbols, rituals and narratives: storytelling and drama constitute key features of contemporary management culture (czarniawska ) , while 'fictional expectations' shape the functioning of capitalist systems (beckert ) . accordingly, death ( : - ) criticises what he terms the 'anti-theatrical prejudice' in social science scholarship. 'symbolic aspects of summitry are not sideshops', he contends, 'but essential to the manner in which summits govern the conduct of global politics'. instead of opposing 'symbolic' politics to a hypothetical 'real' politics, we should accept that symbols and narratives form part and parcel of contemporary liberal governmentality (blühdorn ; death ) . the imminent conclusion of the regime building process therefore represents a critical juncture not only for un climate governance, but also for social science research. what are the prospects, risks and caveats of the new approach? as the focus shifts from negotiation to implementation, a new chapter opens for the unfccc and its annual cops. while a thorough assessment of the effectiveness of the new governance approach would be premature, developments since the paris cop point towards two main issues with the new approach. president donald trump's decision in june to withdraw from the paris agreement represented a severe test for the post-paris process. given the historical responsibility and political weight of the usa, the decision weakened the unfccc as the central forum of global climate governance. by sending a very negative signal, the us administration's retreat also threatened to undermine the 'paris prophecy', which, as we have shown, forms a crucial part of the post-paris climate governance framework. to uphold the momentum, it therefore became essential to show that the international community-state and non-state actors alike-was still committed to the goals laid out in the paris agreement, with or without us federal support. in response to trump's decision, the international climate community coordinated a series of high-profile initiatives. notable examples include the #wearestil-lin and america's pledge initiatives. in both cases, the idea was to reaffirm the fact that despite trump's decision, the usa, through the combined efforts of business leaders, university chancellors, mayors and state governors, would fulfil-and even surpass-its paris commitments. in addition to mobilising non-state and sub-state actors, the priority was also to find a new 'climate champion' and saviour of multilateralism to fill in the gap created by the us exit. despite his status as relative newcomer to the climate cause, french president emmanuel macron was rapidly elevated to the rank of 'champion of the earth'. the organisation of a press conference at the elysée palace the day after trump's announcement in june and the hosting of the one planet summit in december were coordinated efforts to retain control of the overall climate narrative and through this, keep the 'paris prophecy' alive. in our view, these and other concerted efforts to 'save' the paris agreement and 'ramp up ambition', by being almost exclusively centred on the production of narratives and signals, pose the risk of further 'virtualising' global climate governance (carrier and west ). moreover, the paris approach's 'performative' dimension complicates the task of publicly recognising that targets-such as the . °c target-are out of reach (geden a) . faced with the need to uphold a positive storyline, stakeholders of global climate governance are incentivised to 'move the goal posts' through 'creative accounting' or unproven techno-fixes, as exemplified by the massive amounts of 'negative emission technologies' included in global decarbonisation scenarios (anderson ; geden b) . by doing so, they risk delaying the necessary acknowledgement that current modes of development are inherently unsustainable. in other words, there is a real danger of deepening the rift between an 'international community' seemingly committed to ambitious climate action and the reality of 'business as usual' in a rapidly warming world. this discrepancy is not unique to the current period. the last decades saw a growing disconnect, or 'schism' (aykut and dahan ; aykut ) , between, on the one hand, a slow and procedural un arena focused on negotiating carbon emission reductions, and on the other hand, a staggering acceleration of a series of phenomena that are at the heart of the climate crisis but outside of climate governance's remit. chief among these are the dynamics of economic and financial globalisation, the expansion of extractivist development models and the global spread of western consumerist lifestyles. indeed, 'climate policy' is an inherently crosscutting policy domain. it touches on a range of very different issues, from development and energy policy, to trade and financial regulation, as well as agriculture and urban planning. yet, the governance of these issues follows very different logics. the voluntary and soft-law approach to climate governance contrasts with the situation in other issue areas. some of these are regulated through 'hard law', enforced by international organisations, while others are exempt from global regulation, and governed instead through global market dynamics and power relations (kingsbury ) . each governance arrangement draws on specific tools and mechanisms to exert influence on relevant actors and practices. the shift in global climate governance brought about through the paris agreement has exacerbated these differences. indeed, while it is very ambitious in terms of its global temperature targets, the paris agreement is evasive when it comes to spelling out the changes that will be required to attain them. there is no mention, for instance, of phasing out fossil fuels or 'decarbonising' the global economy, nor, for that matter, of encouraging renewables or energy efficiency. another important issue that is completely absent from the text is international trade regulation (brandi et al. ) . this links back to the 'fragmentation' of global governance, whereby the management of a problem falls upon diverse international organisations with potentially contradictory objectives (biermann et al. ). these fragmentations owe nothing to chance but are rather the product of structural 'selectivities' that are rooted in the global order and protected by powerful interests (brunnengräber ) . saudi arabia and other fossil fuel interests, for instance, actively worked to prevent any discussion on energy questions within the climate convention, so as to thwart any international regulation in that domain (aykut and castro ; depledge ) . the same applies to trade, whose absence from the climate negotiations is due to the active efforts of a coalition of industrialised and emerging economies (luterbacher and norrlöf ) . it is worth noting, however, that the similarities between the two cases stop there. unlike energy, international trade is regulated through a fairly robust international organisation, the world trade organisation (wto) and a number of legally binding bilateral treaties (mattli and woods ). from 'non-governed' issues where the strongest get their way (such as energy), to issues that are regulated through legally binding treaties (such as trade), to those managed through soft law (such as human rights and most environmental issues), we are in the midst of an increasingly complex global governance landscape. this landscape is not set in stone but is the product of political strategies and historical struggles that continue to act as barriers to an effective management of the climate crisis. the multi-dimensional nature of the problem calls for an in-depth rethinking of the established global order, beginning with the existing division of labour and hierarchy between international organisations, and the regulatory void when it comes to strategic domains such as fossil fuel production and trade. scholars of international relations generally agree that a central feature of international regimes is that actors' expectations converge in a given area of international relations (krasner ) . while it has generally been thought that such convergence is best reached through binding regulations and the building of strong international organisations, this no longer holds for paris-type governance arrangements. a growing body of scholarship therefore examines the paris shift and considers its consequences. in this article, we argued for the need to broaden the perspective adopted in this literature along two broad lines. we first suggested to re-embed the voluntary turn in climate governance within broader transformations in the ways that global problems are governed. in the course of these transformations, neo-managerial tools and techniques are increasingly adopted by international organisations. second, we argued that the new climate governance is not only characterised by institutional innovations. it also builds on narratives and signals as central means of implementation, by aligning actors' expectations and coordinating their behaviour towards a low-carbon future. based on these observations, we suggest the term incantatory governance to characterise the paris framework. the term highlights the iterative nature of the new 'bottom-up' and voluntary governance process. it also points to the increasing prominence of communicative devices and marketing techniques in global climate governance. our analysis suggests that both of these evolutionsthe rise of 'soft law' approaches and the widespread deployment of communicative techniques-reflect a much broader process: the entry of management culture, techniques and actors into global environmental governance. having said this, we consider the analyses laid out in this article as no more than a starting point. we hope that they will inspire further research on the discursive and performative dimensions of the new climate governance, but also beyond. indeed, given the climate arena's central position in global politics, one can expect other governance spaces to draw inspiration from it. this makes it all the more important to scrutinise the mechanisms of post-paris climate governance, evaluate their effectiveness, signal potential drawbacks and understand the governance shift's wider implications. one final observation should be made relating to the challenges facing those who express more fundamental reservations about the brave new world of 'performative' or, as we have termed it, 'incantatory' governance. critical perspectives are important in order to both problematise the selective and fractioned geographies of global regulation and highlight the shortcomings of a climate governance architecture that brushes aside issues that are key to solving the problem. and yet, there is little room for radical or fundamental critique under the current climate governance since such critique risks undermining the 'paris prophecy' by sending negative 'signals'. how then can we avoid both complacent self-censorship and a sterile, and potentially destructive, critical stance? we can perhaps begin by recognising that in a fractured and divided world, and in the face of multiple and interrelated crises, the paris agreement provides a snapshot of what can presently be expected from the un system and the unfccc process. it therefore goes without saying that the climate problem cannot be solved within the un system alone and that the paris agreement only forms one piece of a much larger puzzle. solving this puzzle will require actions at multiple levels and in a wide range of arenas. while this includes businesses, it also encompasses states and regulations, other international organisations, as well as collective mobilisations and social movements, which appear as key to shifting current power relations in favour of transformative change. it will also involve long and arduous efforts to re-politicise the climate debate and show the connections between climate change and other important issues that have traditionally been ignored in, or excluded from global climate governance. with their own rituals, heroes and discourses, recent and innovative climate protests, from 'fridays for future' to extinction rebellion, can be interpreted as attempts to do just that. our concluding remark is inspired by the current situation of global confinement and lockdown in the struggle against the covid- virus. in many countries, and especially of the global north, the measures imposed by governments to address this global health crisis are unprecedented since world war ii. the command-and-control approach combining quarantines, curfews and emergency laws stands at the antipodes of the managerial and 'incantatory' governance approach that we just analysed. it is too soon to say how this crisis and its political and economic consequences will affect the prospects of global decarbonisation. however, the contrast between these two governance models-one centred on transnational coordination through signals and narratives, the other on commandand-control and the sovereign power of nation-states-is striking. it could well be, therefore, that the covid- experience deeply affects and transforms, yet again, 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makes good business sense the business brief. shaping a catalytic paris agreement make law, not war: internationale und transnationale verrechtlichung als baustein für global governance two's company and more's a crowd. the complexities of multilateral negotiation the conundrum of corporate social responsibility: reflections on the changing nature of firms and states key: cord- -yrca hij authors: winkelmann, ricarda; donges, jonathan f.; smith, e. keith; milkoreit, manjana; eder, christina; heitzig, jobst; katsanidou, alexia; wiedermann, marc; wunderling, nico; lenton, timothy m. title: social tipping processes for sustainability: an analytical framework date: - - journal: nan doi: nan sha: doc_id: cord_uid: yrca hij societal transformations are necessary to address critical global challenges, such as mitigation of anthropogenic climate change and reaching un sustainable development goals. recently, social tipping processes have received increased attention, as they present a form of social change whereby a small change can shift a sensitive social system into a qualitatively different state due to strongly self-amplifying (mathematically positive) feedback mechanisms. social tipping processes have been suggested as key drivers of sustainability transitions emerging in the fields of technological and energy systems, political mobilization, financial markets and sociocultural norms and behaviors. drawing from expert elicitation and comprehensive literature review, we develop a framework to identify and characterize social tipping processes critical to facilitating rapid social transformations. we find that social tipping processes are distinguishable from those of already more widely studied climate and ecological tipping dynamics. in particular, we identify human agency, social-institutional network structures, different spatial and temporal scales and increased complexity as key distinctive features underlying social tipping processes. building on these characteristics, we propose a formal definition for social tipping processes and filtering criteria for those processes that could be decisive for future trajectories to global sustainability in the anthropocene. we illustrate this definition with the european political system as an example of potential social tipping processes, highlighting the potential role of the fridaysforfuture movement. accordingly, this analytical framework for social tipping processes can be utilized to illuminate mechanisms for necessary transformative climate change mitigation policies and actions. there is growing concern that global climate change is reaching a point where parts of the earth system are starting to pass damaging climate tipping points ( ): in particular, part of the west antarctic ice sheet (wais) appears to already be collapsing because of irreversible retreat of grounding lines ( , ) which in turn is expected to trigger loss of the rest of the wais ( ) . other tipping points may be close: a recent systematic scan of earth system model projections has detected a cluster of abrupt shifts between . and . °c of global warming ( ) , including a collapse of labrador sea convection with far-reaching impacts on human societies. the abrupt degradation of tropical coral reefs is projected to be almost complete if warming reaches . °c ( , ) . the possibility of the global climate tipping to a 'hothouse earth' state has even been posited ( ) . against this backdrop, there is a growing consensus that avoiding crossing undesired climate tipping points requires rapid transformational social change, which may be propelled (intentionally or unintentionally) by triggering social tipping processes ( , ) or "sensitive intervention points" ( , ) . examples for such proposed social tipping dynamics include divestment from fossil fuels in financial markets, political mobilization and social norm change, socio-technical innovation ( - , , ) . equally, if human societies do not act collectively and decisively, climate change could conceivably trigger undesirable social tipping processes, such as international migration bursts, food system collapse or political revolutions ( ) . social tipping processes have received recent attention, as they encompass this sort of rapid, transformational system change ( , , , ) . here we develop an analytical framework for social tipping processes. drawing upon expert elicitation and a comprehensive literature review, we find that the mechanisms underlying social tipping processes are categorically different from other forms of tipping, as they uniquely have the capacity for agency, they operate on networked social structures, have different spatial and temporal scales, and a higher degree of complexity. following these distinctions, we present a definitional framework for identifying social tipping processes for sustainability, where under critical conditions, a small perturbation can induce non-linear systemic change, driven by positive feedback mechanisms and cascading network effects. we adopt this framework to understand potential social tipping dynamics in the european political system, where the fridaysforfuture movement ( ) pushes the system towards criticality, generating the conditions for shifting climate policy regimes into a qualitatively different state. the proposed framework aims to establish a common terminology to avoid misconceptions, including the notions of agency, criticality as well as the manifestation and intervention time horizons in the context of social tipping. in this way, the framework can serve to connect literatures and science communities working on social tipping, social change, complex contagion dynamics and evidence from behavioral experiments (e.g. , ). we start by reviewing the characterization of tipping points across the natural and social sciences. over the last years, a suite of concepts and theories describing small changes with large systemic effects has been developed at the intersection of natural and social sciences. more recently, the concepts of tipping points and tipping elements have been broadly adopted by both natural and social scientists working within the field of climate change. while the concept of 'tipping' originated in the natural sciences ( , ) , social scientists made extensive use of the idea in the th century, often without using the terminology of tipping. famously, schelling ( ) , following grodzins ( ) , developed a theory of tipping processes to explain racial segregation in us neighbourhoods. granovetter ( ) modeled collective behavior as a tipping process that depends on passing individual thresholds for participation in riots or strikes. kuran ( ) described political revolution in terms of tipping dynamics, while gould and eldridge ( ) distinguish phases of policy change and stability in terms of 'punctuated equilibrium'. gladwell ( ) popularised the concept of 'tipping points', exploring contagion effects ("fads and fashions"), sometimes triggered by specific events. several recent studies have examined tipping processes within contemporary social systems. homer-dixon ( ) and battison ( ) explored the financial crisis as a tipping phenomenon. nyborg ( , ) discussed shifts in norms and attitudes, for example regarding smoking behaviors. centola ( ) associated tipping points with the "critical mass phenomenon", wherein - % of a population becoming engaged in an activity can be sufficient to tip the whole society. similarly, rockström et al. ( ) highlighted this so-called pareto effect in the context of decarbonization transitions. kopp et al. ( ) distinguished different social tipping elements within the realm of policy, new technologies, migration and civil conflict that are sensitive to "climate-economic shocks". here, a tipping element is a system or subsystem that may undergo a tipping process. since the mid s, ecologists and social-ecological systems (ses) researchers have also developed an extensive body of research on tipping processes using the terminology of 'regime shifts' and 'critical transitions' (e.g. [ ] [ ] [ ] . recognizing the impacts of human development on various ecosystems, this body of work often models ecological regime shifts as a consequence of social drivers. less attention, however, has been paid to sudden changes in social systems triggered by ecosystem changes. there is a rich literature on the collapse of past civilizations (e.g. , ) and the potential role of tipping points in that ( ) . recently, cumming and peterson ( ) brought this together with work on ecological regime shifts, proposing a "unifying social-ecological framework" for understanding resilience and collapse. further, rocha et al. ( ) noted that tipping dynamics can be produced by the interactions between climatic, ecological and social regime shifts. the concept of climate tipping elements introduced by lenton et al. ( ) and schellnhuber ( ) , has been increasingly adopted within earth and climate sciences. climate tipping elements are defined as at least sub-continental-scale components of the climate system that can undergo a qualitative change once a critical threshold in a control variable, e.g., global mean temperature, is crossed. positive feedback mechanisms at the critical threshold drive the system's transition from a previously stable to a qualitatively different state ( ). other scholars, e.g., levermann et al. ( ) , suggest a somewhat narrower definition of climate tipping elements by introducing additional characteristics, such as (limited) reversibility or abruptness. the tipping elements identified so far include biosphere components such as the amazon rainforest ( ) ( ) ( ) and coral reefs ( , ) , cryosphere components such as the ice-sheets on greenland and antarctica ( ) , and large-scale atmospheric or oceanic circulation systems including the atlantic meridional overturning circulation ( , ) . their tipping would have far-reaching impacts on the global climate, ecosystems and human societies (e.g. , ). in response to the concept of climate tipping points, social scientists are re-engaging with this concept yet again, creating an additional layer of tipping scholarship with an emphasis on the need for and possibility of deliberate tipping of social systems onto novel development pathways towards sustainability (e.g. , ) . scholars argue in particular that the rapid, non-linear change of social tipping dynamics might be necessary to speed up societies' responses to climate change, and to achieve the goals of the paris agreement. it is this element of acceleration, propelled by positive feedbacks, that makes the concept of tipping particularly interesting. for example, otto and donges et al. ( ) reported expert elicitations identifying social tipping elements relevant for driving rapid decarbonization by . rapid-paced changes are a distinctive feature potentially differentiating tipping dynamics from many other forms of social change, including incremental (policy or institutional) changes, or more radical (socio-technical) transitions or societal transformations. over the last decade, the literature on deliberate transitions and transformations towards sustainability has expanded significantly, exploring the dynamics that lead to the reorganization of social, economic or political systems (e.g. , ) . in many ways, this literature and the emerging work on social tipping are interested in very similar phenomena: fundamental shifts in the organization of social or social-ecological systems -a movement from one stable state to anotherincluding a change in power relations, resource flows, as well as actor identities, norms and other meanings ( ) . transformations can be fast, but speed is generally not one of their defining characteristics. this temporal feature of social tipping points -rapidity of change compared to the system's normal background rate of change -combined with the fact that tipping processes can be triggered by a relatively small disturbance of the system is motivating scholarship on leverage or 'sensitive intervention points', e.g. farmer et al. ( ) , who identified such potentially high-impact intervention opportunities, e.g., financial disclosure, choosing investments in technology and political mobilization that may be key for triggering decarbonization transitions. based on a bibliometric and qualitative review of these various bodies of literature across the natural and social sciences, milkoreit et al. ( ) proposed the following general definition of (social) tipping: "the point or threshold at which small quantitative changes in the system trigger a nonlinear change process that is driven by system-internal feedback mechanisms and inevitably leads to a qualitatively different state of the system, which is often irreversible." milkoreit et al. ( ) further noted there is a need to recognize and identify potential differences between climatic (or ecological) and social tipping processes to gain a deeper understanding of these phenomena. given this diverse and nascent field, there is a clear need for consensus as to what defines social tipping processes, as well as an understanding of how these processes are similar and diverge from dynamics in other non-social systems. further, there are currently limited examples of social tipping elements in the context of sustainability transitions presented within the broader literature ( , , , ) . here we explore the characterization of tipping processes within the natural and social sciences, examining how social and climate tipping processes are differently conceptualized. we draw upon a mixed qualitative methodological approach to illuminate these differences and key distinctions. initially, core differences were identified and discussed via expert elicitation ( ) . a selected group of experts from across the climate and social sciences were invited to take part in an expert elicitation workshop, that focused on identifying a common definition for social tipping processes, as well as the characterization of their dynamics. this workshop was convened in june in cologne, germany. the workshop participants were split into cross-disciplinary breakout groups, to independently identify the dynamics of social tipping processes. then, each of these groups reported their findings to the broader plenary, for discussion, consolidation, reconciliation and clarification. the process was then repeated for further clarification within the breakout groups. through this iterative inductive and deductive process, several unique themes and characteristics were identified from the broader set of codes, resulting in the key differences in and definition of social tipping processes presented below. drawing upon the differences identified in the expert elicitation workshop, we then review and synthesize the emerging field of social tipping processes, particularly in comparison to the related climate and ecological tipping dynamics. we then draw upon these unique characteristics to develop a common definition for social tipping processes, which we explore using the example of the fridaysforfuture student movement. social and climate systems' tipping processes exhibit several broad, fundamental differences in their structure and underlying mechanisms: (i) agency is a main causal driver of social tipping processes, (ii) the quality of social networks and associated information exchange provides for specific social change mechanisms not available in non-human systems, (iii) climate and social tipping processes occur at different spatial and temporal scales, and (iv) social tipping dynamics exhibit significantly more complexity than climatic ones. the most important characteristic differentiating social from climate tipping processes is the presence of agency. while a significant body of work (e.g. ), including latour's actor-network theory ( ) , addresses different forms and effects of non-human or more-than-human agency, here, we focus on a more narrow understanding of agency that is based on consciousness and cognitive processes such as foresight, planning, normative-principled and strategic thinking, that allow human beings to purposefully affect their environment on multiple temporal and spatial scales. while humans have a generally poor track record of utilizing their agentic capacities especially with regard to shaping the future (e.g. [ ] [ ] [ ] , they appear unique in their capacity to transcend current realities with their decisions. agency in this more narrow sense can be understood as the human capacity to exercise free will, to make decisions and consciously chart a path of action (individually or collectively) that shapes future life events and the environment ( ) . the notion of intentionality inherent in the idea of agency implies that human actors are not only able to adapt to changes in their environment, but also deliberately create such changes. non-human life forms can also be engaged in deliberate changes of their environment (e.g., beavers building dams), but the cognitive quality of these actions differs from those of humans, which can be based on different forms of knowledge and meaning about the world, moral norms and principles, or ideas about desirable futures. agency allows individuals and societies to be proactive rather than merely responsive in their relationships with other humans or the environment through planning, goal setting and strategic decision-making, which links decisions and behaviors in the present with consequences and realities in the (distant) future ( ) . governance scholars address this social-cognitive capacity for forethought and goal-pursuit in terms of anticipation ( ) and imagination ( ) , which can be tied to a set of futuring methods ( , ) . the ability to anticipate and imagine futures enables humans and their societies ( , ) -as opposed to animal communities or ecosystems -to transcend the present and shape the future according to our values and goals ( ), possibly increasing the prospects for human survival in times of fast and significant environmental change ( , ) . although this ability has been underutilized in the past, especially in the context of responding to climate change ( ), it is a crucial dimension of the human repertoire of tools to create change and to ensure its long-term well-being. agency interacts with many of the additional differentiating characteristics we identify below in important ways. for example, agency plays a role in the creation of social networks, institutions and meaning, i.e., the production of the structures of social systems. these network structures in turn enable and constrain agency (e.g. , ) . physical climate tipping elements, such as ice sheets or ocean circulations, lack that ability to intentionally act and adapt. however, the adaptive capacity of ecosystems can be interpreted as a form of non-human agency and learning mechanism ( ), see also supplementary information s . while scholarship on non-human agency, including that of animals, inanimate objects, landscape features or ecosystems (e.g. , ) might expand our understanding of agency, the cognitive abilities that characterize human agency, especially long-term and strategic thinking, do not exist in the non-human or inanimate worlds. understanding the nature of social networks is crucial for studying social tipping. while both natural (including physical and ecological) and social systems can be structurally characterized as networks and studied using a network science approach ( ), social systems differ from natural systems in the quality of the networks' nodes and interconnections and the processes and dynamics facilitated and impacted by these particular network characteristics. social systems feature additional network levels of information transmission (cultural and symbolic) that are largely restricted to human societies compared to natural systems ( ) . network qualities unique to social systems: networks in social and natural systems share various commonalities such as the existence of fundamental nodes and links ( ) . in contrast to most natural systems, however, social networks have the capacity to intentionally generate new nodes, which include socially constructed entities such as organizations and movements ( ) . new nodes can be created through cultural, political or legal means, as can the rules for their interactions with other existing nodes. social system nodes are unique in that they have richer cognitive realities, particularly agency and forethought. these nodes often have conflicting vested interests, which may be more short-sighted than future oriented. relationships in social networks can consist of shared meanings -especially norms, identities and other ideas -and a vast variety of cultural, economic and political relationships (e.g., employment, citizenship), all of which are not as pronounced or non-existent in less complex human societies and nature. hence, social network links are more diverse than links in natural systems and enable different kinds of network processes. for example, links between nodes in social networks are not necessarily dependent on physical co-presence, due to technologically enabled connections or the presence of more abstract interrelations such as shared norms, values or interpersonal relationships. social network dynamics can be of a purely ideational nature (e.g., the subject of the study of opinion and belief dynamics), but also involve material changes (e.g., resource extraction, movement and transformation for economic purpose). markets are unique social networks, involving both ideational and material network processes. in the anthropocene, the intensity and speed of socially networked interaction has increased dramatically, largely due to new media, digitalization, more efficient means of transportation, lower travel costs, and overall increased mobility, which is likely to increase spreading rates, while at the same time affecting the stability of the network itself ( ) ( ) ( ) . generally, social tipping can either occur on a given network (e.g., through spreading dynamics changing the state of nodes ( ) or change the network structure itself (see figure ). the structural network changes generated by social tipping processes include transitions from centralistic or hierarchical to more polycentric (neuromorphic) structures in urban systems, energy distribution and generation networks ( , ) . structural changes can manifest on large and small-scale spatial networks across multiple social structure levels. in order to capture these network tipping processes, quantifiers from complex network theory such as modularity, degree distribution, centrality or clustering can be used ( ) . , to january , ) . node colors indicate different party membership and links between nodes are drawn if the corresponding members agree on % of all votes in the considered two-year period. the lower network shows the same for the th united states congress (january , , to january , ). the transition from a closely entangled to an almost fragmented topology indicates a polarisation between democratic and republican party members over time ( ) . temporal and spatial scales: scales can differ greatly between social tipping and climate tipping processes and are more ephemeral for social tipping than for climate tipping. temporally, tipping in social systems manifests more commonly on the scale of months to decades, while for the climate tipping elements range from years to millennia. human actors tend to focus on more short-term consequences or outcomes, as complex issues (such as climate change) with longer timeframes are often harder to assess ( ) . within social systems, fund manager performance is evaluated quarterly, politicians often think in electoral cycles, business operates with annual or five-year forecasts, while individual practices and dispositions are constantly evaluated and reevaluated ( ) ( ) ( ) . in natural systems, however, it might take decades, centuries or even millennia for outcomes of change processes to become detectable (see figure ). both social and climate tipping elements can be ordered spatially ( , , ) , although social tipping elements cannot always be precisely located geographically. social scientists and economists have long grouped systems and processes as existing on the macro-, meso-and micro-levels (or some variation thereof), whereby some social systems (e.g., financial markets, political systems, technologies) consist of interdependent subsystems existing on multiple spatial levels. social tipping processes can also display spatial-temporal ephemerality. while climate tipping elements have a known spatial extent and dimensionality (with often a comparable extent in latitude and longitude and a generally much smaller extent in altitude) and have persisted in their current stable state for thousands (if not millions) of years, social tipping processes do not have a spatial extent or effective dimensionality that is known ex-ante and they can emerge (move into a critical state) and disappear (move out of a critical state) over time. ( ) , tabara ( ) and lenton ( ) . complexity: social tipping processes occur in complex adaptive systems ( - ) as opposed to the complex but non-adaptive physical climate system. as such they can exhibit comparatively greater complexity in the (i) drivers, (ii) mechanisms and (iii) resulting pathways of social tipping processes, as well as the aforementioned ephemerality in their spatial-temporal manifestations, including a potentially fractal and varying dimensionality and a more complex interaction topology ( , ) . social tipping processes can rarely be linked to a single common control parameter, such as is the case with global mean temperature in climate tipping dynamics. for most of the climate tipping elements like the ice sheets or the atlantic meridional overturning circulation, the control variables such as local air temperature, precipitation or ocean heat transport, can often be translated or downscaled into changes in global mean temperature as one common driver ( , ). however, for social tipping processes, multiple, interrelated factors are often identified as forcing the critical transition. for example, shifts in social norms regarding smoking ( ) can be linked to several, entwined factors, such as policies, taxation, advertising and communication, social feedbacks (e.g., via normative conformity), or individual preference changes. centola et al. ( ) show that tipping in social convention is possibly explained by a single parameter: the size of the committed minority). at larger scales, the collapse of complex civilizations has been linked to multiple interacting causes, and whilst disagreement abounds over the balance of causes in particular cases, there is general agreement that multiple factors were at play ( ) . this kind of causality -multiple interacting, distributed causes across varying scales -are a key characteristic of complex systems ( ) , contrasting starkly with conventional notions of causality involving bivariate relationships (one cause and one effect). further, due to their potential for agency and adaptive plasticity, social systems are open to a larger number of mechanisms that could cause a tipping process and various pathways of change that a tipping process could follow towards a greater number of potentially stable post-tipping states ( ) . climate tipping processes are often modeled as bi-or multistable, where the directional outcomes of forcing are to some extent known or knowable, e.g., based on paleoclimatic data and processbased earth system modelling. given a specific forcing change, one can predict in what state the element will restabilize as well as the "net" effects of the tipping process on larger earth systems. based on this understanding, the tipping of climate system elements is generally perceived as undesirable and often as part of pushing the earth system out of the "safe operating space for humanity" ( , ) . in contrast, for social systems, it is often unclear what a final stable state of the system will look like, or even whether the changes resulting from a tipping process will be normatively considered "positive" or "negative". as clark and harley ( ) point out, the characteristics of complexadaptive social systems, including the diversity of actors and elements and the different outcomes generated by local and global interactions, imply that the development pathways of these systems are less predictable. further, a social tipping process can generate new and destroy existing actor types (e.g., identities, institutions) and their behaviors. cross-scale dynamics and local differences are important to understand the emergent system structure and change dynamics, but predictive capacities, e.g., regarding the timing of a social tipping point or the boundaries between different stable states, do not yet exist ( ) . hence, the term 'managing transitions' is less useful than the idea of navigating a transformation pathway. the political nature of social change processes ( ) -different actors within a social community pursuing different, sometimes opposing, interests and visions for a reorganization of a social system while bringing to bear different resources and strategies -further exacerbate this situation. actors can deliberately generate new feedback dynamics that support or slow change, even after a tipping point has been passed, and they can actively work to adjust the direction of change. from the discussion above, it follows that a definition of social tipping process should take a microperspective and incorporate network effects and agency in addition to common tipping characteristics already explored in the review by milkoreit if a tipping element is already in a critical condition, where the stability of its current state is low, there may be a time window during which an agential intervention might prevent an unwanted tipping process by moving the system into an uncritical condition (see also si text s ). alternatively, if a tipping element is not already in a critical condition, there may be a time window during which some intervention might move it into a critical condition in order to bring about a desired tipping process. the small change triggering the tipping process could be either (i) a localized modification of the network structure (e.g., a change on the level of single nodes, small groups of nodes or links) or of the state of agents or subsystems, (ii) small changes of macroscopic parameters or properties, or (iii) small external perturbations or shocks. we deliberately do not require the trigger to be a single driving parameter. this is because we expect that a social tipping process could be triggered by a combination of causes rather than a single cause. furthermore, a social tipping element may be tipped by several different combinations of causes. consequently, for social tipping elements we cannot always expect at this point to identify a common aggregate indicator (such as global mean temperature in the case of climatic tipping elements) and a well-defined 'threshold' for this indicator at which the system will tip (see also the discussion on complexity above). note that social tipping as defined here is a unique form of social change, e.g., distinct from climate economic shocks ( ) and more specific than socio-technical transitions ( , ) . further, social tipping also denotes a shift to a qualitatively different state, and such, is different from standard business cycles or causes of seasonality. as such, social tipping presents a particular process of social change, where a system undergoes a transformation from one qualitatively different state to another, after being in a more critical state and affected by a potentially small triggering event. we propose several filtering criteria to focus on social tipping processes (i) that have the potential to be relevant to global sustainability in future earth system tractories and (ii) where human interventions can occur within a pertinent intervention time horizon on the order of decades and will have consequences within a political/ethical time horizon on the order of hundreds of years. (i) relevance of social tipping for global sustainability the social tipping process can impact a wide array of social systems, such as technological or energy systems, political mobilization, financial markets and sociocultural norms. we consider social tipping processes to be relevant here that have an impact on the biophysical earth system or on macro-scale social systems. the qualitative change in a 'relevant' social tipping process significantly affects the future state of the earth system in the anthropocene directly or indirectly through interactions with other social tipping processes. relevance can hence be defined in terms of impacts on biophysical earth system properties such as global mean temperature, biosphere integrity or other planetary boundary dimensions. for example, tipping dynamics to a political system could result in policy regime changes, affecting substantial reductions in greenhouse gas emissions ( , ). furthermore, we consider social tipping processes that have relevant impacts on macro-social systems and can be triggered by changes in the same biophysical earth systems, for example, mass migration due to climate impacts ( , ) . we are interested in potential social tipping processes in which humans have the agency to substantively intervene. for example, such interventions could be via technological or physical capacities of agential or structural actors. this therefore places emphasis on human intervention, such as decreasing the likelihood of extreme weather events via mitigation efforts, or triggering socio-technological changes towards decarbonization. we define intervention and ethical time horizons as follows: intervention time horizon human agency interferes with a social tipping element, such that decisions and actions taken between now and an 'intervention time horizon' could influence whether (or not) the system tips. we suggest to consider only social tipping processes with an intervention time on the order of years ( ), which arguably presents a practical limit of human forethought ( ) and of future-oriented political agency. for example, international governance efforts for global sustainability challenges, such as the ozone regime or the sustainable development goals, tend to work with similar time horizons. similarly, social tipping processes for rapid decarbonization to meet the paris climate agreement would have to be triggered within the next few years ( ), with ambitious emissions reduction roadmaps aiming for peak greenhouse gas emissions in ( , ) . the intervention time horizon is analogous to the 'political time horizon' defined for climate tipping elements in lenton et al. ( ). the time to observe these relevant consequences should lie within an 'ethical time horizon'. this recognizes that consequences manifesting too far in the future are not relevant to the current discourse on how contemporary societies impact earth systems. such an ethical time horizon could consider only social tipping processes which can have relevant consequences within the next centuries at most, corresponding to an upper life expectancy of the next generations of children born. currently, international climate policies, including those of the european union (eu) are insufficient to meet the + . °c or + °c goals of the paris agreement ( ) . while european policy makers presume to lead global mitigation efforts and characterize their actions as ambitious ( , ) , actual policy measures and proposals have been lagging behind this aspiration ( ) . eu countries emit about a tenth of the world's emissions, and a policy change towards more rapid decarbonization would not only have significant direct impacts on the climate system, but likely have indirect effects on the policies of other major emitters. but what kinds of sociopolitical processes can lead to these necessary changes? could such changes result from social tipping dynamics? public opinion is a crucial factor in policy formation, where the public can be understood as a "thermostat" signalling what is politically feasible ( , ) . shifts in public opinion can punctuate previously stable and 'sticky' institutions, leading to policy change ( ) . increased activism and public concern regarding climate change can generate new coalitions, or shift the priorities of existing ones ( , ) . here we examine the european political system as an example of and how social tipping processes could be triggered as a result of large-scale public activism and social movements. the european political system is composed of networks of agents (i.e., activists, decision-makers and organizations) with a range of social and political ties and is structured in nested and overlapping subsystems (i.e., national group, transnational political coalitions). viewed through the lens of social tipping, european political dynamics present a 'social system', embedded within the broader international political and climate change governance community 'environment'. driven by the fridaysforfuture movement ( ) (among other things), a groundswell of bottom-up support for more proactive climate policies has recently developed among european citizens, resulting in routine mass demonstrations and historical wins for green parties in the european parliamentary elections, as well as in federal elections in austria, belgium and switzerland. the european political system could be moving towards a critical 'state', creating the conditions for a tipping process towards radical policy change, bringing european climate policy in line with the paris agreement. accordingly, the european political system could constitute a potential 'social tipping element', where as it nears critical conditions, a small change to the system or its broader environment could lead to large-scale macroscopic changes, affected by cascading network dynamics and positive feedback mechanisms. such transformations could involve establishing more aggressive mitigation strategies that connect goals (such as remaining below + °c, % emissions reductions by , zero carbon emissions by ) with measures and pathways that have a reasonable chance to achieve them (i.e., investment in negative emission technologies, increased carbon taxation policies etc.). the fridaysforfuture movement has been pushing the european political system towards criticality, where it becomes more likely that the system will be propelled into a qualitatively different state. the movement was set off and inspired by a single swedish high school student choosing to protest on the steps of the riksdag for meaningful climate action. greta thunberg's protest quickly spread through the european social-political networks until more than a million students have been participating in weekly protests. this growing bottom-up pressure on the european climate policy-makers ( , ) has created an opening for significant policy change. the european political system consists of embedded subsystems at multiple scales. at the national scale, for example, the german socio-political system responded strongly to the activities of the similarly, several national western european green parties received historically strong electoral support in the may european parliamentary elections (such as in belgium, germany, finland, france and luxembourg). this increased support is also reflected in polling data in germany, where the green party has been effectively equal with the conservative party as the preferred political party of german voters in the latter half of ( figure , panels c and d). subsequently, germany introduced its first ever federal climate change laws, mandating that the country meet its goals (a ~ % reduction in ghg emissions) and establishing pathways to carbon neutrality by . currently, only a limited set of countries have enacted national climate change laws, and germany is one of the largest and most diverse economies to propose such actions. this presents the possibility for policy diffusion and transfer to other states ( ) , particularly considering the influential role germany plays within the european union. climate policy entrepreneurs could build upon momentum to further capitalize on windows of opportunity, pushing climate change proposals prominently into national and supra-national governmental agendas before the ephemeral moment passes ( ) . the covid- pandemic has placed new priorities on the policy agenda, also reflected in issue salience of climate change (see also fig. s in supplementary materials). as political and behavioral responses to covid- have led already to a significant temporary reduction in greenhouse gas emissions ( ) , this shock could be further leveraged to reinforce climate action -future economic recovery packages should set european economies on a pathway towards carbonneutrality, rather than return to the old normal ( , ) . drawing from this social tipping framework, the european political system may remain near a critical state. it remains unclear whether the covid- shock has supplanted climate change, or whether both remain on the political agenda. for example, discussions of a "green new deal" remain at the core of covid- economic recovery plans within the european union. the sociopolitical dynamics have likely moved the germany political subsystem further towards criticality, but it remains largely unknown whether this will result in tipping towards a qualitatively different state, in germany or in the broader european political system. rather, these judgements can likely only be made in hindsight, observing whether the system remained stable, moved towards criticality or experienced tipping dynamics. such an analysis in line with the proposed framework requires specific process tracing, identifying the key moments, actors, networks, mechanisms affecting criticality, the triggering event (threshold), and the positive feedback dynamics propelling the system towards qualitative changes. much attention is often paid to the specific triggering event, but it is rarely one single actor or action which accounts for the entirety of the tipping process. rather a full account needs to be made of all of the previous and related processes that have further placed the system towards criticality, allowing for such changes to become more likely. accordingly, for a tipping process to occur at the scale of the entire european political system, moving it into a state of decarbonization that is aligned with the paris agreement, a series of additional social movements and protests, or other shifts within the system or the environment, may be required. while we identify the role of fridaysforfuture in creating critical conditions, or potentially triggering the social transformations required for global sustainability, recent literature has identified further tipping candidates which could have generally "positive" effects on global sustainability. for example, divestment and reinvestment present candidates for rapid decarbonization and processes to achieve climate targets ( , ). in this case, intervention times range from years to decades, depending on the social structure level ( ). previous studies note that the adoption of technologies and behaviors such as rapid uptake of autonomously driven electric vehicles (if socially licensed), rapid change in dietary preferences reducing meat consumption and associated land-use and climate impacts can follow an epidemic-type model of diffusing across social networks ( , ) . alternatively, social tipping processes can lead to states of criticality with less desirable outcomes: recently it has been shown that climate change has contributed to the emergence of infections carried by mosquitoes, like dengue fever or zika, which could be accelerated further by increased mobility, e.g., through denser air traffic networks ( ) . the thermal minimum for transmission of the zika virus could in fact give rise to a threshold behaviour ( ) . changes to the local environment may enact "push" factors, resulting in large scale migrations ( , ) . further, increased global mean temperature has been suggested to increase the likelihood of civil conflicts ( ) . these social tipping processes are of great interest to policy makers, as it is desirable to potentially trigger or facilitate "positive" tipping ( , ) , while at the same time, mitigating the effects of potential "negative" outcomes. social tipping processes have been recently recognized as potentially key pathways for generating the necessary shifts for sustainability. drawing upon this emerging field, this paper develops a framework for characterizing social tipping processes. we find that mechanisms underlying social tipping processes are more likely to exhibit the unique characteristics of agency, social-institutional and cultural network structures, they occur across different spatial and temporal scales to climate tipping, and the nature of tipping can be more complex. social tipping processes thus present qualitatively different characteristics to those shared by climate tipping processes. accordingly, this paper develops a common framework for the unique characteristics of social tipping processes. we identify social tipping as a process, resultant of a complex system of drivers, resulting in shifting a system into a more (or less) critical state. it can thus serve to structure and inform future data analysis and process-based modelling exercises ( , ) . even so, while there is an emerging focus on social tipping dynamics ( - ), there remains great difficulty in pinpointing tipping events and generalizing the emerging dynamics. drawing from natural tipping dynamics, previous work on social tipping has often focused on identifying specific trigger events or critical thresholds in macroscopic system variables in analogy to identifying for instance critical temperature thresholds in the context of climate tipping ( ) . in natural systems the underlying dynamics are more deterministic and often can be directly observed, allowing for the identification of specific thresholds and events. while social systems comprise a much more open and complex system, one that is constantly adapting and where dynamics are often incredibly complex, interrelated and cannot be directly observed. accordingly, one could observe the same event across ten similar social systems, and could potentially observe ten unique outcomes. as such, anticipating a specific trigger, making causal inferences, or having generalizability in expected effects are all greatly limited within social systems. further, social tipping points are sometimes also understood as a point in time, rather than a point in a complex parameter space. such an approach makes it difficult to identify social tipping processes, as they often do not contain easily observable macroscopic thresholds nor temporal markers for change. rather, a complex adaptive systems viewpoint is required, understanding the multitude of interrelated processes and social structures driving change, and not focusing on a single trigger or threshold. accordingly, our framework proposed here focuses on identifying the processes and mechanisms of such change, and not a single triggering event, where the interplay of micro-level changes embedded within adaptive structural conditions can affect systemic changes. the notion of a critical state is central within our framework. changing conditions to the system's environment can cause it to enter more (or less) critical states, such that a single, or multiplicative action, can effect a systemic change. it is these changing conditions, and specifically the processes and dynamics underlying them, that are of analytical importance. drawing upon the analogy of a tipping coal wagon ( ), it is not the single, specific piece of coal that caused the wagon to tip, but rather the processes by which the wagon was filled with enough coal that any single piece (placed at a number of different locales) could cause such tipping. accordingly, the specific triggering event of a social tipping process could be somewhat random or arbitrary, as the conditions are critical enough such that any event with enough magnitude could have triggered these dynamics. it is therefore key to focus on the processes and mechanisms underlying the nature of such critical states which allow some trigger event to cause contagion dynamics. from social network models, we can deduce which kind of structural features make a system less resilient and thus more prone to social tipping ( ). one example is polarization, where social network models and social mediabased data analyses have shown that in polarized states with nearly disconnected network communities which in themselves are highly connected, contagion processes are more likely to occur ( ) ( ) ( ) . behavioral experiments and corresponding conceptual modelling approaches suggest that minority groups can initiate social change dynamics in the emergence of new social conventions ( , ) . furthermore, a rich social science literature has noted an array of factors (i.e. political institutions, technological or behavioral adaptation, environmental, normative and attitudinal) effective in shifting the social conditions surrounding climate change ( ) . a better understanding of critical states as demanded by our framework may help to identify early warning signals that could possibly indicate that a social-ecological system is close to a critical state in specific situations ( , ) . social tipping processes present a specific type of social change -characteristized by non-linear shifting states driving by positive feedbacks -which is similar to, but conceptually distinct from, other forms of social change. similar to how we explore the differences between natural and social tipping processes, further research should engage with social tipping in comparison to other forms of social change (such as historical institutionalist perspectives, social movements, policy feedbacks, complex systems). one of the greatest challenges lies in dealing with multiple, entangled drivers of tipping processes on different scales -temporal, spatial or social structural levels -and different levels of agency and heterogeneous agents and subsystems. in order to further understand the dynamics arising from these various levels of agency, it is crucial to identify examples from different subfields (economics, political science, demographics). a key current limitation in applying our framework is finding and operationalizing empirical data describing actual spreading processes on networks across these different levels, particularly compared to macro-economic data and public opinion polls ( ) , even though first steps in this direction are being made ( , ) . particularly data on the social structures and networks is notoriously difficult to access. while there have been advances in developing modeling frameworks ( , ) to simulate social tipping dynamics, linking these theoretical modelling to empirical data and behavioral experiments requires more attention. even if predictive modeling (i.e., the kind of deterministic, time-forward modeling we know from earth system models for instance) of such social dynamics in the sense of inferring time trajectories is very difficult or even conceptually unfeasible, such process-based modelling of social tipping dynamics can be very crucial to understand the nature of critical states also in realworld social situations. lastly, we focus here specifically on social tipping processes relevant for mitigating climate change, or sustainability more broadly, fitting within the previous literature. but, such a framework for social tipping dynamics is generalizable to other areas of study and social phenomena (such as the rapid social movements and public opinion dynamics surrounding racial inequality in the united states). while we explore one example of social tipping in detail, further inquiry is required to test the distinctiveness of social tipping processes, as well as the utility of the proposed definition to other social tipping processes. systematizing the types of social tipping processes, and exemplary case studies, would help to further illustrate these forms of change. research is also warranted into establishing typical timescales of social tipping; understanding how network structures affect social tipping dynamics; identifying typical network structures of systems entering critical states; discerning the temporal aspects of how effects travel through different social network structures; and gaining a better understanding of the origin of spreading processes. data acquisition, analysis and process-based modelling could all play a role in this research agenda. a wealth of social media data is available to study potential social tipping processes. however, this kind of data has mostly yet to be adopted within the context of earth system analysis and tipping dynamics. social tipping processes could be decisive for the future of the earth system in the anthropocene: some rapid shifts in social systems are, in fact, necessary to meet the targets of the paris agreement and the sustainable development goals ( ) . while we focus here on processes relevant for future trajectories of the earth system, we suggest that further analysis could use or adapt our definition to characterize other types of general social tipping processes (i.e. revolutions or rapid transformations). we also recognize that tipping processes within ecosystems present an interesting intermediary case between social and physical climate tipping as they typically incorporate characteristics from both realms. they are also crucial in determining future trajectories of the earth system (see preliminary discussion in the si). understanding, identifying and potentially instigating some social tipping processes is highly relevant for the future of the anthropocene, particularly with regard to the potential role in triggering rapid transformative change needed for effective earth system stewardship ( , [ ] [ ] [ ] . resulting trajectory would still move at some time ″ > ′ into some state ″ (which will usually depend on the influence exerted) that is qualitatively different from * . similarly, an uncritical condition, ( ∘ , ∘ ), is tippable by a decision maker if there is a possible trajectory ( ( ), ( )) % % , starting in ( ∘ , ∘ ) at some time ′, that the decision maker can force and to follow, and this trajectory would move into some state ″ at some time ″ > ′ that is qualitatively different from ∘ (a tippable uncritical state roughly corresponds to what others call a 'sensitive intervention point' ). at any time at which the system is not in an unmanageable critical state, the prevention time is the time interval it takes before some quasi-inertial trajectory has moved it into an unmanageable critical state. in other words, at time zero it is the largest time interval so that, when no intervention takes place until time , for all > with < , the system would not be in an unmanageable critical state at time . similarly, at any time at which the system is in a tippable uncritical state, the triggering time is the time interval it takes before some quasi-inertial trajectory has moved it into an uncritical state that is no longer tippable. in other words, at time zero it is the largest time interval so that, when no intervention takes place until time , for all > with < , the system would not be in a tippable uncritical state at time . we only consider social tipping processes for which the prevention or triggering time is smaller than some intervention time horizon. ecosystem tipping processes share properties of physical climate tipping dynamics in atmosphere, ocean and cryosphere in that they can often be described by a common driver, as well as that of deliberative social tipping elements in that they have adaptive capacity, and can therefore be regarded as intermediate. but, as previously noted, human agential capacity is far greater than those of other species. similarly to human social systems, ecosystems are comprised of interacting living organisms, they can be viewed as networks with components that can adapt (e.g., food webs). this is different from physical tipping elements such as the cryosphere elements (e.g., melting of permafrost) which do not typically exhibit the same networked structures. within the nominally 'climate' tipping elements are some major biomes -notably boreal forests, the amazon rainforest, and coral reefs -that are composed of living organisms and exhibit ecological network structures. indeed changing interactions between the living elements of these systems may be key to tipping dynamics -for example epidemic bark beetle infestation of boreal forests triggered by climate warming allowing the beetles to complete two life cycles rather than one within a season ( ) . thus these biotic tipping elements lie towards smaller scale ecosystems in the continuum, and tend to be more closely related to social systems in spatial and temporal scales compared to the typically much larger and more slowly changing physical climate tipping elements. these differences give rise to a proposed ordering of tipping elements, ranging from ( ) the physical climate tipping elements via ( ) ecosystem tipping elements to ( ) social tipping elements (table s ). tipping elements in the earth's climate system retreat of pine island glacier controlled by marine ice-sheet instability marine ice sheet collapse potentially under 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systems futurict: participatory computing to understand and manage our complex world in a more sustainable and resilient way fundamental structures of dynamic social networks interaction data from the copenhagen networks study earth system modeling with endogenous and dynamic human societies: the copan:core open world-earth modeling framework a first assessment of the impact of the extreme summer drought on central european forests general: we are very grateful to william c. clark, anne-sophie crépin, niklas harring, matthew ives, j. doyne farmer, wolfgang lucht, and john schellnhuber, for providing helpful insights and comments. we thank the participants of two dominoes workshops on social tipping dynamics held at gesis leibniz institute for social science, cologne, in summer and for foundational and framing discussions. the authors declare no competing interests. in this section, we give a more formal version of the definition of 'social tipping process' given in the main text, as a reference for mathematically inclined readers.after defining what we mean by a social system and its environment, we first classify their possible states into critical, unmanageable, uncritical, and tippable conditions, and then finally define the notions of prevention time and triggering time. by a social system, , we mean a set of agents together with a network-like social structure, that interacts in some form with the rest of the world, called the environment, , of the system, such that, if no "perturbation" or deliberate "influence" by some decision-maker occurs, and together can only follow certain "quasi-inertial" (or "default") trajectories restricted by the agency of the system's agents. let (%) and (%) denote the states that and are actually in at time .a critical condition for the system is a pair of possible system and environment states, ( * , * ), such that there exists another possible pair of states, ( ′, ′), with the following properties: . the state pair ( ′, ′) is no further away in state space from ( * , * ) than a certain "small" distance, , that represents the possible magnitude of "local" perturbations in (affecting only few agents or network links directly) or small changes in that are considered sufficiently "likely" to care about, with respect to some suitable distance function . in other words, (( ′, ′), ( * , * )) < . . if and were in state ( ′, ′) at any time ′, there is a quasi-inertial trajectory that would move at some later time ″ > ′ into some state ″ that is "qualitatively" different from * . this move represents a "global" (i.e., affecting a very large fraction of the agents) and "significant" change in the system (but not necessarily in its environment).if such a change actually happens, the time point ′ (not the state!) at which it starts may be called the tipping point or less ambiguously the triggering time point, and the system behavior within the time interval from ' to ″ is called the corresponding tipping process. an uncritical condition for and then is any pair of states that is not critical.a critical condition is unmanageable for an actor that may influence or in some way if there exists a possible pair of states, ( ′, ′), with (( ′, ′), ( * , * )) < and the following property:• assume that and were in state ( ′, ′) at any time ' and afterwards the state of and would follow any trajectory ( ( ), ( )) % % that the actor can force it to follow. then the key: cord- - mqn ihm authors: davies, anna; hooks, gregory; knox-hayes, janelle; liévanos, raoul s title: riskscapes and the socio-spatial challenges of climate change date: - - journal: nan doi: . /cjres/rsaa sha: doc_id: cord_uid: mqn ihm anthropogenic climate change is increasing the frequency and severity of the physical threats to human and planetary wellbeing. however, climate change risks, and their interaction with other “riskscapes”, remain understudied. riskscapes encompass different viewpoints on the threat of loss across space, time, individuals and collectives. this special issue of the cambridge journal of regions, economy, and society enhances our understanding of the multifaceted and interlocking dimensions of climate change and riskscapes. it brings together rigorous and critical international scholarship across diverse realms on inquiry under two, interlinked, themes: (i) governance and institutional responses and (ii) vulnerabilities and inequalities. the contributors offer a forceful reminder that when considering climate change, social justice principles cannot be appended after the fact. climate change adaptation and mitigation pose complex and interdependent social and ethical dilemmas that will need to be explicitly confronted in any activation of “green new deal” strategies currently being developed internationally. such critical insights about the layered, unequal and institutional dimensions of risks are of paramount import when considering other riskscapes pertaining to conflict and war, displaced people and pandemics like the – global covid- pandemic. this special issue of the cambridge journal of regions, economy, and society expands and enhances our understanding of the spatial, temporal, economic and sociological dimensions of climate change and riskscapes. riskscapes "play out in time and space" (müller-mahn and everts, , ) , encompassing different points of view on risk that highlight the "real-andimagined geographies based on individual and collective experience, tradition and knowledge" (müller-mahn et al., , . these articulations of riskscapes were preceded by theorists examining the social forces giving rise to diffuse risk distribution and contestation in modernity (see rosa et al., ) . by , empirically inclined social scientists in the usa argued "social, economic, and political forces inevitably create myriad [environmental] riskscapes in which overlapping air pollution plumes emitted by [various sources]…lead to cumulative exposures that pose health risks to diverse communities" (morello-frosch et al., , ; see also fitzpatrick and lagory, ) . the 'riskscape' concept has subsequently been applied in a range of other contexts beyond air quality (müller-mahn and everts, ) . however, explicit examinations of climate change riskscapes and their interaction with other riskscapes remain comparatively understudied (cf. gebreyes and theodory, ) . this is surprising, given the attention to climate change risks and their configuration by leading international agencies, including the intergovernmental panel on climate change (ipcc). in its special report 'managing the risks of extreme events and disasters to advance climate change adaptation' (ipcc, ) , for example, the ipcc acknowledges the challenges of understanding and managing the risks related to climate change. in particular, they emphasise that climate change impacts have social and economic as well as physical dimensions. so, while changes in the frequency and severity of the physical events generated by climate change will affect risk, the spatially diverse and temporally dynamic patterns of exposure and vulnerability also need to be considered. indeed, they recognise that differences in vulnerability and exposure can arise from non-climatic factors and from multidimensional inequalities that are produced by uneven development processes. alongside this, the ipcc conclude, with a high degree of confidence, that climate-related hazards can exacerbate other stressors, often with negative outcomes for livelihoods, especially for those living in poverty. contemporary events, like the - global covid- pandemic, underscore the importance of understanding the layered dimensions of risks. as with climate vulnerabilities and public and environmental health (faber, ; gebreyes and theodory, ; klinenberg, ; solomon et al., ) , emerging accounts of the covid- pandemic indicate that communities facing elevated threats to their lives and livelihoods are those who are elderly, experience chronic medical conditions, and are socially, politically and economically marginalised (cdc, ; manderson and levine, ; raffaetà, ) . this special issue demonstrates how analysing riskscapes can advance our understanding of climate change dynamics. these insights can be extended to anticipate dynamics at work on the covid- pandemic. physically, climate change will have greatest effect on communities with high population density. these communities are often near coastlines and in flood plains where they may face vulnerabilities of exposure to rising sea levels and other threats (see liévanos, ) , or have overbuilt environments to a degree that greater meteorological variability has outsized effect (for example, superstorm sandy in new york) (faber, ; see also taylor and weinkle, ) . the high density of people in such spaces also make these communities more vulnerable to pandemics like covid- . indeed, as of may , confirmed ) and associated deaths ( , ) within the usa were concentrated in the major population centre of new york city (dong et al., ) . in both the case of climate and covid- vulnerability, the local overbearance of human settlements on the environment makes adaptation and mitigation still more difficult. overlap in terms of community economic risk and resilience between covid- and climate change is also substantial, highlighting the kind of 'double exposure' risks initially flagged by o'brien and leichenko ( ) and elaborated in leichenko and o'brien ( ) . cascading economic effects from covid- (lockdowns) and climate change have the greatest effect on the poorest and socially marginalised communities. similar to the realm of climate-related inequalities in the usa (faber, ; klinenberg, ) , the disparate impact of covid- on low income and under-represented minority communities has been extreme, with some states in the us reporting up to a third of deaths being among racial and ethnic minorities, and projections that the economic implications of the pandemic will also hit minority communities the hardest (cdc, ; hale, ) . the disparate outcomes from underlying inequalities are projected to extend across the global south (wood, ) . in separate realms of inquiry (for example, environmental studies, health disparities, economic geography, criminology, security and terrorism, disasters and spatial inequality), scholars have already documented that place matters when considering risk. exposure to risk and its consequences varies by where a social actor lives, works and the multiplicity of other contexts in which they engage in social interaction. too often, however, these risks have been studied in isolation, for example, heightened environmental exposure studied in isolation of elevated exposure to crime, elevated exposure to health risks without concern for heightened economic risks and so forth (muller et al., ) . the ramifications of this approach have been laid bare and not least during the covid- crisis, which has had substantial interwoven health, economic, political and social implications. in the reshaped world responding to this crisis, considering the complexities, nuances and place-specificities of riskscapes and climate change is now more important than ever. using the concept of riskscapes highlights the social, temporal and spatial texture of risk (neisser and müller-mahn, ) and calls attention to interactions among risks and their "cumulative impact" across several dimensions of human life and the biophysical environment (renn, ; solomon et al., ) . entire nations-concentrated in the global south-are at heightened risk of repeated cycles of war (collier, ) . these conflicts scar the environment in profound and enduring ways (smith et al., ) ; these wars are often precipitated by environmental dislocation, with climate change playing an increasingly prominent role (dunlap and brulle, ) . whether internally displaced or migrating across national borders, those forced to flee violence live with multiple risks and face an uncertain future (hooks, ; united nations high commissioner for refugees, ) . these communities are also at risk of comparable long-term impacts (stalled economies, overtaxed medical systems and nutritional shortages) of pandemics like covid- . the origins of these risks and the forces that sustain them often operate on and across multiple spatial scales-from the local to the global. by studying climate change and riskscapes, it becomes possible to understand the "interdependencies and spillovers between risk clusters" (renn, , ; see also beck, ; neisser and müller-mahn, ) . prominent social theorists-most notably beck ([ ] ) and giddens ( )have drawn attention to the pervasive and growing importance of risk in contemporary societies. this concern extends beyond the academy-it is of concern to the general public and policy makers. the growing reflexivity of late-modernity-made possible by an unprecedented capacity to gather, analyse and share information-not only creates unprecedented opportunities, it also creates unprecedented threats to individuals and entire societies. moreover, this capacity to compile and analyse information allows for a discourse centred on identifying, mapping and managing risks (beck, [ (beck, [ ] (beck, [ , giddens, ) . efforts to identify, avoid, mitigate and manage risks are transforming political and social institutions. building on his earlier work (beck, [ ] ), beck's ( ) "world risk society" thesis highlights the growing prominence of large-scale technological and industrial processes in modernity that has given rise to unstable global financial markets and climate change and associated threats for the broader public. extant social and political institutions are not equipped to manage such risks. these trends pose threats to the legitimacy of science and of political institutions because accurate risk analysis is often hindered by the indeterminable and uninsurable nature of human "manufactured" risks. because the "dangers posed by global warming aren't tangible, immediate or visible in the course of day-to-day life" , our collective response will be halting and insufficient (giddens, , ) . furthermore, the physical, social, political and economic risks are fundamentally interwoven together. communities with lower social resilience-for example, those divided by substantial class, racial, ethnic, gender or cultural cleavages that undermine shared trust-lack the cohesion to effectively understand and respond to crisis (gotham and greenberg, ) . the identification and response to risk occurs in an institutional context. experts and political economic elites are often entrusted with the authority to classify and organise risk for the broader public (beck, [ (beck, [ ] (beck, [ , clarke, ; freudenburg et al., ; perrow, [ perrow, [ ] . in the context of environmental risk, it has been shown that scientific, corporate and state actors are tightly coupled in decision-making processes that are predicated on the dynamics of maintaining the prestige and objectivity of scientific inquiry, capital accumulation and state legitimacy (beck, ) . furthermore, the institutional context of government policy and professional associations "incubate" the expert and elite organisation of risk in taken-for-granted norms of safety and "acceptable" codes of conduct-all of which are monitored and enforced by experts and elites (beamish, ; clarke, ; perrow, [ perrow, [ ] turner, ) . for example, political and economic actors and institutions across the world are refashioning previous capital accumulation strategies and their spatial and ecological "fixes" through financial instruments and market-based mechanisms that seek to mitigate against and adapt people and places to environmental disasters, terrorist threats and the climate crisis (castree and christophers, ; gotham and greenberg, ; knox-hayes, ; ouma et al., ) . these dynamics-that is, elite domination and the downplaying, normalising and obscuring of environmental risks-extends to military organisations and warfare (bonds, ) . risk is culturally embedded. research into this aspect of risk illustrates the importance of attending to the local "historical legacy and interpretive contexts to perceptions of risk" (beamish, , ) . auyero and swistun's ( ) ethnographic and historical study of flammable, a poor and heavily contaminated argentine shantytown, is instructive for understanding how the normalisation of risk can shape how it is subsequently understood and responded to. in this case, the normalisation of exposure to environmental health risk was associated with an institutionally organised confusion over the cause of environmental contamination and how to motivate and articulate collective solutions to that contamination. alternatively, as norgaard ( ) illustrates in the context of local responses to climate change, normative modes of thought and practice normalise risk-perpetuating practices even in the face of mounting evidence of the dangers of climate change. similarly, in her work, knox-hayes ( demonstrates that cultural knowledge and practice shape the adoption of universalistic solutions to climate mitigation, even in economic domains such as with the creation of emissions markets. in order to be more effective, policy makers must consider the way different communities and societies make value judgements, assess risks and devise strategies to respond (knox-hayes, ) . this special issue brings together international scholars at the forefront of empirical and conceptual thinking about riskscapes. their research refines and sharpens our understanding of climate change risk and riskscapes, integrating understandings of risk from across diverse realms on inquiry under two, interlinked, themes: governance and institutional responses and vulnerabilities and inequalities. this also acknowledges how governance and state in/action can exacerbate risks, a theme that is addressed by several of the articles in this issue. it is no surprise to find a strong focus across the articles in this special issue on how different locations and communities attempt to manage the sum of complex combinations of risks. understanding the form, dynamics and impacts of governing riskscapes lies at the heart of much intellectual inquiry and practical action. ravi raman's tightly contextualised article is focussed on the rebuilding of post-flood kerala, india (raman, ). the physical scars of flood events are visible reminders of not only risks and their spatiality, but also of how institutions respond to that spatiality. raman documents how various agencies, including local people and state and non-state actors, influence each phase of rescue, relief and rebuilding. local fisher-folk communities, for example, draw on their cultural knowledge of climate change and risk to rescue flood victims just as others have done elsewhere (see knox-hayes, ) . in addition to these local governing alliances, raman also flags the role of international institutional alliances-for example with the un agencies-in supporting humanitarian interventions. he argues these diverse but coordinated responses create a state-society synergy sensitised to the "ecospatiality" of riskscapes in kerala. the ecospatiality concept recognises that building resilience in the aftermath of an extreme event requires new consideration of the arrangement and assemblage of spaces that make dwelling and habitation more attuned to the specific geographical features and potential risks of a region. rather than relying on rehabilitation and restoration to previous conditions, the goal of the ecospatiality state-society synergy is to redevelop while laying the foundation for a more resilient, egalitarian and ethical society. raman's article has broader implications, as its findings align with the broader suggestion that the covid- pandemic, for example, presents societies with the opportunity to move forward with new technologies for enhanced energy efficiency and resilience (invest away from fossil fuels where markets have experienced collapse) rather than to return to the old normal (worldand, ) . meanwhile, iain white and judy lawrence explicitly focus on the governance challenges posed by climate change in new zealand (white and lawrence, ) . they emphasise that as climate change impacts are dynamic, uncertain and contested, they pose significant challenges to the ways in which policy actors imagine and manage risks across space and time. identifying and applauding major efforts to reflect the latest insights from risk research in national policy in new zealand, they nonetheless find significant challenges remain to be resolved if appropriate governance and implementation strategies are to be successfully designed and implemented. white and lawrence demonstrate how tensions emerge between the theory of riskscapes, which emphasises that risks are always in a state of becoming, and the practices of risk management which seek to periodically "fix" risks, through plans, for example, in order to address them. while this process is a familiar feature of public policy design and implementation in many arenas, time lags between establishing scientific consensus for a particular course of action, developing policy and implementing that policy tend to be extensive with respect to climate change. positively, white and lawrence's historically situated paper identifies a greater impetus for policy development, more extensive political consensus on action and widespread use of the language of contingency, uncertainty and dynamism in new zealand now than ever before. while this is narrowing the gap between riskscape theory and climate change policy practice, issues remain with regard to connecting complex climate change riskscape imaginaries-comprised of an assemblage of biophysical, social, economic or political forces-to governance arrangements that are able to address them. there have long been academic calls for anticipatory risk governance (see fuerth, ; rosa et al., ; quay, ) that can recognise and address the dynamism and uncertainty of climate change riskscapes. however, the means and mechanisms for operationalising anticipatory governance remain unclear. nowhere is the art of anticipation more foregrounded than within the realm of re/insurance. in their paper examining the riskscapes of re/ insurance in florida, zac taylor and jessica weinkle use riskscapes theory to draw critical insights from existing re/insurance debates (taylor and weinkle, ) . they extend müller-mahn et al.'s ( ) argument that riskscape thinking must directly contend with machinations of power, working to reveal the asymmetrical, ongoing and always-political nature of re/ insurance. ultimately, taylor and weinkle argue against the expansion of re/insurance markets to govern climate risks precisely because the riskscapes approach demonstrates the importance of geographical contingencies and the limits to marketisation given the contested and shifting nature of "extra-market" considerations. jonathon everts and katja müller reveal the pivotal role that extra-market considerations played in the dynamic german coal industry (everts and müller, ) while building on recent calls to bridge conceptualisations of riskscapes and scale (aalders, ; müller-mahn et al., ) . climate change appears more and more like a "boundary object" (star and griesemer, ) , a common reference point for conflicting parties who invoke different meanings about climate change for different reasons. in this article, the authors examine the intertwined scales of riskscapes of coal mining, regional economic development and climate change in germany. the authors bring brenner's ( ) notion of "politics of scaling" into the analysis of riskscapes and look at the ways in which coal mining structures and embeds deep socio-ecological vulnerabilities across time. they argue that understanding the intricate relationship within and between different riskscapes and practices of scaling (from the local to the global) provides us with an analytical handle for deciphering the complexities of economic and environmental politics. further, they argue that doing so points us toward the transformative potential that lies within rescaling risks. risk has temporal implications through the politics of scaling. detlef müller-mahn, mar moure and million gebreyes also take on the themes of multiscalar politics and anticipation within governance in their study of riskscapes and climate change in the african cases of ethiopia and côte d'ivoire (müller-mahn et al., ) . they argue for greater scrutiny of how space is structured by multiscalar connections and uneven power relations. in particular, they urge practitioners and scholars to give increased consideration to how the future is made present through risk management. this includes taking into account how futures are envisioned differently by diverse actors both substantially and in terms of time horizons, the extent to which different visions incorporate risk (or not), and ultimately, how visions and risk assessments translate into agency. comparing and contrasting the politics of anticipation with a riskscapes framing, müller-mahn and colleagues conclude that thinking in terms of riskscapes with its focus on the nested and contested nature of the future, better acknowledges the diversity of material conditions, discourses and practices. the work is particularly trenchant for efforts to address climate change. to succeed, communities will need to adapt governance frameworks and social policies to address present conditions. the structure, distribution and flexibility of governance have a profound impact on the capacity of communities to conduct successful climate change mitigation and adaption. governance must be attentive to the social, political, economic and environmental dimensions of crises like climate change to assess the risks that these bring and to generate integrated responses. the capacity of governing institutions to think systematically and holistically in rapidly evolving situations and to do so across a range of socially and politically constructed temporal and spatial scales is also critical. the successes and failures of governing structures are addressed throughout the above articles in this special issue. however, the efficacy of various responses must be evaluated holistically and across different dimensions of risk, and for different groups given their relation to a given risk. here the human and social dynamics of riskscapes come to the fore. prior research debates the nature and durability of vulnerable social and spatial positions within climate change riskscapes and other socio-spatial manifestations of risk. models of the "world risk society" (beck, ) or analogous "urban risk societies" (elliott and frickel, ; romero-lankao et al., ) posit and observe a "social boomerang" dynamic of diffuse risk distribution in the post-war era. in contrast, five contributions to this special issue illuminate the significance of local and regional "risk settings" (müller-mahn and everts, ) or "contextual environments" (leichenko and o'brien, ) that structure and interlock multiple exposures to climate change-related risks and other risks over time. these articles highlight the stark inequalities and differentiated vulnerabilities that individuals and communities face from climate change riskscapes. ann tickamyer and siti kusujiarti interrogate three disasters experienced in indonesia to identify how socio-spatial risks are differentiated within particular contexts (tickamyer and kusujiarti, ) . power and gender roles, relations and practices are shown to be significant in mapping the socio-spatial relations of the resulting riskscapes. tickamyer and kusujiarti use their empirical analysis to demonstrate how such insights might inform plans for climate resilience in indonesia. using three case studies from indonesia, the aceh tsunami, the bantul earthquake and the merapi eruption, the authors illustrate how spatial, social, cultural, religious and political structures affect the experience of disaster. across the cases, gender relations, social capital and community resources are intertwined as drivers of risk and resilience across varying riskscapes. in particular, where women are empowered, have greater equality and participation in public spheres as well as opportunities to develop social capital and leadership, their families and communities have greater response and resilience to hazards. building resilience therefore requires great social and gender equality. from the standpoint of governance, it also requires a shift from market-based policies with hierarchical and predatory political systems to systems immersed in civic engagement and community cohesion. in a similarly nuanced fashion, jesse divalli and tracy perkins analyse neighbourhoods in southwest washington, dc, usa as sites of disparate expert and lay risk identification and mitigation practice (divalli and perkins, ). they find that this context results in what müller-mahn and everts ( ) describe as a "space of tension" . highlighting the disproportionate power held by the city, which has resulted in development plans that rarely account for residents' visions for their homes, divalli and perkins argue that the neoliberal growth strategy is being resolved largely in favour of the district and developers the district favours. while this effort is likely to produce a more resilient city in some ways, according to certain metrics they may also displace many current residents in the process; perhaps illustrating the birth of a new form of climateproofed gentrification. as documented widely within climate change policy (see hügel and davies, ) , divalli and perkins find tensions between the rhetoric of planning strategies that claim to speak for all residents and the reality of limited public participation and engagement within them. this is particularly the case for low income, residents of colour who have long experienced disadvantage in other contexts. a false picture of increased resilience will be generated as people become displaced through the district's strategies, pushing vulnerable residents beyond their administrative borders through what divalli and perkins call "resilience through attrition" . conceptualising resilience-related redevelopment as a risk to vulnerable populations in this way pushes considerations of climate change and riskscapes into social interactions at the neighbourhood scale. policy and design professionals are increasingly urged to consider and mitigate these risks within resilience planning as is seen in the metro vancouver region in canada examined by lily yumagulova in this special issue (yumagulova, ) . this article uncovers barriers and enablers for resilience planning across canada's multiscalar governance systems. in particular, yumagulova uses empirical material to unpack the underlying mechanisms for producing, reproducing and disrupting unequal patterns of risk across the region in british columbia. she does this by examining the role of the historic and existing flood management regimes in enabling and constraining collaborative planning capacities to address future climate risks such as sea-level rise. yumagulova finds clear evidence of historically differentiated treatment of indigenous communities in terms of flood risk transfer in the area. in particular, the analysis shows that the flood management regime favoured investments in structural flood protection (such as dykes) for colonial settlements, while leaving indigenous communities exposed to flood risk. these historical decisions left a legacy of underdevelopment for contemporary indigenous residents that led to further inequalities. yumagulova makes a strong call for a greater presence of indigenous voices in future risk and resilience planning in the region if these inequalities are to be addressed. raoul liévanos's case study of stockton, california, usa offers three main contributions to the climate change and riskscape literature (liévanos, ) . first, it synthesises a conceptual framework from prior research that attends to how elites' use of racial categories and racist real estate investment and development patterns structure the spatial concentration of separate and interlocking climate, environmental and economic risk exposures over time in what he calls "high-risk neighbourhoods" . the study conceptualises climate, environmental and economic riskscapes, respectively, "as spatially varying vulnerabilities of exposure to sea-level rise, flooding, and adverse housing market incorporation and displacement" . liévanos draws on archival sources spanning - and an innovative coupling of geographic information systems and qualitative comparative analysis. he uncovers how different "configurations" or "recipes of risk" (grant et al., , ) involve the devaluation of particular racial groups and racially classified spaces, threatening housing market positions, unequal flood protections and elevated risk of exposure to climate-related sea-level rise in stockton's high-risk neighbourhoods. moving continents, but staying with the theme of vulnerability and inequalities, yvonne braun draws on the concepts of riskscapes and "syndemics" (de waal and whiteside, ; singer, ) to explore the (un)intended consequences of development, which can exacerbate existing vulnerabilities for communities in the southern african country of lesotho (braun, ) . braun finds that poverty, food security, inequality and health risks co-occur, particularly in relation to regional climate stressors and to the impacts of large-scale infrastructural development such as the lesotho highlands water project (lhwp), one of the five largest transnational construction projects active in the world. in lesotho, it was largely small-scale farming families who absorbed the most direct losses and stresses to their livelihoods from the lhwp project, and yet it is these same people who experience the greatest risk from current and future climate changes (twomlow et al., ) . braun argues that it is international and national development agendas which have created a series of displacements to increase rather than reduce vulnerability and risks. instead she urges those who govern to adopt a more holistic approach to their work; an approach that deliberately seeks to anticipate and mitigate interactive, syndemic relationships and their consequences. even as we applaud the contributions of this special issue, we recognise gaps and challenges. some of the most daunting challenges and disruptive changes that are being set in motion by climate change and the arc of human history have received too little attention. while climate change is a central theme throughout this special issue, several topics associated with it are not fully addressed. in this section, we consider connections between climate change riskscapes and three of these: conflict and warfare; migration and displacement; and pandemics. in the first decades of the st century, wars have been fought by and in middle income countries (for example, iraq, colombia and syria) and among the world's least developed countries (for example, central and eastern africa) (collier, ; hooks, ; kaldor, ; mann, ) . there is a growing body of literature which identifies the risks of climate change as a threat multiplier, linking the onset and dissemination of warfare and conflict with rapid environmental change (barnett and adger, ; gleick, ; kelley et al., ) . beyond the human suffering and infrastructural damage, wars degrade the social capital and institutional integrity needed to secure the peace. as a result, cycles of violence bring repeated bouts of conflict to people and places who can least afford it (collier, ; hooks, ) . multi-sided wars and conflict among irregular armed forces create overlapping risks, including (but not limited to) environmental degradation, predatory commandeering of the economy, gender and age-related coerced labour and enslavement, and systematic degradation of the infrastructure (health, transportation and communication). these threats intersect with existing riskscapes, amplifying risks, heightening inequality and crippling efforts to mitigate risks. to be sure, the risk society literature has focussed on the social, political and economic dynamics of war, militaristic, and terrorist threats and risk management strategies (for example, amoore and de goede, ; beck, ; heng, ; rasmussen, ; rosa et al., ; williams, ) . in addition, recent riskscape literature has begun to illuminate the reproduction of social vulnerabilities and ensuing uneven redevelopment trajectories following terrorist attacks (gotham and greenberg, ) , and it features propositions about the salience of war games and analogous performative exercises and simulations for making future riskscapes present and the target of anticipatory action (neisser and runkel, ). yet the riskscape literature has not explicitly addressed war and conflict. because riskscapes highlight the temporal and spatial texture of risk and because they shed light on the multiplicity of perceptions and meaning assigned to these risks, adapting the riskscape framework to the study of war and conflict offers great potential. this is particularly the case in the context of climate change, as there is widespread concern and mounting evidence that climate change stresses will exacerbate distributional and political tensions, making wars more likely still (see dunlap and brulle, ) . a second gap centres on the issues of displacement and migration. people are on the move. in sympathetic accounts of globalisation, this mobility allows migrants to seek out economic opportunities and more hospitable political contexts to pursue their aspirations. but this geographic mobility reveals the darker side of globalisation. in the first decades of the st century, many migrants have been forced to migrate, are fleeing intolerable oppression and are escaping dangerous war zones (hooks, ) . the united nations high commissioner for refugees (unhcr) reports an alarming increase in the total number and a rapid increase of "people of concern" . from to , there were approximately million persons of concern, but this number more than tripled by . as of , there were more than million persons of concern (united nations high commissioner for refugees, ). the rate of growth is not only striking but the total now represents a significant share of the world's population. if displaced persons constituted a country, it would rank as the th most populous in the world. for both scholarly and substantive reasons, it is unfortunate that the riskscape literature has not been deployed to understand this humanitarian challenge. these mass migrations pose conceptual challenges that the concept of riskscape is well-suited to address. in the riskscapes literature-including the contributions to this special issue-there is a strong tendency to focus on a specific geographic area, the people who reside there, and/or institutional dynamics that contribute to the displacement of people from those areas. displaced people (internal and external) and migrants are moving across riskscapes at a variety of scales across the globe. what risks do they perceive? how do they (attempt to) cope with them? what voice do they have in identifying risks and institutionalising mitigation? how and why do elites, experts and risk management institutions respond to such migrants? taking full advantage of the fluidity and flexibility of the riskscape concept, and its attention to multiple and interlocking risks, would help us better understand this startling increase in displacement and forced migration on their own terms and in relation to climate-induced displacement and migration. indeed, sea-level rise is displacing vulnerable social groups and coastal settlements (hardy et al., ; maldonado et al., ; shearer, ) . further, overtly and covertly racist and nativist state policies, organisations and narratives threaten the lives and livelihoods of climate-change migrants, particularly from the global south, sometimes under the guise of resilience-based climate change adaptation and mitigation (baldwin, ; methmann, ) . because it is likely that the number of climate refugees will continue to grow in coming decades, these contributions will be all the more valuable in coming years. for example, the amazon rainforest is a riskscape. the indigenous peoples who have long lived in the rainforest are being displaced. these encroachments impose multiple layers of vulnerabilities, threatening their culture, their livelihood and the health. the risks are perceived and can be examined-as the contributors to this special issue have done in a range of settings. but the amazon rainforest is a riskscape with global implications. in the riskscape literature, including several contributors to this special issue, emphasis is placed on the varied meanings and perceptions of risk. in the case of climate change and pandemics, these differences can have global implications. in the amazon rainforest, cutting a tree or clear-cutting a hectare of forest may seem insignificant in the context of a vast-seemingly endless-rainforest. on the grounds that it is emblematic of and a requisite for progress, brazil's president, jair bolsonaro, aggressively promotes and defends this clear-cutting. this clear-cutting may push the deforestation to a tipping point that changes regional weather patterns and the global climate (piotrowski, ). furthermore, in the anthropocene epoch, just as climate change can be attributed to human activities, so human activity accounts for the increasing rate of zoonotic spillover (wood et al., ) . and, if these practices lead to zoonotic spillover, they could set in motion one or more global pandemics. current risk management organisations and institutions cannot see viruses and cannot detect the spillover from one host to the next. nor can these organisations and institutions immediately perceive the connection between individual acts and the global climate. for both climate change and pandemics, we are at risk of calamity. by the time that the effects are sufficiently "visible and acute" to spur concerted action, "it will, by definition, be too late" (giddens, , ) . the events of spring bring to the fore the dynamics and disruption of pandemics. the emergence, impact and aftermath of pandemics intersect with and transform existing riskscapes and the people who navigate them. in her contribution to this special issue, braun ( ) discusses syndemics (a concept emerging from the public health literature) and explores implications for riskscapes. syndemics draws attention to the multiple and overlapping factors that shape health disparities. by weaving in the concept of riskscape, she highlights spatial and temporal processes that reinforce and exacerbate syndemics. while braun's focus was on a megadevelopment project, the theoretical synthesis that she advances provides guidance for understanding the origins, context and aftermath of the covid- pandemic. the spillover of viruses from animal to human populations is and has been a threat to human health. before the covid- pandemic, this threat was accelerating. over the many millennia that the human species has existed, there were viruses known to infect humans, as of (woolhouse et al., ) . given this modest total, the rate of novel infections in recent years is striking. as reported by the world health organization ( ) there is every reason to believe that this alarming rate of novel disease emergence will continue and may well accelerate. it is estimated that there are over . million unknown viruses in animal reservoirs; it is believed that over , (perhaps as many as , ) of these viruses have the potential to infect humans (carroll et al., ) . various social, political and economic activities are encroaching on and destroying fragile ecologies around the globe, and in so doing, they are at the same time, (i) stressing mammalian and bird populations that are host to hundreds of thousands of viruses, (ii) dramatically increasing interactions between domesticated animals and these mammalian and bird populations and (iii) increasing direct human interactions with these animals and the viruses they host (carroll et al., ) . consider the dynamics underway in the amazon basin. vast tracts of the amazon forest are being cleared (often burnt) to make way for large agricultural operations-ranching prominent among them. the amazon rainforest is an ecological hotspot, thousands of species are found in this forest-and only in this forest. as their unique ecosystem shrinks or disappears altogether, animals will be stressed (many will go extinct) and they (and the tens of thousands of viruses they host) will come into sustained contact with livestock and with people. since so few of these viruses have been identified and studied, it is impossible to predict the potential for zoonotic spillover and the emergence of a dangerous pandemic (carroll et al., )-but it is certain that the risk of spillover is heightened by the destruction of the rainforests and other such biodiverse habitats. moreover, these encroachments and destructive practices-and the associated risks-are taking place around the globe. as with climate change, pandemics such as covid- tend to bring less attention to the destructive practices and behavioural changes needed to shift course, and instead draw attention towards technological solutions. managing covid- , including the closure and reopening of communities, depends on the rate and capacity to develop, manufacture and disseminate technologies including testing capabilities and vaccines. shortages in critical medical equipment like personal protective equipment including n masks and respirators exacerbated the medical crisis in countries like italy, and these shortages forced reconsideration of the operation of global supply chains (raffaetà, ; zhou, ) . while in some instances, political, economic and social institutions may be adequate, the covid- pandemic has shed light on the areas where social, political and economic institutions need improvement. further, the covid- pandemic may illuminate how communities with low levels of trust and social solidarity may not sustain lockdowns, allowing the virus to spread or rebound (manderson and levine, ; raffaetà, ) . in the case of climate change, low levels of trust and an inability to commit to and implement shared sacrifice will impede or delay the painful physical (energy transition, re-zoning) and economic measures necessary for mitigation and adaptation. the poor and socially marginalised also have least capacity to work remotely or relocate. further, they have limited financial reserves to overcome the effects of covid- (for example, purchase staple goods at inflated prices) and climate change (repair buildings after harsh storms). these concerns are particularly daunting for the communities around the globe that are, (i) currently locked down in response to the covid- crisis and (ii) also exposed to overlapping climate-related environmental risks such as flooding, fires, hurricanes and extreme heat events. these events will require considerable institutional flexibility and rapid political response. from the political standpoint, resilience is not an attribute possessed by a community in isolation. in the context of these multiple challenges, resilience will likely depend on communities gaining access to state resources and their needs being anticipated and addressed in state policies. community resilience is of decisive importance. where the challenge is of a global scale-as is the case with covid- and climate change-community resilience can be magnified or undermined by the larger state. in the case of covid- , hard-hit communities cannot secure the inflow of needed medical equipment on their own. in some instances, national-level responses have exacerbated these shortages. in other instances, the national response has procured needed supplies and bolstered local efforts, thereby strengthening community resilience. as effective countermeasure (testing regimes, lockdowns and contact tracing) must be organised at higher levels (state/ province or national), community resilience will be amplified or undermined by the larger political context. in a similar fashion, those communities experiencing the worst effects of climate change may well lack the ability to secure the inflow of resources needed for mitigation (energy reduction and shifts to renewables) and adaptation (anti-flood measures, increased water storage capacity). community resilience can minimise these shortfalls. but, once again, community-level options will be limited or augmented by the larger political context and the state's commitment to systematically address climate effects. as such, the concern for risk goes well beyond the realms of environmental issues and climate change, issues of crime, terrorism, economic (in)security and health equity are increasingly framed in terms of risk, and efforts to mitigate risk. in the immediate context of the covid- pandemic, the biomedical impact of the pandemic is transforming riskscapes around the globe. the facility of contagion, the severity of illness and likelihood of death vary by where one lives, who one is, and one's socioeconomic resources. writing in the spring of , it is impossible to predict the long-term impacts of the pandemic (assuming optimistically that a vaccine successfully tamps down infection in and thereafter). businesses will fail, unemployment has soared and may remain extremely high, the food supply and household-level food security are at risk, and global trade and travel may fall precipitously. each of these developments will play out unevenly across human societies; each will heighten vulnerabilities for many people. together this collection demonstrates, both empirically and conceptually, the relevance of adopting a riskscapes frame when considering climate change risks and their governance. it extends our understanding of riskscapes with respect to territorial coverage, with articles focussing on case studies drawn from diverse territories including india, new zealand, usa, indonesia, canada, germany and lesthoto. conceptually, in several respects, contributors have critically engaged with and have extended the riskscape concept. first, contributors have developed explicit connections between the temporality and spatiality of riskscapes (everts and müller, muller-mann, moure and gebreyes, and white and lawrence) . second, they have displayed a concern with social inequalities and pushed the riskscape literature to come to terms with gender (tickamyer and kusujiarti), race (divalli and perkins, liévanos), indigeneity (yumagulova) and class (divalli and perkins, liévanos, braun) . third, in different ways, each contributor to this special issue displayed a concern for power differences, highlighting the manner in which some individuals, social groups and organisations exert disproportionate influence in the definition of risk and characterisation of risk in time and space. fourth, they have drawn out the linkages between and among understandings of riskscapes, imagining alternatives and social justice. these insights include envisioning a more equitable and more inclusive planning of: (re)insurance markets in florida (taylor and weinkle), megadevelopment projects in africa (braun; muller-mann, moure and gebreyes), climate change mitigation in british columbia (yumagulova) , efforts to anticipate and "fix" climate change risks in new zealand (white and lawrence), urban renewal and gentrification in washington, dc (divalli and perkins), understanding the layers of risks in stockton (california) (liévanos) , coming to terms with climate change risks for small-scale agriculture (braun; müller-mann et al.; tickamyer and kusujiarti) and energy transition challenges (everts and müller) . these insights into social justice further enrich and add texture to the concept of riskscapes. the riskscape literature in general-and contributors to this issue specifically-have emphasised social justice. it is not simply the case that there are distinct, at times incompatible, interpretations of risks and riskscapes. social justice focuses on the institutionalised recognition of risks, steps taken (if any) to mitigate risks and imposition of costs for these mitigation efforts. the articles in the issue draw together lessons from cases around the globe. although riskscapes highlight the unique characteristics and the context of specific places, they also draw together important lessons of governance, planning and socio-ecological engagement that are critical to building resilience at the local, regional, national and global scales. communities need governance structures that are adaptive, inclusive and forward thinking to build resilient systems. they also require economies that empower different segments of society and build long-term value across multiple domains. these systems are essential for crises arising from issues such as climate change, war, displacement and migration or the current covid- pandemic. they bring to bear considerations of risk and resilience not only across space, but also layered through time. the contributors offer a forceful reminder that social justice principles cannot be appended after the fact. the covid- pandemic is creating and will leave multiple, profound and overlapping scars. recovering from this pandemic will require biomedical reforms, health care enhancements, job creation and economic renewal. as this special issue has emphasised, the recovery from this pandemic will play out across time and space-amplifying or dampening vulnerabilities of extant riskscapes. if these efforts are inclusive and infused with social justice, the post-pandemic social world could be marked by greater resiliency and enhanced social wellbeing. in a similar vein, climate change adaptation and mitigation pose complex and interdependent social and ethical dilemmas. if megadevelopment projects create winners and losers-and they do-global climate change mitigation and adaptation will do likewise, on a much larger scale. calls for "green new deal" resonate because the term references both the environmental (green) and the social justice dimensions (new deal). examining and advocating a calls for a "green new deal" through the lens of riskscapes offers a reminder and a tool to consider the interplay between environmental and social justice interventions across space and time, and from the individual to the national and global scales. the scale of risk: conceptualising and analysing the politics of sacrifice scales in the case of informal settlements at urban rivers in nairobi risk and the war on terror flammable: environmental suffering in an argentine shantytown premeditation and white affect: climate change and migration in critical perspective climate change, human security and violent conflict environmental hazard and institutional betrayal: lay-public perceptions of risk in the san luis obispo county oil spill silent spill: the organization of an industrial crisis risk society: towards a new modernity. thousand oaks world at risk the knowledge-shaping process: elite mobilization and environmental policy environmental change, risk and vulnerability: poverty, food insecurity and hiv/ aids amid infrastructural development and climate change in southern africa the limits to scale? methodological reflections on scalar structuration the global virome project: expanded viral discovery can improve mitigation banking spatially on the future: capital switching, infrastructure, and the ecological fix coronavirus disease : racial and ethnic minority groups. center for disease control and prevention acceptable risk? making decisions in a toxic environment the bottom billion: why the poorest countries are failing and what can be done about it new variant famine: aids and food crisis in southern africa they know they're not coming back": resilience through displacement in the riskscape of southwest an interactive web-based dashboard to track covid- in real time climate change and society: sociological perspectives the historical nature of cities: a study of urbanization and hazardous waste accumulation riskscapes, politics of scaling and climate change: towards the postcarbon society superstorm sandy and the demographics of flood risk placing' health in an urban sociology: cities as mosaics of risk and protection catastrophe in the making: the engineering of katrina and the disasters of tomorrow foresight and anticipatory governance understanding social vulnerability to climate change using a 'riskscape' lens: case studies from ethiopia and tanzania the politics of climate change turbulent and mighty continent: what future for europe water, drought, climate change, and conflict in syria crisis cities: disaster and redevelopment in bringing the polluters back in: environmental inequality and the organization of chemical production the economic impact of covid- will hit minorities the hardest. personal finance racial coastal formation: the environmental injustice of colorblind adaptation planning for sealevel rise the 'transformation of war debate': through the looking glass of ulrich beck's world risk society war and development: questions, answers, and prospects for the twenty-first century ) wars, states and political sociology: contributions and challenges public participation, engagement, and climate change adaptation: a review of the research literature, wires: climate change managing the risks of extreme events and disasters to advance climate change adaptation: a special report of working groups i and ii of the intergovernmental panel on climate change new and old wars: organized violence in a global era climate change in the fertile crescent and implications of the recent syrian drought heat wave: a social autopsy of disaster in chicago the spatial and temporal dynamics of value in financialization: analysis of the infrastructure of carbon markets the cultures of markets: the political economy of climate governance technocratic norms, political culture and climate change governance environmental change and globalization: double exposures racialized uneven development and systemic risk: sea level rise and high-risk neighbourhoods in the impact of climate change on tribal communities in the us: displacement, relocation, and human rights covid- , risk, fear, and fallout have wars and violence declined? visualizing climate-refugees: race, vulnerability, and resilience in global liberal politics environmental justice and southern california's 'riskscape': the distribution of air toxics exposures and health risks among diverse communities environmental inequality: the social causes and consequences of lead exposure the risk society at war: terror, technology and strategy in the twenty-first century risk governance: coping with uncertainty in a complex world exploration of health risks related to air pollution and temperature in three latin american cities managing the risks of climate change and terrorism the risk society revisited: social theory and governance the social construction of alaska native vulnerability to climate change toward a critical biosocial model of ecohealth in southern africa: the hiv/aids and nutrition insecurity syndemic the war on drugs in colombia: the environment, the treadmill of destruction and risk-transfer militarism cumulative environmental impacts: science and policy to protect communities institutional ecology, 'translations,' and boundary objects: amateurs and professionals in berkeley's museum of vertebrate zoology the riskscapes of re/insurance: mapping contested practices of catastrophe future-making in florida riskscapes of gender, disaster, and climate change in indonesia man-made disasters building adaptive capacity to cope with increasing vulnerability due to climatic change in africa: a new approach united nations high commissioner for refugees (unhcr) continuity and change in national riskscapes: a new zealand perspective on the challenges for climate governance theory and practice in)security studies, reflexive modernization and the risk society think , ventilators is too few? try three. ideas, the atlantic a framework for the study of zoonotic disease emergence and its drivers: spillover of bat pathogens as a case study human viruses: discovery and emergence world health topics what coronavirus means for the possibility of a carbon-free economy disrupting the riskscapes of inequities: a case study of planning for resilience in canada's metro vancouver region the global effort to tackle the coronavirus face mask shortage. the conversation key: cord- - k vtho authors: nissen, e.; kallberg, a.; usa, a. simonsson thomas jefferson national accelerator facility newport news va; sweden, stockholm university stockholm title: first direct observations of gear-changing in a collider date: - - journal: nan doi: nan sha: doc_id: cord_uid: k vtho in this work we perform the first ever demonstration of gear-changing in a real world collider. gear-changing refers to a collision scheme where each ring of a collider stores a different harmonic number of bunches. these bunches are kept synchronized using different velocities. such a system has been theorized, but has now been demonstrated using the double electrostatic ion ring experiment (desiree) in stockholm sweden. the experiment was able to demonstrate a gear-changing system, with both four on three bunches and five on four bunches. we determined a measurable parameter that shows a gear-changing system out to $ $ turns of the slow beam. we also developed new insights into how to control this type of system, opening up new possibilities for research. gear-changing refers to a system where two collider rings have different harmonic numbers and different bunch numbers while still having all bunches in the two rings collide with each other. for example, a collider could have four bunches in one ring and three bunches moving at / the velocity in the other ring. the synchronization could also be achieved with an appropriate pathlength difference between the two rings. a system utilizing gear-changing allows for a wide range of energies and particle species without changing the size of the rings. it can also reduce systematic errors in some experiments, i.e. where spin polarization is important. a schematic of gear-changing is shown in gear-changing also has some drawbacks. since each bunch interacts with each other bunch, each bunch now has to come into equilibrium all the other bunches in the machine. in systems like super kek-b or the jefferson lab eic proposal the number of bunches is in the thousands, and can be approximated by a linac-ring type system [ ] [ ] . this type of system leads to stability issues and cross resonances [ ] [ ] . there is also a problem caused by each beam's abort gap which means that when gearchanging reaches these gaps the beams will not receive any type of beam-beam effect. a theoretical treatment was performed for possible use in rhic to reduce systematic errors [ ] . an experiment to determine the effects of abort gaps was performed using an electron lens to simulate the interactions [ ] . demonstrating a full gear-changing system is possible in rhic, though it is invasive and has not been tried. another machine is able to demonstrate gear-changing at lower energies, the double electrostatic ion ring experiment (desiree), in sweden [ ] . desiree is made up of a pair of electrostatic storage rings that can merge up to kev in one ring and up to kev in the other ring beams of a variety of mostly singly charged ions [ ] . these beams merge with each other while moving in the same direction. typically velocities are matched to measure mutual neutralization reactions like those that would be found in the interstellar medium. the construction allows the use of a wide range of relative two fast choppers bunch the beams to get one or several bunches with a square timecurrent bunch shape, with a . µs pulse length. to get the required revolution frequencies, corrections were done in two steps. first the ion source platform voltages were adjusted, including rescaling of all bends and quadrupoles. next the voltages on the drift tubes were changed to locally vary the velocities of both beams and thus also the revolution frequencies. the frequencies were measured with a fourier transform of the pick-up signal without rf. rf is not normally used in desiree, but here it was applied on two cm long drift tubes which otherwise are used as schottky detectors. this was used to maintain the bunch structure over long periods of time [ ] . in this experiment we used a sinusoidal waveform with amplitudes of - v. to make the bunches of the two rings overlap in the common straight section, the timing of the choppers and the rf phases were adjusted. the core of this experiment is a four on three gear-changing system. carbon was chosen for the slow (four bunch) and nitrogen was chosen for the fast (three bunch) beams. since these bunches are oppositely charged, their oscilloscope signals will cancel each other in the pickups. the pickup amplification is - , so a bunch of n + gives a negative signal, while a c − bunch gives a positive signal. because the nitrogen beam is % faster than the carbon beam there will be an overlap pattern in pickup (pu ) and a mirror of this overlap pattern in pickup (pu ). this occurs because the carbon bunch starts out ahead of the nitrogen bunch, but since the nitrogen bunch is faster it passes through the carbon bunch and exits before the carbon. the bunches start out with a square longitudinal profile which evolves into a gaussian. the exact pattern will evolve into something similar to what is shown in if we assume that the bunches are perfectly timed such that they cross at the center of the merger region then the profiles in pu and pu will be symmetric. the distance between the center of the carbon and nitrogen beams is referred to as the overlap parameter. the demonstration of the gear-changing system has two parts. first, we will use an rf bucket filling scheme where the last bunches of each ring will be left off, this will then have three carbon bunches with the fourth bucket empty, and two nitrogen bunches with the third bucket empty. this will result in a repeating pattern every three carbon turns which will quickly show that gear-changing is occurring. it will also allow us to check how the bunches are evolving, and how that affects our ability to measure the distance between the beams. we call this the missing bunch experiment once everything is measured out properly we will measure the beams with full buckets. this is the second part, the full bucket experiment. the beam-beam tune shift for a machine like this is extremely small, due to the low charge to mass ratio and small particle number. there will be a small boost from having the beams moving in the same direction, however we used an algorithm [ ] to perform some simple simulations. a mm offset in one plane of a carbon bunch would give a . nm offset to a nitrogen bunch in that plane after carbon turns, this is not measurable. the experiment was performed in may of , via a remote collaboration because of covid- related travel restrictions. due to the small size of the offsets in the pickup signal we used an oscilloscope that would take snapshots of the beam at different numbers of turns. we can see an example of the repeating pattern in fig . the symmetric pattern in the collisions is visible, as is the shape of the bunches without collisions, as well as the delay between signals from the pickups before and after the mr. these plots show the pickup signal averaged over test runs. in order to measure the collision patterns we analyzed the data by fitting the oscilloscope signals to double gaussians with a linear baseline. the fit function we use is, where a, b, c, d, e, f and g are fitting parameters. we zoomed in on the relevant collisions for each oscilloscope channel, and used a fitting function with the initial positions and voltages of the carbon and nitrogen bunches. this is the unconstrained fit. we can then plot the differences between the pickup positions of the peaks of the carbon and nitrogen going in and coming out of the merger region. for perfectly adjusted beams these distances should be the same on either side of the merger region. since the bunches are not perfect gaussians, the fits may not necessarily show the reality of the system. one method of countering this is to constrain the possible fit functions. we do this using the magnitude and σ that we obtain from the uncollided bunches in the missing bunch system. we then constrain the fitting function to stay within % of the σ values and % of the magnitude values. this is the constrained fit. we start can systematically work to reduce these errors. another iteration of these measurements used a five on four gear-changing setup. we maintained the same . kev carbon energy and added another bunch. we decreased the nitrogen energy to . kev to match the velocity to % of the carbon velocity and also added another bunch. this leads to a smaller overlap parameter since the velocity difference is smaller. we measured the uncollided bunches in fig , and interestingly, the bunches do not move symmetrically around the expected value. pu 's data shows a larger value than pu . this implies that the timing is incorrect, and that the nitrogen bunch is entering the merger region late. the collision is happening, but is not again we tried to keep the signals symmetric, and after a great deal of scanning phases and frequencies fig shows the best pattern we could get. the smaller overlap parameter makes these measurements difficult, and using the offset data we got from fig would likely have given us timing that wasn't shifted slightly like we have in this system. this type of analysis should provide a more accurate way to synchronize the bunches. for this experiment we did not expect to measure a direct beam-beam interaction due to the low current and the limited number of diagnostics. conventional, high energy, beambeam systems are head-on instead of co-moving, reducing their effects to an impulse, and the beams are usually relativistic enough that the fields can be considered purely transverse. neither of these is the case in desiree. since the energies, and thus velocities are low enough, there will be longitudinal effects on each bunch. two effects can be seen; one is a change to the distribution over repeated collisions, and another is a change in the velocity of the bunches as they move through each other. an example of the change in the structure of the bunches over multiple turns is shown in fig . a future multi-particle simulation of these interactions should give us a better understanding both of the evolution of these bunches as well as how we may be able to measure these interactions, providing an opportunity for future research. to find the time of flight difference between a collided and uncollided bunch, we need only solve the system of differential equations shown below to get a first order guess of how the timing will be altered by the interactions of the bunches. where n i(t) and ca(t) are the positions of the nitrogen and carbon bunch centroids respectively, with n n and n c the number of nitrogen and carbon atoms. the term is the electric permittivity, a is the atomic mass number, and m is the atomic mass, and δ is a value of − to prevent singularities. in the experiment the number of particles per bunch were about for carbon and for nitrogen. this gives an offset less than ns. with a higher current and a more accurate oscilloscope, we expect a shift between the uncollided and collided beams of to ns which would be measurable. we have described a recent demonstration of a gear-changing system for the first time in an operating machine using desiree. the predicted overlap parameter was observed in four on three and five on four setups. there is also evidence suggesting the system has beam-beam interactions occurring, providing an opportunity for future study. we learned how to perform collider experiments with beams moving in the same direction. during the experiments, we checked the timing of collisions by measuring the distances between the positive and negative peaks in the colliding system. looking back it would have been better for us to use the uncollided signals in the missing bunch experiment to fine tune the timing. these types of lessons are important when controlling a system that hasn't been created before. while the timing in the five on four system exhibited some error, it did yield useful information about the fitting methods that we used to measure the four on three system. limited diagnostics are one of the limitations of this experiment, which can be remedied with more dedicated instrumentation, and a more sensitive oscilloscope for future experiments. we are not sure how much higher we can push the number of bunches using desiree. for an n(n− ) system the overlap parameter gets smaller with increasing n to the point that it would become difficult to measure. measuring the uncollided bunches in the missing bunch experiment will help, but it would require a more complicated pattern for the uncollided system. while the rf system is capable of harmonic numbers as high as , the chopper is thermally limited to an unknown degree. another possibility is a five on three system which would increase the maximum n since the velocity difference, and the overlap parameter, would be larger. we also observed synchrotron motion in the bunch lengths which couldform the basis for future measurements. while there is potential for ongoing experiments with gear-changing in desiree, a better understanding of the beam-beam effect requires an experiment in a machine such as rhic, or a dedicated facility. a machine with a similar footprint to desiree but using electrons, and having dedicated diagnostics would not only be able to perform gear-changing, but would have a beam-beam effect sufficient to reach the predicted instability threshold calculated in [ ] . office of nuclear physics under contract de-ac - or the beam-beam effect and its consequences for the modeling of the jefferson lab eic an analytic approach to emittance growth from the beam-beam effect with applications to the lhec barycentre motion of beams due to beam-beam interaction in asymmetric ring colliders beam-beam effects of gear changing in ring-ring colliders beambeam interaction in the asymmetric energy gold-gold collision in rhic cederquist, the double electrostatic ion ring experiment: a unique cryogenic electrostatic storage ring for merged ion-beams studies cryogenic merged-ion-beam experiments in desiree: final-state-resolved mutual neutralization of li + and d − direct observations in desiree of gear-changing events -initial measurements simulations of gear-changing and the beam-beam effect in jleic using guinea-pig the authors would like to thank both andrew hutton and todd satogata for many useful conversations, and a great deal of advice with this. this work was performed at the swedish national infrastructure, desiree (swedish research council contract no. - ). this material is based upon work supported by the u.s. department of energy, key: cord- -z h sc authors: semenza, jan c; ebi, kristie l title: climate change impact on migration, travel, travel destinations and the tourism industry date: - - journal: j travel med doi: . /jtm/taz sha: doc_id: cord_uid: z h sc background: climate change is not only increasing ambient temperature but also accelerating the frequency, duration and intensity of extreme weather and climate events, such as heavy precipitation and droughts, and causing sea level rise, which can lead to population displacement. climate change-related reductions in land productivity and habitability and in food and water security can also interact with demographic, economic and social factors to increase migration. in addition to migration, climate change has also implications for travel and the risk of disease. this article discusses the impact of climate change on migration and travel with implications for public health practice. methods: literature review. results: migrants may be at increased risk of communicable and non-communicable diseases, due to factors in their country of origin and their country of destination or conditions that they experience during migration. although migration has not been a significant driver of communicable disease outbreaks to date, public health authorities need to ensure that effective screening and vaccination programmes for priority communicable diseases are in place. population growth coupled with socio-economic development is increasing travel and tourism, and advances in technology have increased global connectivity and reduced the time required to cover long distances. at the same time, as a result of climate change, many temperate regions, including high-income countries, are now suitable for vector-borne disease transmission. this is providing opportunities for importation of vectors and pathogens from endemic areas that can lead to cases or outbreaks of communicable diseases with which health professionals may be unfamiliar. conclusion: health systems need to be prepared for the potential population health consequences of migration, travel and tourism and the impact of climate change on these. integrated surveillance, early detection of cases and other public health interventions are critical to protect population health and prevent and control communicabledisease outbreaks. travel medicine will increasingly see the health consequences of significant changes associated with global change, particularly climate change and socioeconomic development. together these changes mean more opportunities for people to travel to pathogen-endemic countries and for pathogens to be imported to new locations, with the potential for unexpected communicable disease cases and outbreaks. moreover, people who migrate or are displaced can also be at increased risk for chronic disease and mental health issues. thus, travel medicine needs to take a broader perspective than just asking patients where they are going or where they have been, considering how the ongoing and projected shifts in the magnitude and pattern of disease could affect the health and well-being of individuals and populations. modifications to the approaches used by health systems to manage adverse health outcomes are needed to ensure health care providers have the most up-to-date information. migration describes the movement of a person away from their usual residence whereas travel describes a person who passes from place to place, for any reason. migration and travel have shaped the history of humanity and enriched societies economically, socially and culturally. multiple and interacting factors drive migration in particular. climate change, including increasing climate variability, can be one of these factors. we discuss the potential impacts of climate change on migration and travel and the implications for travel medicine and public health practice. following a brief review of climate change, we discuss the potential health implications of migration and travel and the way in which they are affected by climate change and provide suggestions for health systems to better manage the potential impact of climate change, migration and travel on population health. the intergovernmental panel on climate change special report on warming of . • c concluded that human activities have caused ∼ . • c of global warming since pre-industrial times and that, if it continues to increase at the current rate, warming is likely to reach . • c between and . the special report concluded that climate change is increasing, and will continue to increase, land and ocean temperatures and the frequency, intensity and duration of heat waves in most land regions (high confidence). it also concluded that climate change will continue to increase the frequency and intensity of heavy precipitation events globally and the risk of drought in the mediterranean region specifically (medium confidence). climate change is a long-term process, but the extent to which individual weather events are influenced by climate change can increasingly be estimated. , migration the greatest human migration started or years ago when homo sapiens left africa for other parts of the world - ; a series of 'mega-droughts' may have contributed to this. [ ] [ ] [ ] migration has continued since and has become a defining characteristic of our times, affecting every inhabited continent. migrants may move internally, settle in neighbouring countries or move to other continents. currently, europe, north america and oceania are the main destinations for migrants although, overall, europe and asia have received the greatest number of migrants in recent decades. migration has political, social, economic, cultural, environmental and public health implications. migrants generally contribute more in taxes and social contributions than they receive in benefits. [ ] [ ] [ ] [ ] but migration can also pose chal-lenges, particularly when there is a rapid influx of large numbers of people. the international importance of migration was recognized in by the united nations general assembly when the agenda for sustainable development was adopted; of the sustainable development goals (sdgs) directly or indirectly relate to migration (table ). in , the united nations general assembly adopted the new york declaration for refugees and migrants to support a comprehensive approach to migration. these international accords were also thematically linked to other major international agreements, including the sendai framework for disaster risk reduction and the paris agreement under the united nations framework convention on climate change. , the drivers of migration are complex and include political, economic, social, cultural and environmental factors (figure ). these same factors can determine whether migration is permanent or temporary. the drivers of migration can be grouped into five categories - : ) push factors that force people to migrate including war, unemployment, food scarcity, over-population or prosecution. ) pull factors that attract people to a new destination including work and educational opportunities, political or religious freedom. ) network drivers that facilitate the migration process such family ties, social support, kinship, safety and feasibility of transport. ) national policies that allow or prohibit migration. ) personal aspirations and motivations. these drivers are often interconnected and situation specific ( figure ). they can also interact with climate change, which can itself be a trigger for migration ( figure ). , although environmental drivers do not appear to have been the most significant contributor to international migration to date, , migration in response to the consequences of climate change can be a pragmatic adaption strategy that balances the risks of staying and the risks of moving. , [ ] [ ] [ ] [ ] for example, for the tonga-speaking people in southern zambia, migration is an adaptation strategy to climate variability when the availability of water affects both agricultural production and livestock survival. additional warming of . • c is projected to further exacerbate climate-related drivers of migration; studies suggest that an increase in extreme weather and climate events and disasters associated with climate change could trigger an increase in migration. [ ] [ ] [ ] [ ] climate change impacts on migration and health. the complex and multiple drivers of migration mean that it is difficult to attribute specific migrations to climate change (figures and ) . as a result, projections of migration associated with climate change vary widely, ranging from million to million, with these numbers based on extrapolations. , in , a world bank report on the impact of climate change on migration estimated that by -in just three regions-climate change could force more than million people to move within their countries. table summarizes the world bank findings from three case studies in sub-saharan africa (ethiopia), south asia (bangladesh) and latin america (mexico). evidence for the possible role of climate change on migration comes from different sources (figure and ) . these include studies of the extent to which 'temperature and precipitation' are associated with out-migration. for example, in recent decades, temperature had a statistically significant effect on out-migration from agriculture-dependent countries. there is a nexus between climate change and migration in that temperature and precipitation are positively associated with migration: a • c increase in average temperature in the sending country is associated with a . % increase in bilateral migration flows; an additional millimeter of average annual precipitation is associated with an increase in migration of . %. another study suggests that an increase in precipitation anomalies from the long-term mean may also be associated with an increase in out-migration. under a moderate climate change scenario, if global mean temperature rises by • c, tropical populations would have to move considerable distances in order find places where temperatures are similar to those in places they currently live in and to preserve their annual mean temperatures. 'extreme weather events' linked to climate change, such as heat waves, droughts, storms, floods and wildfires, cause shortterm population displacement ( figure ). in , disastersmost of which were related to extreme weather-displaced . million people in countries ( figure ). in , california only low-income countries show marked increases in population under spp . b the higher number of climate migrants under the more climate-friendly scenario in ethiopia is in part driven by the regional climate models, which project lower water availability by in general compared with the other two scenarios (pessimistic reference and more inclusive development scenarios), which are coupled with higher emissions. experienced the most destructive wildfire season on record with more than , people being evacuated from their homes. a number of factors led to these fires, including accumulated natural fuel and compounding atmospheric conditions that were linked to climate change. based on the experience of other events in the usa, some people will eventually return to their communities, but others will permanently relocate. for example, following hurricane katrina, it was estimated that between % and % of the population returned, with young adults being more likely to have moved further away. , in , after hurricane andrew in florida, % of displaced residents relocated within the same county, % moved within the state and % moved out of state. in some cases, extreme weather events can cause longerterm migration. for example, an increase in the frequency and intensity of droughts in tanzania has killed livestock and forced the maasai people to migrate from rural to urban settings, with documented implications for their mental health. similarly, prior to the syrian migrant crisis in , the fertile crescent was affected by a severe and sustained drought associated with anthropogenic increases in greenhouse gas emissions and this, combined with unsustainable agricultural and environmental practices, contributed to internal migration, political instability and conflict and eventually to an exodus of one million migrants to europe. 'river flooding and erosion' has adversely affected agriculture in bangladesh and resulted in population migration to india ( figure ). climate change models project an increased frequency and likelihood of severe storms, with implications for sea level and flooding, in the ganges-brahmaputra-meghna delta, and this is likely to displace a large number of people. in pakistan, the adverse economic impact of heat waves on agricultural and non-agricultural activity has increased outmigration. in contrast, flooding in pakistan was not associated with significantly increased migration, possibly due to the larger scale of the response by relief agencies to floods compared with heat waves. in other regions, food scarcity due to flooding has contributed to internal migration. in in mali, flooding destroyed fields, damaged grain stores and impeded livestock production and, as a result, an estimated . million people are food insecure and internally displaced. 'gradual changes' due to climate change, such as changing rainfall patterns, rise in sea level, increased salinization and decreased soil fertility, can also be a driver for migration ( figure ). practice. migration has implications for health and public health practice. , migrants can be exposed to health risks in their country of origin and country of destination (figure ) ; they may be particularly at risk during their journey, due to lack of shelter, exposure to harsh climatic conditions, heat and overcrowded, inadequate transit and detention centres. public health interventions need to target migrants in all of these settings, and especially during migration, in order to minimize associated health risks (figure ) . sudden displacement because of a disaster or other event can be very stressful and manifest in post-traumatic stress disorders, depression or anxiety that can continue for months or even years. other factors that can affect the mental health of migrants include family separation, acculturation, job insecurity, restricted mobility, dangerous border crossings, stigmatization and marginalization. stress is associated with higher rates of depression and anxiety disorders in the short term and with cardiovascular disease in the longer term. [ ] [ ] [ ] epidemic intelligence data from the european centre for disease prevention and control (ecdc) suggests that migration is a relatively infrequent driver of communicable disease threat events in europe, compared with travel and tourism. , there was no evidence during the migrant crisis in europe that migrants posed a risk to european union citizens from communicable diseases, and despite the barriers to accessing immunization that migrants experience, the contribution of migrants to the current measles outbreaks in europe is minimal. however, migrants may be at increased risk of communicable disease due to exposure or conditions during migration and in the country of destination (table ) . , , , [ ] [ ] [ ] [ ] for example, there is evidence that tuberculosis transmission occurs on migration routes to europe, including during long-term stays in refugee camps. overcrowded and inadequate refugee camps, detention centres and housing can spread vectors (e.g. lice and fleas) that transmit diseases such as relapsing fever, trench fever and epidemic or murine typhus. poor and overcrowded living conditions can also increase the risk of respiratory diseases, such as seasonal influenza. migrants living in overcrowded conditions with poor sanitation may also be at risk of cholera outbreaks. overcrowding, poor sanitation and limited access to health services can also facilitate the emergence of antimicrobial resistance (amr) among migrants, particularly refugees and asylum seekers. migrants may be at increased risk of communicable disease in their country of destination due to factors including lack of vaccination, low socioeconomic status and poor living conditions and limited access to health care (table ) . recent evidence from the european union (eu) and the european economic area (eea), based on cd cell counts and hiv-rna trajectories from seroconversion also suggests that migrants may be at risk of hiv transmission in their country of destination; two-thirds of a sample of hiv-positive migrants from sub-saharan africa, latin america and the caribbean acquired their hiv infection in the country of destination. essential public health measures include ensuring adequate living conditions, access to health care in refugee camps, detention centres, screening for communicable diseases and assessment offer serological screening and treatment (for those found to be positive) to all migrants from countries of high endemicity in sub-saharan africa and focal areas of transmission in asia, south america and north africa. offer serological screening and treatment (for those found to be positive) for strongyloidiasis to all migrants from countries of high endemicity in asia, africa, the middle east, oceania and latin america. offer vaccination against mmr to all migrant children and adolescents without immunization records as a priority. offer vaccination to all migrant adults without immunization records with either one dose of mmr or in accordance with the mmr immunization schedule of the host country. offer vaccination against diphtheria, tetanus, pertussis, polio and hib a (dtap-ipv-hib) to all migrant children and adolescents without immunization records as a priority. offer vaccination to all adult migrants without immunization records in accordance with the immunization schedule of the host country. if this is not possible, adult migrants should be given a primary series of diphtheria, tetanus and polio vaccines. see table for details. cxr means chest x-ray; ltbi, latent tb infection; tst, tuberculin skin test; igra, interferon-gamma release assay; mmr, measles/mumps/rubella. source: ecdc. public health guidance on screening and vaccination for infectious diseases in newly arrived migrants within the eu/eea. https://ecdc.europa.eu/sites/portal/files/documents/public% health% guidance % on% screening% and% vaccination% of% migrants% in% the% eu% eea.pdf. vaccination against hib is only recommended to children up to five years of age. of vaccination status on arrival in countries of destination (table ). available evidence shows that it is cost-effective to screen child, adolescent and adult migrants for active and latent tuberculosis, hiv, hepatitis c, hepatitis b, strongyloidiasis and schistosomiasis (table ) . targeted hiv prevention, testing and treatment programmes are also required in countries where migrants are at risk of hiv acquisition after arrival. there is also a clear benefit from ensuring that migrants are included in vaccination programmes and providing catch-up vaccinations where needed (table ) . particular attention should be given to vulnerable children, women and the elderly, who might also suffer from non-communicable diseases, such as cardiovascular diseases, diabetes, cancer, chronic lung diseases, mental health and trauma. sudden migration can result in complications from noncommunicable diseases if treatment and care are discontinued or if access to medications is interrupted. these conditions can cause a life-threatening deterioration and acutely exacerbate preexisting conditions in the event of sudden migration (table ). travel, including for business and tourism, has increased dramatically in recent decades. globally, in , commercial airlines carried more than billion passengers; this is projected to have risen to . billion in and to continue to increase in the coming years. air travel contributes to greenhouse gas emissions and, hence, to climate change. at the same time, the effects of climate change on tourism assets, such as biodiversity, coral reefs, glaciers and cultural heritage, are leading to an increase in 'last chance' travel to destinations before they are further degraded. [ ] [ ] [ ] for example, in the southern hemisphere, sea level is projected to rise by - cm by the end of this century, submerging much of the maldives. in northern latitudes, climate change is already having an adverse impact on the winter tourism industry in lapland and may make it impossible to maintain its snowcovered winter wonderland in future. in many ski resorts, artificial snowmaking, which has high energy costs, is increasingly being used to supplement natural snow, to improve reliability and to extend the season. climate change can be a factor in creating conditions suitable for vector-borne disease transmission. importation of pathogens from endemic areas can then result in the subsequent spread of these diseases under suitable climatic conditions. extended seasonal transmission seasons due to climate change can also increase the window of opportunity of pathogen importation. climatic suitability, including lower and upper temperature thresholds, affects the growth, abundance and survival of mosquito vectors such as aedes aegypti and aedes albopictus that can transmit dengue, zika and chikungunya. in europe, a warmer climate appears to be associated with more frequent outbreaks of dengue and chikungunya are projected to become more suitable for onward transmission of the chikungunya pathogen. climate change may have contributed to the explosive spread of zika in brazil. the epidemic was preceded by 'record warmest' climatic conditions, accompanied by a severe drought. the utilization of home water storage containers during the drought might have provided breeding sites for aedes mosquitoes and increased exposure to mosquitoes. virus importation into a susceptible population could then have triggered the epidemic. precipitation, another important environmental factor affected by climate change, also influences the availability of conditions suitable for mosquito vectors. for example, heavy rainfall increases the abundance of a. albopictus and, hence, the risk of transmission of dengue and chikungunya. climate change can increase the risk of other vector-borne diseases, including tick-borne and rodent-borne infections, and can also influence the range, seasonality and incidence of water-borne diseases such as cholera. , global air travel may facilitate the spread of 'resistant pathogens'. for example, it is thought to have facilitated the rapid dissemination of methicillin-resistant staphylococcus aureus from the uk and north america across europe and then to asia. vancomycin-resistant enterococci and klebsiella pneumoniae carbapenemase-producing k. pneumoniae followed a similar trajectory. the burden of antibiotic resistance in destination countries can also be exacerbated by climate change, because an increase in ambient temperature is associated with a significant increase in antibiotic resistance for common pathogens, including k. pneumoniae and s. aureus. it is hypothesized that temperature accelerates horizontal transfer of resistance genes or the uptake of genetic material. ambient temperature may also facilitate environmental growth of resistant strains and enhance transmission from food, agriculture and environmental sources. climate change impacts on travel: implications for public health practice. air travel can facilitate the spread of communicable diseases around the globe. passenger volume has been identified as a significant driver of the importation of viremic passengers. , flights directly connect millions of passengers between europe and asia, africa, north america and south america, with many of these locations 'hot spots' for the emergence of communicable diseases. a disease outbreak in one part of the world can quickly spread to another part via air travel. , for example, in-flight transmission of severe acute respiratory syndrome (sars) in resulted in the rapid spread of sars-associated coronavirus (sars-cov) around the world. international air travellers departing from mexico in unknowingly carried and disseminated the novel influenza virus a(h n )v around the world. similarly, air transport networks contributed to the spread of middle east respiratory syndrome from saudi arabia in , chikungunya dispersion in the americas in , ebola from west africa in and zika spread in the americas in . [ ] [ ] [ ] [ ] more specifically, air travel can increase the risk of importation of pathogens from endemic areas into regions with competent mosquito vectors and suitable climatic and environmental conditions for vector-borne diseases. dengue, a mosquito-borne viral disease, is a significant public health concern, threatening almost half of the world's population. table . vaccinations to be offered to migrants in the absence of documented evidence of prior vaccination within the eu/eea priority vaccinations mmr administer to individuals ≥ months of age. two doses of mmr a should be administered at least month apart but preferably longer according to national guidelines. measles vaccine provided before months of age does not induce protection in all and should be repeated after months of age. loss of access to medication or devices, loss of prescriptions, lack of access to health care services leading to prolongation of disruption of treatment degradation of living conditions loss of shelter, shortages of water and regular food supplies and lack of income add to physical and psychological strain interruption of care due to destruction of health infrastructure, disruption of medical supplies and the absence of health care providers who have been killed, injured or are unable to return to work interruption of power supplies or safe water life-threatening consequences, especially for people with end-stage renal failure who require dialysis source: http://www.euro.who.int/en/health-topics/health-determinants/migration-and-health/migrant-health-in-the-european-region/migration-and-health-key-issues# transmission occurs largely in tropical and sub-tropical regions, but outbreaks have occurred in europe around the mediterranean where a. albopictus is present. infected air passengers from endemic areas can arrive in europe during their viremic period, be bitten by local mosquito vectors and transmit the dengue virus locally and trigger an outbreak due to a warming climate. other means of transport are also implicated in the spread of disease-causing pathogens. for example, ballast from cargo ships played a role in moving pathogenic vibrios to new locations that were environmentally and climatically suitable, even in northern latitudes. travel and tourism can also facilitate the importation of pathogens from developed countries with low vaccination coverage, to developing countries with high rates of susceptible individuals due to lack of health care access. , responding to the public health challenges associated with travel and climate change requires robust national surveillance systems, including effective tracking of vector location and disease importation. integrated surveillance of invasive and endemic mosquito species is crucial for effective prevention and control of vector-borne diseases. early detection of outbreaks can be aided by seasonal surveillance in areas where competent vectors are active and in close proximity to airports with a large influx of passengers from endemic areas. for example, the risk of dengue importation can be described by a model that relates air travellers from dengue-affected areas to dengue importation. this approach can delineate in space and time where the risk is the highest, and target seasonal surveillance to high-risk areas, for early case detection and intervention. an important consideration when projecting risk of disease transmission associated with climate change and travel is that the timing of risks may be under-estimated, i.e. transmission may occur sooner than models suggest. for example, the modelled future environmental suitability of a. aegypti in the contiguous usa in the period - projects potential suitability in southern areas of mid-western states, such as illinois, indiana and ohio. however, breeding colonies of a. aegypti were found in ontario in southern canada in , indicating that the risk of transmission of diseases such as dengue fever and zika may occur earlier than the projections suggest. the importation and spread of tropical pathogens in temperate regions due to climate change is another aspect to consider for public health practice. it is a potential threat to the safety of the blood supply and, hence, of blood transfusions, particularly with respect to pathogens for which there are no diagnostic tests. [ ] [ ] [ ] [ ] [ ] expansion of areas with an increased risk of communicable diseases due to climate change also increases the risk of blood bank contamination with communicable pathogens as a higher number of prospective blood donors are exposed to potential infections for a longer period, e.g. if climate change alters the length of the annual mosquito activity season. moreover, the asymptomatic phase of many infections, even if it is relatively short, increases the potential for transmission by transfusion. climate change projections of the probability of infection should therefore be used for preparedness activities. in addition, certain pathogens, such as the west nile virus, can persist in stored blood components and subsequently cause infection through intravenous application. pathogen reduction technology that inactivates pathogens in donated blood is a strategy that could be used for pre-emptive risk reduction. the world is warmer today than in pre-industrial times, and warming is projected to continue. the frequency, duration and intensity of extreme weather events have increased, with implications for migration in particular. global warming is associated with migration from farming communities and from small islands and coastal areas. evidence of climate change impacts on migration in other settings is less clear. the combined impact of travel and climate change can favour the importation, establishment and spread of tropical diseases in temperate regions. these developments require public health action, including targeted and culturally appropriate interventions and novel technologies, to prevent and control emerging health threats associated with the interaction between climate change, migration and travel. it calls for training of health care professionals to provide appropriate health care (prevention, screening, and treatment) for migrants and for risk assessment of diseases among travellers/ tourists. adequate living conditions, screening and vaccination programmes, and medical interventions for migrants can prevent outbreaks of communicable diseases and the spread of resistant pathogens. assessment and interventions also need to encompass chronic conditions, mental health and trauma due to migration. targeted hiv prevention, testing and treatment programmes are also required in countries where migrants are at risk of hiv acquisition after arrival. effective national surveillance systems that include tracking of vector location and disease importation can inform targeted prevention and control interventions. novel approaches including modelling the arrival of air passengers into environmentally and climatically receptive areas, to improve assessment of the risk of importation of pathogens from endemic areas, and assessment of climatic suitability, can inform seasonal surveillance or active case finding. specific measures may be required to ensure blood safety, including pathogen reduction technology, where there is a risk of the importation and spread of tropical pathogens in temperate regions. the authors report no financial interests or connections, direct or indirect or other situations that might raise the question of bias in the work reported or the conclusions, implications or opinions stated-including pertinent commercial or other sources of funding for the individual author(s) or for the associated department(s) or organization(s), personal relationships or direct academic competition. j.c.s. wrote the first draft of the paper. both authors approved the final paper. herein are the authors' own and do not necessarily state or reflect those of ecdc. ecdc is not responsible for the data and information collation and analysis and cannot be held liable for conclusions or opinions drawn. we should like to thank wei-yee leong from the lee kong chian school of medicine, nanyang technological university, singapore for her help with the figures. none declared. an ipcc special report on the impacts of 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population changes on human exposure to the virus vector mosquito aedes aegypti county health unit. aedes aegypti mosquito climate change projections of west nile virus infections in europe: implications for blood safety practices blood supply under threat emerging infectious threats to the blood supply emerging infectious diseases that threaten the blood supply current perspectives in transfusion-transmitted infectious diseases: emerging and re-emerging infections emerging infectious disease agents and their potential threat to transfusion safety we would like to thank the editor and the three anonymous reviewers for detailed and constructive feedback on the manuscript. we also appreciated the insightful comments and suggestions from teymur noori, pontus naucler and senia rosales-klintz at ecdc. the views and opinions expressed key: cord- - if qquj authors: nan title: perspectives on the economics of the environment in the shadow of coronavirus date: - - journal: environ resour econ (dordr) doi: . /s - - - sha: doc_id: cord_uid: if qquj nan the environmental and resource economics special issue "economics of the environment in the shadow of coronavirus" comes at a hugely critical time for environmental economists and policy makers alike. we are in a situation of significant social change, a change that could potentially lay the foundation for mankind's future in the years to come. as part of this special issue, ere is trialling a novel, experimental form of article, drawing together short, focussed pieces from a wide group of authors addressing the plethora of issues which such a fundamental challenge as the coronavirus pandemic generates. these provide critical and reflective perspectives on the environmental, socio-economic and policy paths that may be taken in the near and further future-strategies that could lead mankind either on roads to a much more sustainable development, or along paths that could bring about more instability, inequality and further environmental pressures. this innovative article combines short, policy-relevant and less technical papers that deal with specific aspects and provide clear recommendations for policy makers and suggestions for future research alike. the target audiences are policy makers and companies, but also researchers who want quick yet sufficiently detailed knowledge about particular analyses relating to covid- and issues in environmental economics. we hope that the articles contained within this perspectives collection provide the necessary information for policy makers to take wise decisions for our future, and for researchers the knowledge to help guide policy makers in their decisions. humankind has been very fortunate to have lived through a period of sustained economic growth pretty much since the agricultural revolution, with especially high rates of growth starting from the second half of the twentieth century. this economic progress has allowed us to make unprecedented improvements in consumption, in health, in education and in addressing inequality. many of us have been fortunate enough to have lived without a war for the past years, which is widely believed to be due to the development of international institutions and a deepening of international trade that led to widespread cooperation and, with it, it brought a new era of global stability. at the same time, the rapid increases in humankind's population, from around billion in to . billion in , coupled with an increase in real-world gdp by a factor of roughly during the same period, have led mankind to progressively push closer to the boundaries of planet earth. to provide additional food for the surge in population, agricultural land use has increased by %; to provide goods and services for the surge in demand, the material footprint of our production increased by an estimated factor of ; and to provide energy for our lifestyles, our use of non-renewable and polluting resources (coal, oil, gas) increased by a factor of . this increase in consumption coupled with a similar increase in input use has transformed the face of the planet earth and has given rise to unwanted side effects and new challenges. some of these challenges are well known, such as local and global pollution, problems of waste and certainly climate change. another, often neglected challenge, has been a consistent pressure on biodiversity due to our increase in land use. a mixture of burgeoning population and increasing resource use that carved deeply in nature's pristine areas has led to species conflict manifested not only in the rapid loss of other species, but also in a much ignored yet increasingly visible negative feedback in the form of viral crossovers (smith et al. ). the linkages between economic development, viral crossovers in the form of communicable diseases and environmental issues in particular have, up to now, seen little attention from environmental economists. as we have now seen, it was worryingly neglectful on our part to not consider these feedbacks more seriously. the greater interconnectedness via global trade and international migration, air travel for both tourism and business purposes, as well as the ongoing growth of large city hubs, have made it easy for communicable diseases to transcend local spaces and quickly make their appearance in even remote corners of the world. while the black death and the spanish flu have been among the worst communicable disease outbreaks in recent history, in late a new virus was detected in wuhan, china. identified as a new member of the coronavirus family and subsequently called covid- , within the course of half a year this virus has spread out from the huanan wet market in wuhan across the whole world. even inhabitants from otherwise remote places such as villages in timbuktu, the korubo and yanomami tribes of the amazon, the navajo nation of north america and the arctic inuit have already tested positive for due to initial uncertainty surrounding both the impact of covid- and its spread through society, many policy makers quickly decided to shut down interactions among individuals by restricting local, national and international mobility. these "lockdowns" had pervasive impacts on economic activity across the globe, with significant reductions in production, increases in unemployment, falls in international migration, diminished levels of international trade, significant increases in bankruptcy filings and large ripple effects down supply chains. relative impacts between developed and developing countries are still very much developing. globally, herd immunity is expected to take some time to develop if indeed it ever does. a vaccine that has the potential to be potent and widely available may need at least another or years for development and broad deployment. some countries are already close to a second wave-this pandemic is here to stay for a while. the question is as to how we shall deal with it. while reducing physical contacts to "flatten the curve" of disease and death has been the preferred policy to slow down the spread of the were advocating for. it is at this point where the contributions selected for this special issue provide first thoughts, first answers and first suggestions for policy makers from the cuttingedge research of environmental economists. in particular, the arguments forwarded support a strengthened focus on economic recovery that, first and foremost, should not undermine the green transition, while also, if possible, provide measures to advance the green transition. the articles then discuss the approaches and potential difficulties that policy makers will be faced with when being confronted with the precise means to implement these green recoveries. as a first step, due to unprecedented levels of unemployment in places such as the usa and significant contractions to economic growth in most countries of the world, an important consideration is that the focus of the stimuli packages should be the economic recovery, i.e. to predominantly deal with the direct impact of the lockdowns on economic activity. once the virus is contained and the short-run recoveries are under way, then it is, however, important to quickly integrate longer-term factors into policy making (borghesi and co-authors). here, it is vital that, in contrast to the stimuli in the aftermath of the financial crisis, policy makers also address inequality (koundouri and co-authors). a more specific focus on furthering a green transition should only be placed once a certain level of economic recovery has been achieved. this is especially vital as the disruptions to supply chains can have fundamental and unpredictable consequences, as often even companies themselves are not fully aware of their complete supply chains. cazcarro and co-authors estimate some of the impacts of these trade-related supply chains and, for example, show that the european demand changes due to covid- have, in total, larger impacts on the rest of the world than on europe itself. requirements for successful green stimuli are that these policies are implemented in a clear and transparent manner (rickels and peterson). in this regard, ing and nicolai argue that companies are likely to prefer stimuli packages that are tight to some environmental efforts rather than to new environmental regulations. on the converse, linking stimuli with environmental efforts is more costly for policy makers and likely to be less efficient. several of the articles in this special issue draw particular attention to the fact that the green stimuli are not enough to successfully further a green transition. what is also necessary is to couple this with a price on carbon and a restructuring of the subsidies paying attention to both the green and fossil industry (gawel and lehmann). a stronger social contract with a higher degree of citizen involvement will furthermore help gain public support but also strengthen social norms and thus decentralized internalization of externalities. lopez-feldman and co-authors discuss the policy responses to covid- with a focus on latin america and argue that, to minimize the likely rebound effect, policies need to be much better coordinated. we have seen that international cooperation quickly breaks down when a crisis looms, so that it would make sense to design international institutions with binding laws and penalties in case of non-compliance. what we have seen so far is that covid- has the potential to become a game changer when it comes to combining stimulus packages with the green transition. that this is a sensible strategy derives from the observation that restricting global warming to . °c requires efforts that go beyond what countries were willing to do so far, and that the stimuli provide the needed opportunity. while the articles contained in this special issue already provide many reasons for policy makers to push for green stimuli, they also clearly point out the difficulties associated with implementing these well. some articles in this special issue also show limitations of current policies or research approaches. for example, borghesi and co-authors discuss that during the covid- crisis the market stability reserve helped to stabilize the eu ets price, but imperfectly. it is, therefore, important to consider ways in which these imperfections can be redesigned. on a different topic, laude explains how the covid- crisis has brought to light both advantages and problems with having local, short supply chains for food, and that there is a substantial lack of research directed towards the impact of crises on the agricultural sector. another example is a more cautionary tale and deals with the covid- cases data. here, cohen and co-authors show very clearly that researchers must be careful with simply using these data as there are many problems in the data collection processes, which differ across countries and also time. as a final remark, we would like to observe that this special issue not only comes at a very turbulent time for mankind in general, but it also comes at a special time for environmental economists in particular. the covid- crisis gives the opportunity to invest significant amounts of money towards aiding the green transition, and the widespread public support is there. we need to now be able to advise policy makers on efficient, reasonable and relevant policies that they may implement as part of the green stimuli packages. however, these policies also need to be well structured and grounded in good research. the peer-reviewed articles in this special issue provide suggestions and articles with these features and will thus, hopefully, serve as a first benchmark in this endeavour. in this paper, we use a multisectoral and multiregional model of the world economy to evaluate the short-term effects that the covid- crisis may have on environmental pressures and resource consumption (measured in terms of water, air emissions and materials extraction). specifically, we focus on the relationship between current and forecast changes in demand and mobility patterns in the eu + uk for and its effect on global resources through global supply chains. this integrated analysis could answer urgent research questions: what are the short-term impacts on emissions and resource consumption of the current and predicted declines in final demand? do the impacts differ among environmental pressures? how elastic are environmental responses to demand drops? on the basis of these short-term responses, what is the relationship between economic growth and environmental pressure? do these effects go beyond european countries through global supply chains? in order to evaluate the short-term effects of changes in aggregated sectoral consumption and demand on environmental pressures, and their diffusion through global supply chains, we develop an environmentally extended multiregional input-output model (see miller and blair , or recently hubacek et al. ; guan et al. ), using the exiobase . database (stadler et al. ) . we focus on water consumption (blue and green), mineral extraction and emissions (co eq , sox, nox, nh and co). we rely on the estimates from the eurostat spring forecast (ec a) which estimate for the eu + uk a change of − % in consumer expenditure, . % in government expenditure and − . % in investment for . sectoral changes in private consumption are estimated based on ec ( b) and oecd ( ), assuming different sectoral sensitivities to the covid- crisis. specific details and the complete matching process can be found in online appendix. our results are as follows: figure shows the eu + uk (light blue bars) and global (dark blue bars) change in different environmental pressures associated with the shock to final demand and household mobility in eu + uk, as a consequence of the covid- lockdown. the expected percentage impacts are much larger in the eu + uk, as the simulation of final demand and mobility directly affects these countries, given the high level of intra-eu dependence. however, the european lockdown also impacts foreign resources due to the interlinkages throughout the production chain. in general, the largest decline happens to gas emissions, both in eu + uk and worldwide. the average . % fall in the aggregate of the three cited components of european final demand (households, government expenditure and investment, representing a . % fall in total final demand) that globally represents a . % decline, would involve a . % decline in global water consumption and . % in mineral extraction. the global fall in emissions from the slowdown in economic activity and european mobility restrictions would be around . % (co eq ). comparing the effect in eu + uk with the global impact, the percentage change runs from four times larger in europe, in the case of water, to times greater for co eq emissions. we find a drop of more than % for mineral extraction and emissions in eu + uk. emissions are reduced, and by more than water and minerals. also, even when no direct electricity demand change is assumed, given the reduction in sector activity, indirect energy demand diminishes and so do emissions. specifically, our model indicates that the production and supply of electricity explain approximately % of the greenhouse emissions fall worldwide and % in the eu + uk. in line with the results of le quéré et al. ( ) , our estimates confirm that the reduction of emissions associated with private mobility restrictions would account for % of the total fall in emissions in europe, whereas the reduction linked to air travel reaches . %. worldwide, these drops would be % and %, respectively. the european lockdown affects the iron and steel industries globally, representing % of the global fall in greenhouse emissions. several other resources are less dependent on the most-affected sectors. the supply chain of food clearly depends on the supply of water. it is difficult to estimate the reductions in the horeca sector based on the available data. that sector, as well as many others that indirectly require these natural factors throughout their supply chains, exhibits significant reductions in resource use. our data show that the largest decline in water consumption (both blue and green) is associated with the primary sectors, which, as expected, experience slight negative growth rates around − . %, given the relatively stable and anticyclical nature of food demand. analysing the changes induced by the different components of final demand, the decrease in household consumption and investment drives environmental impacts to a lesser extent. worldwide, the fall in investment mainly affects environmental impacts (except for water consumption). reductions in investment have a larger role in reducing emissions in small countries like croatia and malta, and also in eastern europe, such as in hungary, latvia, and slovenia. in the eu + uk, household consumption is the most significant element of final demand driving the changes in environmental pressures (with the exception of mineral extraction), explaining % of the european greenhouse gas emissions fall. as for minerals, their strong dependency on gross capital formation destinations, such as construction, manufacture of machinery and equipment, computer and related activities, explains that the . % fall in investment worldwide (- . % in eu + uk) triggers a . % drop in their global extraction (− . % in europe). these changes are distinct by country, with implications for different areas of the world. the intra-eu trade is revealed to be highly important, showing that the main changes occur within the eu + uk countries (even if the individual shock of a given country is not so significant). figure displays the percentage falls in co eq , so x , no x and nh emissions by eu + uk country. according to the projected changes, the greatest cuts are expected in co eq emissions. almost a quarter of the estimated reduction in co eq emissions occur in germany (figure si ) . the reductions in co eq emissions in smaller economies, such as ireland, austria, greece, and cyprus exhibit the largest percentage falls. reductions in mediterranean and eastern countries are high, mainly in large countries like france, romania, portugal, and spain. we note the potential reduction in no x emissions as a consequence of the production shutdown in france and germany (as it occurs with nh emissions), but the largest percentage drops occur in smaller economies, like austria, ireland, and greece. france also shows the highest reductions in so x emissions, together with poland and germany, ireland and smaller countries. our model also allows us to evaluate how the reduction in the eu + uk demand modifies the pressures on environmental resources outside europe, by considering transmission effects through global supply chains. the largest percentage reduction in domestic impacts arising from the lockdown and subsequent situations in europe occurs in non-european developed countries, with smaller effects in developing economies (fig. ) . we find considerable indirect declines in mineral extraction (larger than %) and gas emissions (between − . and − . %) in the usa and japan. these are mostly associated with linkages with italy, germany, uk and france. in countries such as china, brazil and india, none of the falls in environmental pressure exceeds . % (with the exception of a % fall in mineral extraction in india). again, we find important declines related to the commercial linkages of developing countries with european powers, such as france, germany and the uk, and with other european countries severely affected by the pandemic (and thus their final demand), such as italy and spain. these results support the statements of baldwin and mauro ( ) on the existence of a "supply-chain contagion" related to the covid- lockdown (in europe in this study). however, the "environmental supply-chain contagion" is modest outside the eu + uk. one of the main implications of our work is that the changes occurring in in the eu + uk are not, in and of themselves, able to sufficiently reduce global environmental pressures. these changes have entailed reductions in domestic activities, which have also affected other eu partners, given the high level of intra-eu trade. although some of those changes do not have strong impacts on domestic environmental pressures, transport restrictions within the eu have notably reduced co eq emissions and, even more positively, reduced other pollution-induced health damage. changes outside europe occur due to spillover effects, being relatively more notable for minerals. however, the demand for goods, which ultimately depends on resource use and pressures external to the eu + uk, has not fallen so clearly, and the lion's share of the pressures has not been reduced as much as final demand. in short, we have shown the importance of intersectoral relationships and multipliers to understand demand changes, which are uncertain along , and which we anastasios xepapadeas on december , the european commission presented the european green deal (egd )-a roadmap for making the eu's economy sustainable by turning climate and environmental challenges into opportunities across all policy areas, and making the transition just and inclusive for all. the central objective of the egd is to attain a climate neutral eu by , which means that the eu will aim to reach net-zero greenhouse gas emissions by that year. the actions required to reach this target include decarbonizing the energy sector, which accounts for more than % of the eu's greenhouse gas emissions; renovating buildings to help reduce energy use which currently accounts for % of the eu's energy consumption; supporting industry so it can innovate and become a global leader in the green economy; and promoting cleaner transport, which constitutes an important source of the eu's emissions. in terms of resources needed, at least € trillion (european commission ) are projected to be necessary over the next decade according to the european commission, with sources including the eu budget, national budgets and the private sector. furthermore, the egd encompasses the so-called just transition mechanism whose objective is to help reduce the negative impacts on coal mines or steel factories associated with decarbonization. the roadmap for the egd includes actions related to climate ambition, clean energy, circular economy, smart mobility, greening the common agricultural policy, preserving and protecting biodiversity, and attaining a toxic-free environment and was planned to commence during the spring of . the covid- pandemic which appeared in europe in early , and the containment measures taken in order to control the pandemic and reduce the transmissibility of the virusthe r -below , have had a profound impact on the economy. in terms of macroeconomics, the covid- shock on the economy can be regarded as a keynesian supply shock in a multi-sector economy which triggers shortages in aggregate demand larger than the shocks themselves (guerrieri et al. ) . policies to deal with immediate impacts of covid- are aimed at a fast recovery from the recession, so that the policies are designed and implemented in a short-run context. the question which this note seeks to explore is whether the policies undertaken as a response to covid- could have long-run implications-positive or negative-in terms of sustainability and the objective of carbon neutrality. arrow et al. ( ) state that economic development is sustained at a given point in time if intergenerational well-being is non-declining at this point in time. intergenerational well-being is non-declining if the comprehensive wealth of the economy is non-declining. comprehensive wealth is the value of the assets of an economy, with the asset base or productive base consisting of reproducible capital, natural capital, human capital and health capital. social capital can also be included in the productive base. thus, the issue of sustainability can be analysed in terms of non-declining comprehensive wealth or productive base. the implications of the covid- shock on sustainability should therefore be examined in the context of its impacts on the productive base of an economy. the impact of covid- on the productive base is realized directly through morbidity and mortality and indirectly through the recession that is induced by policies implemented to contain the pandemic and the positive effects of public spending aimed at recovery. r is the basic reproduction number which is defined as the average number of secondary infections produced when one infected individual is introduced into a host population where everyone is susceptible. in a fully susceptible population, an infection can get started if and only if r > . if r < , a typical infective replaces itself with less than one infective, and the number of infectives tends to zero with the passage of time (e.g. hethcote ) . see, for example, the world economic outlook (imf, ) in which the projection for the percentage change in output in the euro area in is − . % with a rebound to + . % in . it should be noted, however, that in the current world of deep uncertainty, these predictions could be inaccurate. greenstone and nigam ( ) indicate that moderate distancing policies have substantial economic benefits in terms of mortality benefits and avoided hospital intensive care unit costs. the valuation is performed in shadow prices, with the shadow price for an asset being the present value of the contribution to well-being from one additional unit of the given asset. sustainability can also be defined in a similar way in terms of comprehensive investment. thus, the virus does not destroy reproducible capital per se, but it reduces its utilization through containment policies. however, the pandemic could affect capital accumulation if the recession slows down investment in reproducible capital. covid- seems to be beneficial for natural capital, at least in the short run. coal use fell by % at china's six largest power plants between the last quarter of and march , while in europe satellite images showed nitrogen dioxide (no ) emissions fading away over northern italy, with a similar picture in spain and the uk (henriques ) . according to a recent international energy agency ( ) report, global co emissions are expected to decline during to . gtco , which is . % lower than in ( . gtco ) (global carbon project ). this would be the lowest level since , and six times larger than the previous record reduction of . gt in due to the financial crisis. however, the international energy agency ( ) report also indicates that if efforts to contain the virus and restart economies are more successful, the decrease in energy demand could be limited to less than %. since the international energy agency's ( ) april report, data show signs of a recovery in carbon emissions. a very recent reportin june -states that new data show a v-shaped recovery in carbon emissions, with carbon emissions declining from february , reaching a minimum in april and then recovering slowly towards the february levels (domjan ) . regarding human capital, defined in terms of changes in the work force and education level, the pandemic is having a short-term negative effect on work force, but in the long term it might change educational patterns and the geographical structure of the supply of education if extensive on-line teaching is established. health capital is, of course, negatively affected through the value of statistical life. the above discussion suggests that the pandemic has a profound impact on the factors characterizing sustainability. thus, policies to start up the economy after appropriate containment of the pandemic and return to long-term desired growth paths will be beneficial for productive-based sustainability components such as health, human and social capital and reproducible capital through the increase in its utilization. the issue of primary interest is the impact of recovery-related policies on natural capital and climate change. this issue relates to the way in which the egd might be adjusted during the start-up period and whether the current beneficial impact of the virus-induced recession on emissions signals a long-term impact. recent results in climate science have established an approximately proportional relationship between the change in the global average surface temperature relative to the preindustrial period (the temperature anomaly) and cumulative co emissions relative to the same base period (e.g. matthews et al. ). with cumulative emissions since being approximately ± gtco (friedlingtein et al. ) , the expected reduction of approximately . gtco relative to due to the covid-related recession will have negligible effects on the temperature anomaly. furthermore, if the recovery projections in are realized, then it is reasonable to expect that the pre-pandemic situation will reemerge with regard to greenhouse gas emissions, unless the pandemic continues in strong recurring waves which make extended and persistent lockdowns necessary. this situation, however, cannot be regarded as the most likely scenario. if emissions recover in the short or even medium term, the projected time period for crossing the . °c threshold-according to the business-as-usual or alternative emissions paths-will not be affected in a significant way (ipcc ) . in this context, the covid- event is expected to have a negative impact on the global productive base (health, human, social and reproducible capital) and therefore on the global sustainability conditions, while the seemingly beneficial impact on natural capital, and especially climate, will be temporary and a return to pre-pandemic paths is most likely. this means that in the post-pandemic world, risks related to climate change damages, including risks from tipping elements and crossing of climate thresholds, are not expected to change. how does this picture fit with the european response to covid- in the context of the egd? the eu is currently developing and implementing a number of policies and stimulus packages to address the recession. the purpose of this note is not to analyse these measures, but rather to explore induced adjustments to the egd. it has been reported that there will be some reprioritization of edg initiatives as a result of the eu response to the pandemic (involved in europe ). some of the initiatives such as the renewed sustainable financial strategy, which aims to increase private investment in sustainable projects, or the "renovation wave", will remain since they are expected to stimulate economic activity; others such as "offshore renewable energy" or "the biodiversity strategy for " might be delayed, but initiatives such as the new eu strategy on adaptation to climate change and the new eu forest strategy will be delayed to . it is clear that the covid- shock and the need to start up the economy will make substantial policy changes necessary. looking at sustainability in terms of natural capital and the environment, it should be clear that any short-run improvement is transient, while looking at sustainability in terms of climate change, it is most likely that there will be no change in the long-term trends. what is important is that, after the shock, the start-up will be based on environmentally friendly policies. thus, maintaining initiatives such as the renovation wave, or promoting the pillar of cleaner transport, is important. on the other hand, delaying initiatives like the strategy on adaptation to climate change may need to be reconsidered. this is because the rationale behind delaying such strategies seems to be that recovery from the recession is expected to be rapid and therefore the delays will be of short duration and no significant time will be lost. however, recovery might be impeded or delayed by issues such as new, possibly weaker waves of the pandemic or more technical issues such as fiscal multipliers. fiscal multipliers during a recession when shocks are concentrated in certain sectors are not expected to be operational, with the multiplier for government spending being around one and the multiplier for transfers likely less than one. these factors might result in the delays-initially projected to be of short duration-extending for a much longer period. the important point here is that the covid- shock will have a negligible effect on the evolution of the temperature anomaly. thus, if adaptation activities and decarbonization do not proceed rapidly, the risks of a climate shock will not be sufficiently mitigated. the need for strong action now is exemplified by the fact that the emissions gap in between current policies and the emissions necessary to keep the temperature anomaly below . °c in is approximately - gtco (unep ). the covid impact on emissions in is expected to reduce the gap by approximately gtco , which falls very far short of the gap that needs to be closed. the important aspect of a climate shock is that in addition to the negative impacts on the productive base of the economy-human, health and social capital-it will have a much more serious negative impact on the reproducible capital relative to the pandemic. this is because the climate shock will not just reduce the utilization of this type of capital, it will destroy part of the capital stock, since it will affect infrastructure, equipment, buildings and so on. recovery in such a case will clearly be slower and more difficult. this creates a serious argument against delaying adaptation programmes which could provide substantial benefits in the presence of climate shocks. typical adaptation projects (e.g. early warning systems, water resource and flood-risk management, sustainable agriculture, strengthening the resilience of existing infrastructure making new infrastructure resilient) are characterized by high benefit-cost ratios (fankhauser ; gca ) . under the resource constraints imposed by pandemic containment policies and the deep structural uncertainty characterizing the situation, the prioritization of these adaptation policies could necessitate the use of max-min expected utility criteria in decision making. as second-round benefits, adaptation programmes which involve investment could stimulate the economy and provide new jobs. to summarize, three important points should be taken into account: (i) that the indications thus far suggest that the benefits associated with the reduction of greenhouse gas emissions because of the covid- are transient, which means that the long-term trends associated with climate change are not expected to change, and therefore, mitigation and adaptation strategies should be strongly pursued in the post-covid- era ; (ii) that there is a need for an ambitious and comprehensive european economic recovery plan from the covid- crisis (european economic and social committee b); (iii) that climate-change-related investments, in particular in adaptation, are expected to deliver significant economic benefits. based on these points, it becomes clear that a green recovery plan with resources directed towards achieving the combined objective of both providing the necessary economic stimuli for recovery and also promoting the transition to a low-carbon economy and adaptation to climate change along with investment in natural capital and increase in comprehensive savings could be a feasible and efficient plan. the egd is an important strategy for securing the sustained development of the eu and protecting climate as a global public good. at the same time, it is clear that addressing the pandemic requires action now in the form of new policies and changes in priorities. however, although covid- is of necessity in the spotlight at present as a major global threat, it should not displace action aimed at an equal or greater global threatthat of climate change-under the misconception that the temporary drop in emissions during the pandemic allows us to delay climate change action now. a green recovery plan could realistically provide the double dividend of helping the eu economies to recover from the covid- crisis and, at the same time, promote the attainment of a climate-neutral eu. according to the gca ( ) study, adaptation investment of $ . trillion in the areas mentioned will provide total net benefits of $ . trillion by , with benefit-cost ratios ranging between and . see, for example, the recent eu european economic and social committee ( a) opinion about the transition to a low-carbon eu and the financing of adaptation to climate change. involved in europe ( ), leaked: full list of delayed european green deal initiatives-euractiv.com. available at https ://europ e.vivia nedeb eaufo rt.fr/leake d-full-list-ofdelay ed-europ ean-green -deal-initi ative s-eurac tiv-com/. ipcc ( ), global warming of . °c. an ipcc special report on the impacts of global warming of . °c above pre-industrial levels and related global greenhouse gas emission pathways, in the context of strengthening the global response to the threat of climate change, sustainable development, and efforts to eradicate poverty. masson-delmotte, v., zhai, p. rebound effect studies have been generally focused on energy use (sorrell ) , although some studies for other natural resources have recently emerged (freire-gonzález and font vivanco ). rebound effect occurs when the use of resources is not reduced as expected after a resource efficiency policy or a specific behaviour. empirical rebound studies aim at capturing the secondary effects of policies and behaviours in order to obtain more adjusted assessments of policies and actions. it is well known in the rebound literature that, counterintuitively, resource efficiency may not reduce the use of these resources, but the contrary. this extreme case is known as backfire, khazzoom-brookes postulate, or jevons' paradox. rebound effects are not usually observed by policy-makers, as it requires different perspectives and approaches coming from social, behavioural and environmental sciences. environmental and social sciences show us that human-environment systems are deeply interconnected. this way of thinking has, however, still not fully permeated in mainstream policy decision circles, which are largely rooted in old intellectual paradigms and other short-term interests. the pandemic has caused many abrupt changes in production and consumption, transport patterns, working conditions, social interaction and many other aspects. most of these changes have been triggered by the policies implemented to contain the pandemic. overall, they have translated into improvements in most environmental indicators, such as carbon emissions, air quality and biodiversity loss (saadat et al. ) . while some authors claim that such changes will not have a lasting impact when the epidemic subsides (mccloskey and heymann ), others argue that aspects related to urban planning, micro-mobility, sharing economy, public transportation, teleworking, tourism, etc., may change for good (honey-rosés et al. ). an important question is, thus, whether covid- will reduce environmental impacts in the future, when economic activity returns to "normality" (in terms of pre-covid conditions). rebound literature shows the importance of considering behavioural and systemic responses to answer this question. beyond other considerations, the pandemic has accelerated some already observed trends, like the pace of implementation and use of digital technologies. one of the most remarkable changes are those related to the impulse of information and communications technologies (ict), due to imposed social distancing rules. there already was a tendency towards an increased use of ict, but its use has been dramatically accelerated due to the pandemic. this acceleration can be observed in many areas, such as teleworking, e-commerce, remote social relationships, virtual sightseeing, surveillance technologies, and other online areas and events (cultural, academic, leisure, educational, etc.) . for instance, in many countries, nonessential workers have been legally obliged to be confined during the pandemic to stop the contagion of the virus, thus promoting telework. despite the potential advantages of teleworking in increasing labour productivity in many industries (harker martin and mcdonnell ) , rigidities in corporate culture and other legal and cultural restraints were hindering and adjourning its consolidation. the use of ict is thought to be environmentally beneficial, largely due to decreased transport, but this premise has been challenged by rebound effect studies. gossart ( ) shows that existing evidence suggests that ict are subject to important rebound effects, mainly because it is a general-purpose technology, and so prone to backfire (sorrell ) . takahashi et al. ( ) calculated the rebound effect of ict services in a case study on videoconferences and found that rebound can reduce up to % of carbon savings. joyce et al. ( ) recently found for sweden strong environmental rebound effects associated with ict use, in most cases far above % (more resources use than before). this backfire effect is strongest for energy use and total material footprint, both close to %. another change may take place in land use and the housing sector, as initial evidence suggests that attributes such as floor space and outdoor space will have elevated importance (mikolai et al. ) . the potential re-distribution of time and expenditures towards resource-intensive sectors, such as construction, water and energy services, will likely cause material, water and energy rebounds. however, the expansion of teleworking can, at the same time, reallocate space and incomes in office rental market. city centres will not need to concentrate workspaces, changing mobility patterns and urban structures in the long term (elldér ). public transport may also be negatively impacted in the short and mid-term, leading to increased private transport (honey-rosés et al. ), another resource-intensive activity. other structural changes may also take place, such as changes in sufficiency measures and broader productivity, leading to macro-economic rebound effects (lemoine ). the pandemic may increase the social acceptance of sufficiency measures such as working less time, spending more time with family and friends, or connecting with nature. these measures have long been proposed to reduce consumption and associated environmental impacts (hayden and shandra ). these measures have, however, been associated with macro-economic price rebound effects as the decreased demand for some products can lower their price and induce additional demand (sorrell et al. ) . moreover, the postpandemic society may likely be a more productive one in labour and capital terms. for example, teleworking (harker martin and macdonnell ) and increased spending in research and development have been associated with productivity growth, which boosts economic growth and resource use. the covid- pandemic will likely cause a range of changes in society, but their permanence and effect on the environment are unclear, especially if we contemplate the secondary effects of behaviour, measures and policies. a key question is whether they will acquire a certain level of permanence, even modifying the mindsets of agents. given the high uncertainty around this aspect, its real dimension could only be assessed ex-post. however, due to confinements, the pandemic has greatly accelerated the expansion and use of general-purpose technologies, like ict. as this has been a long-observed trend, before the irruption of the virus, they have probably come to stay to a large degree. the pandemic offers a great potential to improving (and consolidating) environmental conditions. but beyond what conventional environmental indicators show, additional measures would be needed to counteract hidden rebound effects and, therefore, take full advantage of potential improvements. recent literature shows that different economic instruments like environmental taxation, resource pricing or setting limits to resource use, can be effective for this purpose. this is particularly necessary in this case, given the high risk of backfire due to the high expansion of general-purpose technologies observed. the global financial and commodity markets are facing economic distortions caused by the coronavirus (covid- ) pandemic (fernandes ; irwin ). covid- acts as a negative shock to overall demand of goods and services, resulting in aggravated shortrun volatility in prices (albulescu ). among these products, oil has been dramatically affected due to community lockdown regulations, shutdown of car factories, decline in energy use and increase in unemployment (reed a; the associated press ). however, a decrease in oil consumption may lead to reductions in carbon dioxide emissions (peña-lévano et al. ). data recorded by epa ( ) during march-april show an improvement in overall air quality, especially in high-density populated cities (regan ). air pollution is considered by many scientists as a negative contributor in the coronavirus situation by worsening the susceptibility of infection. a decline in emissions somehow may help prevent mortality temporarily, especially among more vulnerable individuals with underlying health conditions, such as heart and respiratory diseases (conticini et al. ; dutheil et al. ; mooney ; ogen ). thus, in this short article, we discuss the interaction between fossil fuels, air pollution and health risk under the coronavirus pandemic and the lockdown regulations during the period of march-may . the oil market has been dramatically affected by several exogenous factors during the pandemic period: ( ) "shelter in place" mandates aimed at curbing the spread of the coronavirus have decreased people's social mobility and transportation activities. households leave their houses just for short travels for some essential errands, such as shopping groceries and/ or medicine. only essential personnel are required to be physically present at their jobs, whereas the majority are working from home (reed a); ( ) commercial flight demand plummeted globally (reuters ). in the usa, the passenger volume dropped % from a year ago (compared to april ) as estimated by airlines for america, with an overall reduced accommodation of passengers per carrier (rappeport and chokshi ); ( ) tourism activity slumped as many governments banned international travel, heightened border and immigration controls and barred the entrance of foreign visitors to decrease risk of infection. these policies further decreased the demand for taxis and cruise services. for regions such as the caribbean islands (where tourism is a primary economic staple good), this also represents a significant decline in their gross domestic products (semple ); ( ) unemployment has pervaded among the national economies (mazzei and tavernise ). more than million people have filed for unemployment in the usa in a -week period (u.s. department of labor ). this condition is mirrored in other countries. worsening unemployment conditions have substantially reduced consumers' purchasing power. among its consequences is an overall decline in car sales (reuters ; the associated press ); ( ) many industries, restaurants and buildings have been shut down during the lockdown. these closures reduced energy consumption, especially petroleum use, which is a large energy input in the usa and other countries (eia ). meanwhile, global oil supply responded slowly to the decline in demand as refineries cannot abruptly halt production (caldara et al. ; peña-lévano ). the russia-saudi arabia oil price war in march worsened the situation by oversupplying the market and consequently dropping oil prices (irwin ; reed a). in april, the major petroleum exporting countries agreed to decrease the world oil output by % during may-july, which is equivalent to . million barrels a day (krauss ; reed b). two recent studies published in geophysical research letters validate the realized reduction in nitrogen dioxide (no ) pollution in several regions. bauwens et al. ( ) compared no levels in the atmosphere recorded in january to april and for the same period in . notably, no , which is produced by emissions from vehicles and industrial operations, can cause serious lung ailments. their estimates indicate a significant reduction of % in china and - % drop in the usa and western europe. shi and brasseur ( ) estimate a % reduction in no pollution in northern china in january and february . in the same period, they also found a % reduction in particulate matter pollution (particles smaller than . µm). while such cleaner air conditions may persist only temporarily, these trends indicate that the desired environmental gains are feasible and realizable if stringent emission regulations, perhaps mirroring to some extent the pandemic's enforced limits on social mobility and industrial activities, are enforced in the future. several studies relate ambient air quality to mortality and morbidity conditions caused by covid- . this contention echoes an earlier correlation applied to the sars virus outbreak in china in the early s. researchers from ucla's school of public health analysed air pollution levels and sars fatality rates among chinese residents. their results indicate that sars patients' probability of dying would be doubled among residents in areas with high air pollution indexes (cui et al. ) . as applied to the current pandemic, the center for disease control and prevention (cdc) explains that covid- causes a respiratory illness with a heightened risk among people who are years and older as well as those with certain underlying health conditions. latest cdc statistics on the pandemic's severity and fatality indicate that persons with heart ailment, diabetes and chronic respiratory diseases could be at a higher risk of being severely infected by the virus (vogel ). wu et al. ( ) further clarify that pre-existing health conditions identified as relatively more susceptible to contracting covid- are similar to those normally affected by air pollution in the usa. their study estimates that an increase of g m − in long-term exposure to particular matter (pm . ) increases the coronavirus mortality rate by %. isaifan ( ) presents corroborating evidence indicating that % of covid- -related deaths were cases with pre-existing illnesses, with majority of the victims over years of age. conticini et al. ( ) , however, warn that even young and healthy individuals could also be at risk as prolonged exposure to dangerous air pollutants causing chronic respiratory issues could be an additional co-factor that helps increase their vulnerability to being infected by the virus. ogen ( ) establishes that exposure to no may be an important instigator of covid- fatalities according to his research involving four european union countries. these findings are supported by the findings of a study published by the italian society of environmental medicine (setti et al. ) on virus infections in northern italy associated with air pollutants tagged as carriers and boosters. poor air quality has been cited as an aggravating factor in virus transmission. using data from italian province capitals, coccia ( ) notes an accelerated transmission dynamics of covid- leading to his conclusion that the spread of this virus can be considered more as following an "air pollution-to-human transmission" mechanism instead of an interpersonal transmission mode. moreover, fattorini and regoli ( ) analyse long-term air quality data in northern italy and suggest that chronic exposure to a contaminated atmosphere may have created conducive conditions for the spread of the virus. several studies relate the time frame of exposure to toxic air pollutants to mortality and morbidity conditions. lim et al. ( ) establish a significant association between longterm ozone (o ) exposure and elevated mortality risk of certain respiratory diseases. given limited data on the current pandemic, hoang and jones ( ) present emerging evidence on the severity of covid- infection attributed to persistent air pollution conditions, thus suggesting that longer exposure to a polluted atmosphere could aggravate virus infection. zhu et al. ( ) , however, provide concrete evidence suggesting that even short-term exposure to air pollution could increase probability of virus infection. their study analysed daily confirmed cases in cities in china recorded from january to february and found significant relationships between the levels of certain air pollutants and the number of newly identified covid- -infected cases. specifically, their results indicate that a -μg/m increase in the air pollutants' levels was associated with about . % to . % increases in daily new covid- cases. conversely, the health benefits of cleaner air resulting from reduced emissions from fossil fuel during the pandemic's lockdown period deserve attention. several studies recognize that improved air quality during the pandemic temporarily mitigated health risks associated with respiratory illnesses. cole et al. ( ) employed a two-step analytical approach using machine learning techniques and the augmented synthetic control method to estimate possible reductions in death rates in certain regions in china and for the whole country that may be attributed to actual reductions in no concentrations during the lockdown period. isaifan ( ) analyse air quality conditions prior to and during the lockdown period. his results indicate that lives may have been saved due to diminished ambient pollution levels, although eluding possible virus infection still is not necessarily guaranteed. this contention is corroborated by another china-based study conducted by dutheil et al. ( ) . these studies' assertions imply that air quality improvements realized even in such a shorter period of time (spanning less than half of a year) already could have some health benefit potentials, especially in relation to respiratory ailments. the health benefits of the current pandemic's notable environmental gain in air quality, however, will be optimized only if such favourable conditions are sustained over the longer term. the recent lockdown has been short-lived as some communities nowadays have started to revert to normal social routines and regular users of fossil fuels among industries have resumed operations. a brief respite from a usual contaminated atmosphere does not ensure an effective permanent eradication of chronic health conditions. the pandemic experience, however, demonstrates that better health is maintained and ensured not only through medical remedies and but also through more favourable environmental conditions, if sustained over a much longer period. current restrictions on social mobility and economic flexibility under covid- pandemic conditions have actually produced important economic and environmental repercussions that are interestingly contrasting. a general economic slowdown overtly reflected in, among others, reduced consumer demand, spiralling unemployment figures and significant drop in oil consumption causes heightened fears of an imminent economic recession. however, in spite of all the negativity surrounding the pandemic, its environmental consequence of improved air quality is a highly positive note. interestingly, the global community has been trying to accomplish such feat of attaining better air quality over many years of discussions, policy making and policing each other. unexpectedly, it took a serious pandemic to realize such feat. this article traces the interplay of reduced oil consumption with economic issues as well as environmental consequences under pandemic conditions. the more imperative issues now lie on the severity of a looming recession and the global economy's resiliency in transcending the difficult challenges it may bring. should that happen, will the economic cost burdens be outweighed by the realized environmental gains? experts may be quick to assert that improved environmental conditions actually may be short-lived as expected resurgence of resumed economic activities may only quickly bring back pre-covid air conditions. however, proponents of a cleaner world can always draw some inspiration from recent successes in air quality control, especially with the assurance that cleaner air is not necessarily a lofty goal. the challenge in the future lies in achieving such environmental benefit without the need to sacrifice the economic health of the global community. on february , , the brazilian ministry of health confirmed that a -year-old man was positive for sars-cov- : covid- had arrived to latin america. as of july , there have been . million confirmed cases in latin america, compared to . million in the european union and . million in the usa (jh-csse ). furthermore, so far there have been more than , deaths in the region and the trends show that the first wave of the pandemic is far from over (ibid.). there is of course no suitable time for a pandemic to arrive, but these are especially complicated times for latin america. the region is in the midst of a difficult economic situation accompanied by rising social discontent (eclac ; oecd ). moreover, it is characterized by high rates of informality, health systems with limited and unequal capacity, and most of the countries have high levels of debt (oecd ). under these circumstances, covid- is having major short-run socio-economic effects with possible serious long-run consequences, including several potential implications for the environment and the management of natural resources. restrictions of free movement and circulation within and across urban areas of latin america have reduced economic activity as well as the use of motorized vehicles. as a result, many latin american megacities have experienced a short-run decrease in air pollution. concentrations of no have decreased considerably in cities all over the region compared to the levels observed prior to the lockdown measures (iadb ). levels of pm , pm . and co have decreased in bogota, buenos aires and quito (bogota's district secretary of environment, personal communication, june ; roa ; rocha ). nevertheless, the pandemia has not had the same effect on air quality in all the major cities in the region. in mexico city, the reductions in so , pm . and pm concentrations have been modest, and there has been no reduction in ozone. in rio de janeiro, ozone concentrations have increased (dantas et al. ) . furthermore, as the virus and its negative consequences spread across rural areas and make its way through the southernmost part of the region, outdoor and indoor pollution might actually increase. in mexico, as well as in other countries in the region, the use of firewood is likely to rise as rural households try to deal with income reductions (masera et al. ). meanwhile, as winter hits central and southern chile, urban households might increase their use of firewood for heating given that, due to the lockdowns, they have to spend more time inside dwellings (encinas et al. ) . this rise in air pollution could arguably increase the risks associated with covid- . it is too soon to do a formal evaluation of the effects of the pandemic on deforestation and land use change in the region. nevertheless, the available information suggests that covid- is likely to have negative effects on forest cover across the region. early deforestation warnings from peru show that, although deforestation decreased between march and april , since then it has increased surpassing the levels observed during the same period in . according to data from the brazilian national institute for space research (inpe), the first quarter of already evidenced a rise of % in deforested hectares compared to last year's figures. the figures for april reinforce this pattern, with a % increase with respect to april (manzano ). from january to april , deforestation alerts in indigenous territory increased % when compared to the same period of the previous year (greenpeace brasil ). although at this point it cannot be claimed that the pandemic caused the observed increase in deforestation, it certainly does not seem to have provided incentives to halt it. in colombia, contrary to other countries in the amazon region, the trend in showed a reduction in deforestation compared to . however, started with an increasing tendency and the quarantine seems to have worsened the situation (fcds ). the absence of environmental monitoring during the pandemic seems to have encouraged illegal armed groups and regional mafias to take advantage of the situation, exacerbating deforestation with the possible intensification of illegal activities from which these actors derive income, such as illegal mining, land grabbing and illicit crops (bbc ). according to the pan american health organization, in may there were already , covid- -confirmed cases in the amazon basin (martín ). the impact of the pandemic in forest-based indigenous communities is an important source of concern. the spread of the virus in these communities could imply a tragedy that, in addition to the human losses, could affect the traditional knowledge, having negative impacts on the governance of natural resources in the region. this could lead to even more deforestation processes in the future. covid- has caused a disruption in the national and international trade of nature-based goods and services. tourism has come to a halt, affecting the economy of almost all of the countries in the region (mooney and zegarra ). in countries like costa rica, where the touristic industry is intertwined with nature, the shock to the sector could have negative effects for biodiversity and forests. without income from tourism, and given that as a slow recovery process is anticipated, the incentives to protect forests are expected to decrease in the short and medium run. fishing and aquaculture are other industries that have been negatively affected. information for the case of the chilean salmon aquaculture industry suggests that there has been a reduction in demand from international markets (chávez et al. ) . the effect of the shock is being transmitted through the value chain, affecting processing plants and farming facilities. the economic crisis can end up having long-run negative consequences for the environment if, as a result, regulations and environmental policies are relaxed or if institutions are weakened. although at this point there is no evidence of any country in the region purposefully relaxing environmental regulations to promote growth, it is certainly a possibility. what has been observed is that, in order to fund measures to reduce the economic and social impacts of the pandemic, some countries have decided to reallocate funds across the public administration. ecuador, for example, announced cuts affecting the ministry responsible for enforcing environmental regulations (bbc news ). something similar is happening in mexico, where the plan announced by the president is to reduce the operational budget of almost all government entities by % (d.o.f. ). even if countries have a relatively strong environmental legal framework, without a budget to monitor and enforce the regulations, this framework is worthless. countries in the region will very likely incur fiscal deficits and increase their debts in order to fight the crisis. it remains to be seen how the service of the increased debt will impact economic growth and the environment. in the meantime, it seems that the short-run legitimate demands to recover employment levels and improve the health systems might very well push aside the necessary investments to successfully tackle climate change and biodiversity loss. if this indeed happens, it could be the most serious effect of the covid- pandemic in both environmental and social terms. the economic projections suggest that the region will experience a crisis whose magnitude has no precedent in modern history (eclac ). in order to overcome this apparently insurmountable challenge, latin american countries will need well-designed policies that should reconcile economic objectives with social and environmental goals. the social unrest manifested recently in the social mobilizations in the region, should make clear that the apparent trade-off between economic, social and environmental objectives is the result of a false dichotomy between shortand long-run objectives. if environmental objectives are put aside, as has so often happened with social objectives, the economy might recover in the short run but at a very high price. the lockdown measures seem to be having a temporal positive effect on reducing urban pollution in some latin american cities. the challenge now is how to intervene to prevent a return to the same or even higher pre-quarantine emission levels. this is an opportunity to rethink the urban environmental policies while trying to recover from an unprecedented social crisis. at the same time, the observed increase in deforestation reopens political and academic debates about the role of national parks, indigenous reserves and other protection categories in a context of deteriorated livelihoods, illegal economies and a lack of state presence. latin american countries could see this moment as an opportunity to improve regional cooperation in order to design and implement coordinated policy responses not only to the economic crisis but also to the challenges of mitigation and adaptation to climate change. furthermore, countries should coordinate efforts to increase monitoring and presence in the region to effectively reduce deforestation. we have presented an account of some of the most evident environmental effects that the covid- pandemic is having in latin america at this point. considering that we are in the midst of the health crisis and in the beginning of an economic one, it is natural to expect that the trends that we see now will change in the near future and that other environmental impacts will become evident. research that contributes to a better understanding of the environmental impacts and the effectiveness of different policy responses to the pandemic in latin america will be invaluable. there are many potential paths for future research; here, we mention just a few. the consequences of the interactions between poor air quality and covid- on human health are clearly worth studying. this is particularly relevant for the latin american context, characterized by health systems with very limited capacity and high numbers of population without formal employment. results from studies in this area could help us provide better guides to set environmental quality goals, as well as to implement policy interventions that can reduce pollution in the region's context of income inequality and spatial segregation. the short-run environmental effects of covid- show early warnings of an increase in the pressure on forest and other ecosystems across latin america. understanding the impacts of the pandemic on terrestrial and marine ecosystems, as well as on livelihood opportunities for local communities, has the potential to contribute to the design of policies which can improve management and conservation. the pandemic is opening new research questions regarding the impacts of global shocks on natural resource-based industries that participate in international markets. furthermore, the paths that different countries take to get out of the economic crisis might have profound impacts on international trade. if, for example, the world transitions to more reliance upon local production, or if emissions-related tariffs are imposed, large exporters of commodities in the region will be highly affected. the impacts that these potential trade changes could have on the environment are unknown. furthermore, if developed countries implement recovery plans that include provisions to reduce emissions in significant ways, as has been discussed in the european union, will latin american countries be able to respond in the same way? in any case, latin american countries are highly vulnerable to the effects of climate change and some of these effects (e.g. migration) could result in future health crises. a better understanding of the ways in which individuals might adapt to a changing climate, as well as of the barriers that they face to adopt adaptation measures, will be a valuable tool for the design of adaptation policies that prevent future health crises in the region and elsewhere. the distributional and gender-differentiated impacts of the pandemic, and the related environmental policy responses, is another area that deserves attention, especially because early evidence shows that the more vulnerable segments of the population in the region are the ones that are being hit hardest. finally, as has been recently pointed out by , the experience of the pandemic might lead to changes in behaviour and personal choices. it remains to be seen if this is in fact the case, and if so, how are these changes in behaviour modulated by the local context. an even more important issue to consider is what would these changes imply for the design of behaviour-based policy instruments aiming to change consumption and production patterns, as well as transport and land use decisions in latin america. bbc ( ) the place where nature isn´t healing. https ://www.bbc.com/reel/video / p bd wfc/the-forgo tten-peopl e-of-the-globa l-pande mic accessed june bbc news ( ) coronavirus en ecuador: las multitudinarias protestas por las drásticas medidas económicas y recortes de lenín moreno. https ://www.bbc.com/mundo / notic ias-ameri ca-latin a- accessed june chávez c, salazar c, simon j ( ) efectos socioeconómicos y respuestas públicoprivadas de corto plazo ante la crisis del covid- en el sector salmonicultor, una fotografía de la experiencia internacional. interdisciplinary center for aquaculture research-incar, fondap-anid, center, chile. https ://www.incar .cl/wp-conte nt/ uploa ds/ / /pb .pdf accessed june dantas g, siciliano b, frança b. b, da silva c. m, arbilla g ( ) the impact of covid- partial lockdown on the air quality of the city of rio de janeiro, brazil. sci total environ . https ://doi.org/ . /j.scito tenv. . . d.o.f. ( ) decreto por el que se establecen las medidas de austeridad que deberán observar las dependencias y entidades de la administración pública federal bajo los criterios que en el mismo se indican. de abril de . presidencia de la república de méxico. eclac ( ) report on the economic impact of coronavirus disease (covid- ) on latin america and the caribbean: study prepared by the eclac, santiago, p encinas f, truffello r, urquiza a, valdés m ( ) covid- , pobreza energética y contaminación: redefiniendo la vulnerabilidad en el centro-sur de chile. centro de investigación e información periodística. https ://ciper chile .cl/ / / /covid - pobre za-energ etica -y-conta minac ion-redefi nien do-la-vulne rabil idad-en-el-centr o-surde-chile /. accessed june fcds. ( ). cifras deforestación en el bioma amazónico, enero-abril .https :// fcds.org.co/site/wp-conte nt/uploa ds/ / /defor estac ion_ .pdf accessed june greenpeace brasil ( ). desmatamento em terras indígenas aumenta % em . https ://www.green peace .org/brasi l/press /desma tamen to-em-terra s-indig enas-aumen ta- -duran te-a-pande mia-da-covid - / accessed june helm d ( ) . contributing to a global effort. oecd. roa s ( ) medidas para enfrentar al covid- mejoran calidad del aire en dos ciudades ecuatorianas. mongabay-latam.https ://es.monga bay.com/ / /menor -conta minac ion-del-aire-por-coron aviru s-en-quito -y-cuenc a-ecuad or/ accessed jun rocha l ( ) por la cuarentena, la contaminación del aire bajó a la mitad en la ciudad de buenos aires. infobae. https ://www.infob ae.com/socie dad/ / / /por-lacuare ntena -la-conta minac ion-del-aire-bajo-a-la-mitad -en-la-ciuda d-de-bueno s-aires / accessed june resource exporters are now facing new urgent economic policy challenges due to covid- . these challenges are aggravated due to their dependence on finite commodities with volatile prices and demand. in response to the pandemic, resource exporters (such as botswana and saudi arabia) announced cuts in expenditures along with large fiscal (tax relief) and consumption-focused macroeconomic stimulus packages. critically, the novel issues raised by the coronavirus pandemic bring new trade-offs of energy policy between shortterm gains and long-term sustainability, creating an urgent need for critical, quantitative, policy-focused research in the resource exporters-energy policy nexus. a few novel issues emerged in resource exporters during the pandemic that give rise to short-term economic challenges. first, price shocks of unprecedented magnitude for commodities (deutsche bank ; world bank ), from coffee (hernandez et al. ) to hydrocarbons (iea b), causing a large drop in energy investment (iea a). for oil, prices dropped initially due to travel bans and economic activities hiatus, and further with oil price wars following the collapse of opec + agreement. by april , with oversupply, the rise of stockpiles, and the saturation of available oil storage, oil prices reached the lowest level in more than years, and west texas intermediate (wti) reached negative levels for the first time. prices have subsequently partially recovered but are expected to remain low with continuous fears of new covid- waves and uncertain demand. second, unprecedented economic contraction, because the pandemic-triggered decline in economic activity was significantly exacerbated by declines in export revenue. third, unprecedented fiscal pressure, resulting from costs of fiscal and economic stimulus packages plus a simultaneous rise in domestic expenses (especially healthcare and unemployment benefits) and sharp declines in resource export revenue. the effects have been so colossal that states like kuwait are considering halting legally-mandated contributions to the future generations sovereign wealth fund (swf) to ease fiscal pressures (al-zo'bi ). fourth, record and continuous withdrawals from swfs to fund post-pandemic recovery along with reallocation of funds and increased government debt (examples in arabian business ; holter and bloomberg ). even states with the largest swfs such as norway and kuwait have been affected, with the latter expecting depletion of its fiscal stabilization swf (al-zo'bi ). fifth, in oil-exporting gulf states, the unusual stay of millions of citizens and guest workers in the upcoming scorching summer in lieu of usual tourism travel or home-country visits, pressuring existing energy capacity. critically, in oil exporters, these novel issues and policy responses to them further expose existing economic fragilities and challenges, threatening long-term economic and environmental sustainability. for distorted oil economies in urgent need for economic diversification, said diversification and reverse dutch disease are impeded by their existing high distortions in labour, fiscal, industrial and energy markets (shehabi ) . oil exporters suffer from economic inefficiencies resulting from pervasive oligopolies (shehabi ) and market failure in long-run contracts for exploration and development of natural resources (ruta and venables ). fossil fuels are the primary source of energy in most oil exporters. in developing oil exporters, the dominant political economy undergirding policy making is a welfare rentier state, in which maintaining a political equilibrium is central. balancing spending-saving decisions for sustainable resource rents management depends on the sustainability of windfall expectations (gelb and grassmann ). reforming energy policy-which drives fiscal, industrial and environmental policies-is critical for these states' long-term development. yet the novel issues create new trade-offs of post-pandemic energy policy: achieving short-term gains will be at the expense of longterm gains in resource economies. thus, it is critical for fast policy-focused research to address the resource exporters-energy policy nexus, especially in the following two areas. although investments in renewables have been more resilient than in fossil fuels, and while we have seen an increased share of global energy spending on clean energy technologies in (iea a), the reality in hydrocarbon-exporting developing states is different. global oil market supply and demand dynamics have raised their opportunity cost for transitioning away from fossil fuels and for investing in green technology. accordingly, post-pandemic economic stimuli might achieve short-term recovery but harm long-term energy transition. first, the rise in opportunity costs of energy transition will facilitate the reallocation of funds away from renewables projects towards post-pandemic economic stimuli (similar to "green tape" cuts in australia and canada). second, recovering lost investments in energy transition projects is unlikely because the resource rents that fund them are likely to continue to be low in the future, given low resource prices and demand pressures of climate change mitigation. third, continuous withdrawals of diminishing swfs will entail limited resources for future energy transition projects. this is especially so as recovering swfs' withdrawals is unlikely in the light of expected low resource export revenue and the collapse in financial and commodity markets accompanying the unprecedented global recession. finally, economic stimuli expand consumption and welfare redistributive measures, which increase greenhouse gas (ghg) emissions ) and exacerbate existing distortions that have been shown to prevent economic and energy diversification (shehabi ) . new research should investigate the design and implementation of new economic solutions that have at their forefront long-term energy transition goals along with short-term economic recovery. three policy solutions are suggested at the consumer, the energy industry and economy-wide levels. a policy solution to target consumers is technological advancements (both private and public funded) in energy efficiency coupled with economic incentives to rationalize energy consumption. this policy combination can achieve energy transition goals despite expected delays in renewables and without requiring large multiyear investments. it is especially important in gulf oil states where skyrocketing cooling and desalination needs in the summer will be met using fossil fuels (renewables contribute less than % of power generation). a policy suggestion at the sectoral level is investing in clean energy technologies to decarbonize the energy sector itself, namely through carbon capture and storage as well as hydrogen. beyond short-term economic and environmental benefits, these investments can keep oil exporters relevant in a future with a diminishing role of hydrocarbons and collapsing commodity prices. the final policy suggestion is microeconomic and energy policy reform that can moderate effects of export price declines on the economy without the need for additional cuts in renewables investments or further withdrawals from swfs. examples include microeconomic reforms of labour and human capital to increase long-term productive capacity and oligopoly regulation in non-tradables and energy sectors which can increase efficiency and welfare gains that translate economywide (shehabi ) . consequently, resource rents could be salvaged for swfs resources or investments necessary for future development. in the light of the aforementioned novel issues in resource exporters, the postpandemic energy policy and economic stimulus packages are likely to have negative long-term effects on the environment. the reasons are as follows. first, while lockdown measures that minimized transportation and human activity reduced short-term emissions, they also increased power demand which in resource exporters is met mostly through fossil fuels, especially as international demand and prices remain low. second, short-term improvements will be negated upon the resumption of human activity, absent changes in energy policy regimes. importantly, as domestic energy prices remain low and highly subsidized in developing resource exporters, the extent to which covid- restrictions would shift energy consumption habits and behaviours of agents (households and institutions) remains very doubtful. third, there will be limited resources in the future to dedicate towards environmental regulation and ghg emissions reduction. the reason is that funding the post-pandemic recovery will reallocate funds away from environmental projects and savings in swfs. fourth, ghg emissions will increase due to consumption-focused economic stimulus packages and expanded use of fossil fuels to meet rising energy demand. this is particularly problematic because even prior to the advent of the pandemic, resource exporters were among the highest energy consumers and carbon emitters globally. indeed, the ten highest per capita carbon emitters are all resource exporters, with emissions ranging from tonnes (t) per capita (qatar) to . t per capita (kazakhstan), more than . times the global average of . t per capita (ritchie and roser ) . finally, and most importantly, the implementation of policy instruments-namely energy subsidy reform and carbon taxes-to achieve resource exporters' intended nationally determined contributions (indcs), is rendered significantly more difficult post-pandemic. it is a consequence of the magnitude of novel economic contraction and fiscal pressures in welfare-based states. these additional political constraints exacerbate environmental laws' enforcement, for which there already was a widespread failure in resource exporters (unep ). therefore, new research must quantify effects of proposed economic stimuli and of rising use of fossil fuels on resource exporters' environment and economy and accordingly evaluate and design new alternative policies that can achieve short-term recovery and national climate target goals. to that end, four policy suggestions are offered. first, reducing emissions in ways other than the politically difficult tax instruments, mainly through enhancing energy efficiency that reduces emissions at a given consumption level coupled with economic incentives that reduce energy consumption. second, implementing carbon tax instruments in ways that do not harm the most vulnerable of populations and reduce resource exporters' fiscal distortions, thus achieving a "double dividend". third, in the light of the novel issues, a key policy solution is adopting "green welfare expansion": including green options in the typical post-pandemic welfare packages at lower governmental budgetary requirements. examples include expanding subsidies for green technologies (such as solar panels, public transportation or electric cars), as well as excluding from subsidies non-essential high-carbon-emitting products for households above a certain income level. fourth, increasing the political viability of green recovery packages (such as clean physical infrastructure and natural capital investments), including engaging the private sector in initiatives that are typically public-led-such as climate finance, renewables expansion and resource mobilization. this policy is critical because, although green recovery packages may boost economic growth while helping climate change , they are politically contentious in resource exporters where they compete with welfare distribution. critically, applicable to the aforesaid two research areas, the pandemic offers resource exporters an opportunity to engage in a comprehensive reform agenda that addresses short-term pandemic effects while advancing long-term energy transition, economic and resource sustainability. the challenge for policy makers is to avoid implementing policies in haste. properly coordinated policy reforms offer an avenue to address inefficiencies and underlying structural distortions in a way to realize mutual gains and multiple policy objectives at the lowest cost. in designing these reforms and addressing the two areas above, the most suitable research methods are economy-wide general equilibrium models that can quantify effects of shocks and policy solutions in a "second-best" environment. this feature is necessary given the large existing economic distortions in resource economies. these models represent economic linkages and agents and include a wide range of policies (energy, carbon, fiscal, labour or industrial). as such, they can inform evidence-based policy making that accounts for political economic considerations. this research will be critical for filling gaps in the literature on long-term economic and resource sustainability in resource exporters. the current global greenhouse gas (ghg) emissions trajectory indicates that the world is likely to experience catastrophic consequences due to climate change, unless swift action is taken towards funding green solutions and the defunding of fossil fuel activities ( given the ambition of the european union to become a net zero-carbon economy by and the numerous calls to avoid the bailout and stimulus packages towards fossil fuel companies , we examine whether the features of the european central bank's (ecb) € billion pandemic emergency purchase programme (pepp) encourages the resilience of the incumbent fossil fuel sector, or whether it promotes the growth of the emerging low-carbon energy sector during the covid- pandemic and beyond. we draw on a novel dataset of corporate bonds issued in the european energy sector between january and june in combination with the european central bank's (ecb) purchases under the pandemic emergency purchase programme in response to covid- . we show that the likelihood of an energy company bond to be bought as part of the ecb's programme increases with the ghg intensity of the bond issuing firm. we also find weaker evidence that the ecb's pepp portfolio during the pandemic is likely to become tilted towards companies with anti-climate lobbying activities and companies with less transparent ghg emissions disclosure in the event of increased euro-denominated bond issuances in the following months, or re-denominations of non-euro bonds already issued by european energy companies. kainen et al. ). that does not mean, however, that the aim of central banks to remain sector neutral is achievable in practice, as the implementation of ecb's post- quantitative easing shows that assets purchased by central banks to stimulate overall economic growth are benefitting more from the policy than assets which are not purchased by the bank (haldane et al. ; matikainen et al. ) . this means that the choice of asset class through which asset purchasing programs are implemented matters. this is particularly important in the low-carbon economy context, as the fossil fuel energy sector is largely financed through bonds and syndicated bank loans (cojoianu et al. ) , whereas much of the emerging clean technology companies are financed through private equity, equity issuances and asset financing (cojoianu et al. ; gaddy et al. ) . given that the ecb has chosen to enact its asset purchasing program post- crisis predominantly through bonds, this has been shown to favour the incumbent fossil fuel industry (battiston and monasterolo ; matikainen et al. ) , as % of ecb's corporate bond purchases (out of a total of € billion) are in ghg intensive sectorsthough they make up only % of the eurozone area economy and produce % of ghg emissions. the criteria for the corporate bonds bought under the pepp are that: (i) the company must be incorporated in the eurozone and its bond issuance denominated in euro, (ii) the firm cannot be a financial corporation (or a credit institution supervised by the ecb), (iii) it cannot be a public entity, (iv) the bond issuance has to be endorsed by one positive credit rating by an external credit assessment institution accepted within the eurosystem credit assessment framework and (v) they have a maximum maturity of up to years and a minimum maturity of months. in order to understand whether the ecb's bond buying activity during the covid- pandemic has been tilted towards less transparent, more fossil fuel intensive as well as anticlimate lobbying european energy companies, we undertake the following steps. first, we collect all the bonds issued by european energy companies during the period january to june from bloomberg. these span the following energy subsectors as classified by bloomberg industry classification system (bics): power generation, renewable energy, integrated oil and gas companies, oil and gas exploration and production, oil and gas services and utilities. this results in bonds. we then match each bond with ecb's bondholding portfolio, the borrower's record on pro/anti-climate lobbying from influencemap, the ghg intensity of the borrower (collected from bloomberg and measured as thousands tco -e/million eur revenue) and the ghg reporting completeness of the borrower (which is assessed by bloomberg and quantified as if the company is transparent about the organisational boundary it chooses to quantify its ghg emissions and otherwise, bloomberg terminal code es ). we further collect the borrower's revenue (million eur), bond amount issued (million eur) and coupon rate for each bond, also from bloomberg. our resulting dataset with complete data across all variables of interest is comprised of a cross-section of bonds issued across several currencies, and eurodenominated bonds. our dependent variable quantifies the likelihood that the bond of a european energy company is bought by the ecb during the first months of and coded as if it has been bought by the ecb, and if it has not. for our model, we employ a binary logistic regression model with robust standard errors. the full model specification is the following, where ε i is the stochastic error: we show that after controlling for the revenue of the issuer, the bond amount raised and the rate of the coupon, the ecb is statistically significantly more likely to buy the bonds of more ghg intensive european energy companies (models - , table ). on average, a one standard deviation increase in the ghg intensity of an energy company results in a % increase in the likelihood that its bonds are bought by the ecb (β = . , p < . , odds ratio: . , model ). when we consider only euro-denominated bonds (models and ), which are directly under the remit of the ecb, ghg disclosure completeness and pro-climate lobbying are statistically insignificant, yet negative, which suggests that the ecb may be likely to tilt its portfolio towards companies with poorer ghg emission disclosures and less responsible climate lobbying activities. subsequently, we include the bonds issued by european energy companies in denominations other than euro, to account for potential sample selection bias due to the choice of energy companies to abstain from issuing euro-denominated bonds as they may have received discouraging signals from the ecb. in other words, analysing the bond issuance of european energy companies in all currencies considers signals that the ecb may have given to the energy companies prior to issuance, while analysing only euro-denominated bonds only considers the observable decision of the ecb to purchase the bonds of specific energy companies post-issuance. when we do so (model ), it emerges that considering the entire universe of bonds issued by european energy companies, the ecb's portfolio is tilted not only to those energy companies that are more ghg intensive, but also to companies which are less transparent on their ghg performance as well as those companies who are more likely to oppose progressive climate action. having established statistical significance, we investigate the economic and statistical relevance (brooks et al. ) . inspecting the economic relevance of the ghg intensity variable in model , we find ghg intensity to have the largest marginal effects. in terms of statistical relevance, we find ghg intensity to have by far the largest shapley r-squared value, contributing more than % to the overall explanatory power of model . in conclusion, the importance of ghg intensity is underlined by its marginal economic effects and its statistical relevance, as it explains more variation in the dependent variable on its own than all other variables taken together. we also conduct further robustness tests controlling for ecb bond i = + * pro-climate lobbying activities score i + * ghg emissions intensity i + * ghg reporting completeness i + * borrower revenue i + * bond issuance amount i + * bond coupon rate i + i . bond maturity, bond rating and interactions of key variables and find our results to remain statistically significant. in conclusion, drawing on a novel dataset of corporate bonds issued in the european energy sector since january and the database of ecb's purchases under the pepp in response to covid- , we find evidence that the likelihood for a bond to be bought by the ecb increases with the ghg intensity of the bond issuing firm. we also find weaker evidence that the ecb's pepp portfolio during the pandemic is likely to become tilted towards companies with anti-climate lobbying activities and companies with less transparent ghg emissions disclosure. our findings imply that, at later stages of the covid- recovery, an in-depth analysis may be necessary to understand if, and if yes, why the ecb fuelled the climate crisis. even if one accepts that fossil fuel companies were eligible for pepp, then our preliminary evidence still raises the significant question, why the ecb was more likely to directly finance those fossil fuel firms that are likely more harmful to the planet (i.e. have a higher ghg intensity)? the coronavirus covid- pandemic is the defining global health crisis of our time, causing over half a million deaths to date ( july ). but covid- is much more than a health crisis, it has tremendous socioeconomic impact, the scale of which is still hard to assess. measures to address the health crisis generate economic impacts (and vice versa). social isolation measures to "flatten the curve of the pandemic" buy time to increase capacity in the healthcare sector but inevitably deepen the macroeconomic recession. the world bank expect the global economy contract by . % in , approximately three times the size of the - great financial crisis (gfc) and far more widespread. emerging markets and developing economies (emdes) have been severely hit by massive capital outflows, reducing debt servicing abilities (especially for dollardenominated debt) and generating long-term challenges; "with more than % of emdes expected to experience contractions in per capita incomes this year, many millions are likely to fall back into poverty" (world bank ). the economic consequences of the adverse coronavirus shock are: (i) an elevation of uncertainty, which increases precautionary savings, thus reducing consumption and also curtails the appetite for productive investments; (ii) a rise in unemployment, part of which is likely to be permanent; (iii) a decline in the volume of international trade and disruptions in global supply chains; (iv) falls in commodity prices (especially the price of oil), making current account financing of traditional commodity exports challenging; (v) a sharp increase in the required risk premia for holding risky assets. this initially resulted in a plunge in prices of risky assets (e.g. stocks or high-yield bonds) and a sharp increase in financial volatility. however, central bank interventions (especially by the federal reserve of the united states of america-us fed) have seen recoveries from march . extraordinary macroeconomic policy response, on both the fiscal and monetary fronts, has slowed the rates of economic decline. in many countries, fiscal measures have replaced a proportion of lost incomes and mitigated default risk, loan guarantees have helped keep businesses afloat, and liquidity provision by central banks have kept the financial system functional. fiscal authorities in european and the usa took measures that can be classified within three broad categories: immediate fiscal measures and direct transfers to households; tax deferrals and liquidity measures; and loan guarantees. the scale of intervention is truly unprecedented reaching almost % of gdp in germany and % of gdp in the usa. on the monetary front, the fed cut interest rates to zero, announced unlimited purchases of treasuries and mortgage backed securities and started buying corporate debt. moreover, the fed opened debt swap lines with foreign central banks to provide dollar liquidity to the international financial system. the value of fed measures to date exceeds . trillion dollars. the european central bank ecb "has offered low-interest loans to banks, significantly boosted asset purchases, and allayed fears of member-country defaults by lifting distributional restrictions on its bond-buying program" (world bank ). moreover, the eu's recovery plan (proposed may ) mobilizes investments through a recovery instrument of € bn for the period - and a reinforced a long-term budget of € . trillion for the period - . despite these unprecedented measures, the course of the pandemic and its developing economic impact remains uncertain. how can long-term economic dislocation be avoided? a first priority should be to ensure that the work force remains employed. second, governments should channel financial support to public and private institutions that support vulnerable citizen groups. third, small medium enterprises (smes) should be safeguarded against bankruptcy. (the need for taxpayer money to support large nonfinancial corporations is much less obvious.) fourth, policies will be needed to support the financial system as nonperforming loans mount. fifth, fiscal packages, comparable to the loss of gdp, will have to be financed by national debt. while this should be structured to avoid another debt crisis, finance should be directed to investments with positive social, economic and environmentally sustainable profiles, as discussed in the following. there is widespread scientific speculation that economic growth has pushed humanity into new ecological niches wherein humans and animals exchange novel, infectious viruses of which covid- is just the latest in a considerable list of examples. furthermore, the intergovernmental panel on climate change (ipcc) has warned that global warming will likely accelerate the emergence of new viruses. overall, climate change has the potential to end up killing more people than covid- , although this is obtusely referred to as an "increased frequency and severity of natural disasters". such language, the delayed, cumulative nature of the threat and the necessity of coordinated international response, all mitigate against the urgent action that is required. timing is also important. as ipcc ( ) reports, the level and speed of change needed to successfully tackle the climate crisis, is unprecedented; incremental changes will not be enough. that said, there are aspects of the climate change crisis which are less challenging than the covid- pandemic. as sterner ( ) notes, the climate crisis requires policy changes that are less disruptive, economically, socially and culturally, than the measures being taken right now to tackle covid- . for the climate, we do not need to close down the economy. on the contrary, we need a transition to a low-carbon economy that supports public and private investments in renewables, energy efficient and circular, technologies and infrastructure. these technologies exist (wind, solar, etc.) and are becoming consistently cheaper than fossil fuels, while energy storage installations are increasing exponentially. there is growing evidence that green stimulus policies have advantages over traditional fiscal stimulus and that climate-positive policies also offer superior economic characteristics. for example, hepburn et al. run a global survey to assess stimulatory fiscal recovery policies implemented in response to gfc. the economists from countries were asked to ascertain their perspectives on covid- fiscal recovery packages according to: "speed of implementation", "long-run economic multiplier", "climate impact potential" and "overall desirability" according to social, political and personal factors. the responses indicate that green stimulus policies deliver higher multiples due to reduced long-term energy costs and flow-on effects to the wider economy. "relaunching the economy does not mean going back to the status quo before the crisis but bouncing forward. we must repair the short-term damage from the covid- crisis in a intergovernmental panel on climate change, . way that also invests in our long-term future". this code explains the strategy of the eu recovery plan based on three axis: the european green deal, adaptation to the digital age, and a fair and inclusive recovery for all. we must start investing in what makes our socio-economic system resilient to crisis. now is the time to usher in systemic economic change and the good news is that we have our blueprint: it is the combination of un agenda ( sdg) and european commission's european green deal. the european green deal (egd) announced december is the new growth strategy of the european union and is based on four principles: (a) climate neutrality by , (b) protection of human life and biodiversity by cutting pollution, (c) world leadership in clean technology, (d) leave no one behind. the egd investment plan amounts to eur billion per year by . the egd just transition mechanism will help mobilize at least € billion over the period - in the most affected regions. the - multiannual financial framework (mff) allocates an overall target of % for climate mainstreaming across all eu programs. the proposed european climate law aims to turn the egd's political commitment of climate neutrality, into a legal obligation. based in un environment program (unep) emissions gap report , global emissions need to be reduced by % by . unfortunately, the proposed law does not include an ambitious goal with regards emissions, nor does it address the legislative interventions required to achieve climate neutrality by . together with the egd, the sustainable development goals (sdgs) and the paris agreement call for deep transformations. while significant progress is being made on some goals, no country is currently on track towards achieving all sdgs. sachs et al. ( ) identify the major interventions needed to achieve each sdg and group them in six sdg transformations, which operationalize the sdgs at government level and can prove instrumental for egd implementation. the success of the join implementation of the sdgs, the egd and the eu recovery plan will depend on the eu's capacity to engage with its citizens in codesigning the pathways that will allow them to reach the vision, hence the introduction of the european commission climate pact. https ://ec.europ a.eu/info/live-work-trave l-eu/healt h/coron aviru s-respo nse/recov ery-plan-europ e_en. european parliament, committee on environment, public health and food safety draft report on the proposal for a regulation of the european parliament and of the council establishing the framework for achieving climate neutrality and amending regulation (eu) / (european climate law), / (cod). the review of the climate law summarizes the review of the climate change committee of the greek ministry of energy and the environment, the first author of this paper is a member of this committee and co-author of this greek review. united nations environment programme ( ). emissions gap report . unep, nairobi. the proposed climate law does not allow the european commission to impose sanctions on member states (ms), while there is lack of reference to the financial mechanisms that will be required to achieve the goal of climate neutrality. moreover, the law does not consider (a) the well-documented heterogeneity in area-specific climate vulnerability among ms, (b) an eu wide carbon tax and (c) strengthening the european ets. sachs et al. ( ) : the sustainable development goals and covid- . sustainable development report . cambridge: cambridge university press. ( ) education, gender and inequality; ( ) health, well-being and demography; ( ) energy decarbonization and sustainable industry; ( ) sustainable food, land, water and oceans; ( ) sustainable cities and communities; and ( ) digital revolution for sustainable development. https ://ec.europ a.eu/clima /polic ies/eu-clima te-actio n/pact_en. recent generations, including the present, have experienced at least three global crises: the financial crisis - , the ongoing covid- pandemic and the developing climate crisis. if we continue attempting to address the latter two with the same socio-economic model that gave rise to the former crisis, we will fail to find a sustainable and resilient socio-economic-environmental pathway. we believe that we can even do better than just react to crises by adapting to the new crisis-born reality. we can use the integration of scientific, economic and socio-political knowledge to design policies which not only address the immediate impacts of the covid- pandemic, but mitigate the existential threats of future pandemics and the ongoing and unfolding disasters of climate change, biodiversity loss and planetary boundary exceedance. what is needed now is a fundamental transformation of economic, social and financial systems that will trigger exponential change in strengthening social, economic, health and environmental resilience. we need big thinking and big changes. system innovation and transitions thinking can help but calls for intense public participation. now is the time, in addition to directing funds to the control of the epidemic and relevant biomedical research, as well as investing in border security, safe travel and safe trade, now is the time for financial institutions and governments to embrace eu taxonomy for sustainable investments ( ) to phase out fossil fuels by deploying existing renewable energy technologies, eliminate fossil fuel subsidies and redirect them to green and smart climate mitigation and adaptation infrastructural projects, invest in circular and low-carbon economies, shift from industrial to regenerative agriculture and invest in food security, promote european supply chains, reduce transportation needs and exploit the limits of the digital revolution, while ensuring secure information and communication technology networks. a decisive march along this sustainable pathway will enhance economic and environmental resilience, create jobs, and improve health and well-being. the transition should be inclusive and "leave no one behind", hence the need of transforming citizens into codesigners and co-owners of the sustainability transition pathways. kiel institute for the world economy, kiellinie , kiel, germany. wilfried. committing to rigid shutdown measures to contain the spreading of the corona virus has been undertaken on the tacit assumption that these measures will be temporary and can be loosened when the covid- infection rates decrease and discontinued altogether once vaccines are available. mitigating climate change and achieving ambitious temperature targets as set out in the paris agreement require a long-term structural change taking us away from our current carbon-intensive economy to a zero-carbon and then net-negative carbon economy. as current research holds out little hope that a "perfect" vaccine in the form of solar climate engineering will be available in the future, the measures and efforts required must translate into a permanent, ongoing form of commitment. while progressive climate change and the spread of the coronavirus operate on very different timescales, impatience about the duration of corona lockdown has indicated once more a fundamental problem for (long-term) environmental concerns. clearly, the economic and social costs associated with the emergence of the virus and the shutdown are significant , oecd . but any serious cost-benefit analysis would need to take into account not only the fact that different degrees of lockdown are available but also that the overall cost is affected by the expectations of agents regarding possible future re-lockdowns due to insufficient containment of the virus. seen thus, it is anything but clear at which point in time the actual cost of lockdown would have exceeded the economic cost of the virus spreading in an unmitigated (or insufficiently mitigated) way. during the course of lockdown measures, voices calling for a "green" recovery stimulus package centring around low-carbon investments in the aftermath of the corona crisis have make themselves heard. by contrast, advocates of postponing climate mitigation-related taxes, levies, and regulations have also entered the fray, claiming that timely recovery should not be jeopardized by any additional economic burdens. the debate on the relation between (economic) recovery and climate policies has been conducted from three major perspectives. the first of these is largely notable for general statements of intent recommending that the recovery should be "green" and sustainable, that eu climate targets should be supported, and that other environmental targets (maintaining biodiversity, etc.) need to be taken into account when designing recovery measures. such well-meant counsels as the statement issued by the german national academy of sciences leopoldina ( ) are useful in reminding us that recovery from the corona crisis should not come at the expense of neglecting other objectives and that climate policy should not be backburnered, as was the case after the financial crisis in . otherwise, they are of little practical value. the second approach has involved rather detailed proposals calling either for a "greening" of recovery by foregrounding measures to support renewable energies, public transport, energy efficiency, etc. or for a "blackening" of recovery by postponing and/or abandoning climate measures and environmental regulations. predominantly, these proposals are representing the positions of the various interest groups involved. for example, representatives of the aviation industry try to prevent the harmonization of carbon prices on fuels with respect to kerosene and argue against the introduction of kerosene taxes. this idea resurfaces in the discussion on recovery measures by, say, the austrian aviation association ( ). on the other hand, in its comprehensive list of (recovery) demands, the ngo german environment action ( ) urges for example for the abandonment of blue hydrogen projects (though not explaining why this is likely to stimulate economic recovery). various other interest groups are in favour of postponing, suspending or even abandoning existing environmental and climate regulations. for example, janusz kowlaski, the polish deputy minister of state assets urges "…[that] the ets [european emissions trading scheme] should be removed from january , or at least poland should be excluded from the system". clearly, there is no point in discussing nonsensical ideas of this kind. but some of these proposals also make sensible suggestions like adjusting the german cap on renewable energy installations or abandoning the eu average fleet-consumption regulation because the former contradicts german renewable energy targets and the latter is an inefficient instrument for regulating vehicle emissions. however, these suggestions do nothing to provide stimulus for a quick recovery. while specific processes and regulation timelines for regulations may need to be adjusted in the context of the corona crisis, sensible measures of this kind should be discussed and decided upon in the regular political process. confining potential stimulus and recovery measures to their proper purpose does not mean imposing a ban on meaningful (climate or environmental) policies that are not associated with the corona crisis. the third and most sensible perspective replaces specific proposals with (sustainability) assessment guidelines like those suggested by the world bank ( ). while hardly any possible recovery measure would perform well against the comprehensive list of criteria provided by the world bank, such guidelines are helpful in arguing against interest group driven proposals. the world bank has suggested that potential measures up for consideration as part of a recovery strategy need to be assessed against both, short-and long-term criteria, an example for the former being the expected economic multiplier associated with certain measures. bayer et al. ( ) suggest that income transfers (as planned under the us cares package) perform well against this specific short-term criteria: they could help to stabilize private-sector spending and the multiplier could increase to in the case of transfers being conditional-but not related to emissions but to the propensity to consume, i.e. conditional on being unemployed. however, privatesector spending like this should not imply any unintended adverse effects on essential long-term structural change that might arise from such things as (temporarily) adjusted risk preferences. once postponed or stimulated demand and investment take place during the recovery process, carbon-price signals are vital in providing technology-neutral incentives for low-co purchasing and production decisions. overloading stimulus or recovery packages with too many (emission-related) conditions performs poorly against the short-term criteria with respect to a timely recovery. even worse, the inclusion in recovery packages of various detailed suggestions from the various interest groups usually results in a non-transparent, rent-seeking, and political bargaining process in which it remains unclear whether (sensible) individual emissionrelated decisions are being prioritized at the expense of a more challenging long-term climate policy. accordingly, accounting for the long-term criteria requires that existing or planned climate policies providing incentives for emission reductions and technological innovation should remain in place and not be postponed, let alone weakened. otherwise, uncertain (short-term) recovery impulses most likely come at the cost of less efficient emission-reduction paths in the long term. the coronavirus pandemic is having a serious impact on the economy. eurostat estimates that seasonally adjusted gdp decreased by . % in the eu during the first quarter of , compared with the previous quarter. in certain sectors, the covid- crisis has led to the temporary or even permanent closure of sites, a collapse in demand and an increase in production costs linked to the fight against the spread of the virus. this decline in activity can trigger bankruptcies, which result in unemployment and the destruction of physical and human capital, leading in turn to a loss of specific knowledge and skills and to market concentration and the partial relocation of production activities. from an environmental point of view, bankruptcies and relocation may generate higher emissions by increasing production in more polluting sites abroad and by increasing the transportation of goods. in addition, the decline in economic activity may have a negative effect on research and development spending, which is crucial for future growth and the development of more environmentally friendly technologies. many measures have been put in place to limit the economic effects of the covid- crisis. at the european union level, a number of measures such as direct subsidies, selective tax benefits, advance payments, state guarantees for loans and subsidized public loans to companies have been implemented. in addition, many large companies are being bailed out and there is a debate on whether conditions should be attached to this state aid. these conditions could relate to various commitments such as relocating activities, paying taxes, safeguarding employment, and protecting the environment. on may , the eu confirmed that large companies receiving emergency cash during the covid- crisis will not be obliged to devote funds to "greening" their operations. rescued firms will only have to report on their use of the aid and in some cases the aid could be attached to conditions such as a ban on dividends and management bonus payments. nevertheless, member states are free to design national measures in line with additional policy objectives, such as further enabling the green transformation of their economies. many stakeholders are still pushing for conditions to be attached to the bailouts. for instance, the eg think tank proposes making the rescue of airlines conditional on the use of less polluting fuels and tying aid to car manufacturers to the development of electric vehicles. a group of german companies has also requested that state aid be linked to climate actions. this article examines the merits of making aid conditional on environmental efforts. we focus on the rescue plans for companies rather than on recovery plans or the european green deal, which was launched before the covid- crisis. we show that tying aid to environmental efforts is difficult to implement and requires both controls and sanctions, as well as a large amount of information. we also discuss the merits of tying bailouts to environmental efforts compared with implementing more stringent environmental policies (presuming that companies will be bailed out in any case). since environmental efforts can lead to higher product prices and lower profits, two dimensions of acceptability must be taken into account: acceptability from the point of view of companies and social acceptability. acceptability of environmental policies by companies is a necessary condition for their implementation. in the midst of the current crisis, many polluting industries are lobbying to weaken and delay environmental regulations. for instance, the environmental protection agency issued a sweeping suspension of its enforcement of environmental laws, allowing companies to breach environmental standards during the coronavirus outbreak. in indonesia, the trade ministry revoked rules requiring basic certification that wood exports were legally produced, in response to lobbying from the furniture and logging industries. lobbyists may also encourage legislators to compensate companies for losses related to environmental regulation. the implementation of rescue plans, by providing aid and guarantees, could be a tool to make environmental efforts more acceptable for firms. acceptability by society is also central. it depends on the redistributive effects of environmental policies, especially since they are often regressive. in france, the "red caps" and "yellow vests" movements illustrate the difficulty of implementing such policies. both of these movements led to the cancellation and delay of environmental policies (eco-tax applied to heavy vehicles and carbon tax, respectively). the covid- crisis may affect societies' support for more ambitious environmental policies. several observations can be made. first, it appears that there is a confirmation bias, as countries and ngos that had already committed to fight against global warming want to intensify efforts, while countries that were already recalcitrant are calling for a decrease in efforts in the light of the economic crisis. second, the covid- crisis makes the risk of disaster more salient and vivid and may therefore increase the demand for stronger environmental protection. as explained by sunstein ( ) , if a particular risk is cognitively "available" then people will have an increased fear of the risk in question. finally, the crisis has revealed that populations will accept drastic measures (lockdown, wearing face masks) and a rapid change in social norms (social-distancing, for instance), which shows the ease with which individuals could adapt to more ambitious environmental policies. see oates and portney ( ) for a literature review of interest groups and environmental regulation. "polluter bailouts and lobbying during covid- pandemic", the guardian, april , . burkey and durden ( ) and joskow and schmalensee ( ) have detailed how firms can influence the regulator's decisions in the context of pollution rights markets, while bovenberg and goulder ( ) , hepburn et al. ( ) and nicolaï ( ) have shown that few permits are sufficient to neutralize the losses in profits and make the implementation of pollution permits acceptable. on march , the czech prime minister, andrej babis said that the european green deal should be put to one side. during the same period, the green coalition of environmental organizations organized an appeal urging lawmakers to design a green, healthy and just recovery. we study two possible ways of increasing environmental efforts: making aid to companies conditional on environmental efforts or making existing environmental policy more stringent. making aid conditional on efforts to protect the environment can take different forms. commitments may relate, for example, to reducing pollutant emissions, using less polluting production processes, increasing expenditure on research and development, and producing less polluting products. for instance, in france, renault's bailout requires it to increase the share of electric vehicles, while the billion euro air france bailout imposes the use of at least % of alternative jet fuel by , a target of % emissions cuts by , and a % decrease in domestic flights by , especially those that compete with high-speed trains. conditional aid as currently envisaged is equivalent to negotiating and implementing individualized standards on technology, final products, research and development efforts, emissions and performance for each company. the first question that arises is how to determine the conditions. conditional bailouts allow the regulator to individualize the standards applied to a particular firm, making it possible to set the most appropriate instrument and the optimal level of severity for each firm. when making its decisions, the regulator should consider the possibility of a rebound effect as explained by saunders ( ) . an emissions standard may avoid such an effect, but a standard on technology may increase emissions. furthermore, the regulator should take into account its access to information. if the firm commits to finance research and development in green technology in exchange for being bailed out, it will be hard for the government to determine whether the state aid was actually allocated to research. for example, since the results of research are uncertain, a lack of innovation may be explained either by the firm's characteristics (anti-selection problem) or by the misallocation of public funds (moral hazard problem). finally, the regulator should take into account competition, since firms receiving bailouts usually have market power. a government will mainly intervene to save a firm if its bankruptcy would have a significant effect on the economy. the government should hence ensure that the conditions do not further distort competition. a firm with a competitive advantage, for example with cleaner technology, may have an interest in advocating more ambitious environmental efforts for itself but also for its competitors in order to increase its market share by preventing the entry or inducing the exit of such competitors. the effectiveness of conditions will therefore depend on the type of commitment, competition in the market, and the demand elasticity of the final good. another question relates to the duration of the contract. some actions are reversible and after a certain period of time companies can cancel or amend them. for example, the use of cleaner but more expensive inputs is an easily reversible strategy, while a shift in production, for example from internal combustion vehicles to electric vehicles, is a more expensive choice to change. in addition, research and development efforts produce long-term effects with the creation of innovations and possible technological spillovers. the duration must therefore be individualized in function of the company's characteristics. it should also be noted that conditional aid can lead to windfall gains. some companies had already planned emission reductions and changes in strategy. these decisions were aligned with the companies' interests. if the company benefits from the environmental efforts put in place, these can no longer be considered as a counterpart to the company's bailout and the company should then make greater efforts. it seems clear that the success of such schemes lies in companies' compliance with the commitments. companies may nonetheless have an interest in not complying with them. it is therefore necessary to monitor companies' implementation of the commitments and to sanction those that fail to honour them. supervision costs will thus be incurred. one possibility for alleviating control costs is to publicly announce the commitments, which facilitates the monitoring of companies' commitments by society (e.g. by journalists, politicians and non-governmental organizations). this also gives the firm a further incentive to respect the agreement in order to maintain its reputation and to retain its customer base (heyes et al. ( ) and heyes and kapur ( ) ). we now focus on the sanctions to be applied in the event of non-compliance with the commitments. various penalties could be considered, such as financial penalties, a ban on applying for the attribution of public contracts and the state's participation in the company. the sanctions must be explicit, credible and sufficiently significant to discourage companies from breaking their contracts. however, given the current economic situation and the fragility of companies, governments should be cautious when using financial penalties or bans from participating in public contracts since, if effective, they could be fatal to companies. an alternative could be for the state to take a share in the company in the event of non-compliance with commitments. such shareholdings would make it possible to influence companies' strategic choices, but they raise efficiency problems. cavaliere and scabrosetti ( ) , schmidt ( ) and de fraja ( ) , for instance, highlight two effects: production is more efficient in privatized firms because better incentives can be given to managers and employees (productive efficiency), while public firms are more socially efficient because the government cares about social welfare and internalizes externalities associated with firm liquidations (allocative efficiency). existing environmental policies inevitably generate monitoring costs; however, these costs will be borne in any case. increasing the stringency of environmental policy involves determining new levels for existing instruments, which generates costs, but there will be no additional monitoring costs. moreover, opting for a more stringent environmental policy means that market-based instruments can be employed, which is not feasible in the first strategy. due to the principle of non-discrimination, it is unlikely that individualized taxes could be introduced under the negotiated bailout conditions. environmental economists agree that these instruments are more effective than command-and-control instruments since they have the potential to generate revenue and allow for a double dividend, provide incentives to invest in clean technology and require less information to be designed. nevertheless, making aid conditional on environmental efforts makes it possible to individualize the regulatory instruments for each firm, which is not possible in the context of marketbased instruments. the positive effect of the instrumental individualization induced by conditionality only materializes if the existing environmental policy is based on commandand-control instruments. with market-based instruments, individualization is not required to achieve an efficient outcome, whereas it is necessary in the case of command-and-control instruments. companies' negotiation power is an additional argument for increasing the severity of environmental policies rather than conditioning bailouts on environmental efforts. regardless of the policy, firms may use their bargaining power to mitigate the stringency of the regulation or the effort they commit to make. it is clearly easier for a firm to influence its specific negotiated conditions, which are one-on-one relationships, than to influence environmental policies, which would require coordination and a common interest among firms. furthermore, making environmental policy more stringent will affect all companies and not only those receiving bailouts, which increases the attractiveness of this strategy. since they apply to a larger number of companies, these more restrictive policies will have a greater effect on the environment. to conclude, this note analyses the advantages and disadvantages of making bailouts conditional on environmental efforts. we show that such a system could be beneficial for the environment and would be a counterpart to bailing out companies. however, it is costly, requires a large amount of information, and is less efficient than increasing the stringency of existing environmental policies. on the other hand, making aid conditional on environmental effort is more acceptable from the companies' point of view than increasing the severity of environmental policies. companies would rather commit themselves to environmental efforts than pay taxes or buy permits in order to minimize compliance costs. to succeed in making environmental policy more ambitious, the regulator needs the support of society to compensate for companies' reluctance. if there is strong public support, governments should not negotiate conditional aid but should increase the severity of environmental policies. they can integrate such actions into recovery plans or, in the case of the european union, into the revision of the green deal. it is therefore particularly important to study how the covid- crisis has changed society's perception of the need for more stringent environmental policies. for instance, a recent survey by ipsos shows that three out of four people in major countries expect their government to make protection of the environment a priority when planning a recovery from the coronavirus pandemic, although there is considerable heterogeneity across countries. the post-covid- reality is changing the context of most policies, including those in the fields of energy and the environment. the eu green deal objectives should be maintained but concrete policies may have to be adjusted to this new reality. a major energy transition is under way, shaped by political will to tackle climate change. policies have been defined under the paris agreement and geared to a number of targets. the eu has decided to reduce its greenhouse gas emissions by by at least % compared to and has agreed to continue the path towards climate neutrality by . the immediate recession following the covid- crisis drastically reduced energy consumption and greenhouse gas emissions (le quéré et al. ) . the sudden decline in greenhouse gas emissions is the opposite of what a meaningful response to climate change should be, in terms of both quality of life and economic efficiency. the challenge is to structurally decouple economic growth from emissions, not to have them both going down. the lockdown may have some lasting effect in changing some of our habits ), e.g. a generalized familiarity with telework. one can also expect a more general reflection on whether all business and leisure travel, not least by air, is necessary. nevertheless, many changes may not be as extensive and lasting as some would wish. for this reason, it is important to integrate a climate check into the stimulus packages that governments and european institutions are currently designing. the current health crisis and the likely economic downturn could be seen as an unsolicited-and much regrettableopportunity towards a carbon-neutral future koundouri ) . the agreements reached in the context of the eurogroup, the latest turn in the european budgetary discussions, as well as the statements by the commission president all point into this promising direction. the eu green deal has confirmed the eu emissions trading system (ets) as a key element and the price of eu allowances serves as a key indicator worldwide. as could and should be expected, carbon prices have fallen during the lockdown reflecting reduced demand for allowances in line with the drop in power and industrial production. this temporary drop from the € to a € - range, acts as an important automatic stabilizer for businesses in distress. a repetition of the experience of a sharp fall in the see on this issue the important draft report prepared by the european parliament committee on the environment, public health and food safety (european parliament, ). https ://lifed icetp rojec t.eui.eu/ / / /covid - -clima te-polic y-and-carbo n-marke ts/. https ://www.consi lium.europ a.eu/en/press /press -relea ses/ / / /repor t-on-the-compr ehens iveecono mic-polic y-respo nse-to-the-covid - -pande mic/-point : "work is ongoing on a broader roadmap and an action plan to support the recovery of the european economy through high quality job creation and reforms to strengthen resilience and competitiveness, in line with a sustainable growth strategy". https ://ember -clima te.org/carbo n-price -viewe r/. price of eu ets allowances after the recession and a long period of very lowcarbon price levels should be avoided. there are two reasons to believe that this could happen: (i) the eu ets now has a market stability reserve (msr) operating in the short-medium term and (ii) long-term market expectations could be shaped by the carbon neutrality objective. the msr started to operate last year and absorbs a potential oversupply in the market in the short and medium term. if the allowances market is "long" beyond a threshold set in legislation ( million tons, which is approximately % of ets emissions in ), the msr intervenes by withdrawing allowances equal to a percentage ( % up to , % thereafter) of the excess. at the end of , the allowance market was . billion tons "long" and, therefore, the supply of allowances for the period september to august has been reduced by million tons (these allowances are placed in the msr). some question whether the msr, with its delayed adjustment mechanism, will be sufficient to avoid a sharp fall in the allowance price (cf. flachsland et al. ) . in fact, any reduction in the supply of allowances through the msr for the period september -august will still be based on the excess supply of allowances in , i.e. before the current crisis started. in case the msr is unable to absorb the surplus, the eu commission should propose changes in the planned review of the ets in . long-term expectations might overtake this short-term risk. in its european green deal, the commission already indicated its intention to tighten the greenhouse gas emission reduction target from % to %, or even % (with respect to levels). today, the ets sectors face a mandatory emission reduction of % by compared to , and revised targets will likely increase that obligation by at least another or %. such a reduction in the supply of allowances would likely push their prices up, or at least reduce the extent of their decline, in the face of lower demand due to the economic downturn. therefore, carbon market participants as well as those developing innovative clean technologies and products of the future are likely to expect significantly higher carbon prices in the future. this raises the question about a possible loss of competitiveness in europe's traditional industries vis-à-vis competitors located in countries where no similar burden is imposed. some proposed a border adjustment, which would imply both a levy on imports and possibly a rebate on exports. however, such mechanisms are not easy to implement and are subject to criticism on both analytical and political grounds. therefore, it is of crucial importance to realize that a wto-compatible border adjustment mechanism will take some time before it can be implemented. meanwhile, at least equal attention needs to go to domestic policy reinforcement, such as support for innovation and a less rigid interpretation of the state aid rules. governments and european institutions are currently developing a major investment stimulus package. the commission's impact assessments on climate and energy policy serve as useful guidance on where a major surge in investments is needed. we know that energy efficiency requires a major push in the construction sector, both for new buildings and renovations in social housing, hospitals and schools. we know that the energy transition requires more investments in renewable energy, digitised grid infrastructure and energy storage. the significantly reduced electricity demand of today indicates that much more attention should go towards managing flexibility in real time instead of increasing baseload capacity. in transport, electrification is on its way, but investment in charging facilities, traffic management, clean public transport and long-distance rail needs to be ramped up. in industry, not least in power generation and carbon intensive industrial sectors, major efforts are being undertaken to develop new technologies based on hydrogen and carbon capture, use and storage, for example. such investments can create the jobs we need in the post-covid- era and allow to realise the eu green deal objectives at the same time. apart from carbon prices and investment support schemes, it remains of equal importance to maintain a consistent energy price signal throughout the economy. however, the recent fall in the prices of fossil fuels is upsetting the incentives that should support a transition towards sustainability. a reference criterion for economic decisions has been lost and the consequence may be a paralysis, or even a "comeback" of fossil fuels, mothballing or abandonment of investment in renewables. consequently, the business case of the green transition may appear to be weakened in the near term, but a clear policy response may be politically more acceptable because of the price drop in fossil fuels. this raises the question about the other half of europe's emissions, namely those not covered by the ets, in particular the sectors of transport and buildings. the pricing of these emissions can be much improved through the planned review of the eu's energy tax directive. this offers a major opportunity to remove the all too generous exemptions, such as on maritime and aviation fuels, or by adding a co element to the harmonized minimum tax rates. in such a manner, prices of fossil fuels could be stabilized at the pre-covid levels via temporary taxes, whose revenue could help finance the various income-support policies adopted and/ or an acceleration of investment projects for sustainability. in this way the eu should better prepare for a long period of low oil and gas prices and seize the opportunities these can offer. public measures to combat the covid- pandemic have led to a severe economic crisis. in order to cope with this crisis, comprehensive government aid is being requested. accordingly, governments across the world have pledged billions of euros for extensive recovery programs. one of the main questions debated in this context at the moment is how "green" these recovery programs should be ). the expectation of huge amounts of public money being distributed at short notice brings interest groups of every shade to the scene-preferably with old wish lists on hand. consequently, there is a big risk that recovery programs will be captured by interest groups (for an overview of the literature on regulatory capture, see dal bo ). on the one hand, climate change mitigation is put under pressure as being an "extra burden" for industries. for example, european car manufacturers have called for postponing the upcoming tightening of eu emission standards for car fleets (topham and harvey ) . some eu member states call for stalling the eu commission's plan of a european green deal (simon ). on the other hand, many recommend spending the public money mainly on measures that also help mitigating climate change-among them frans timmermans, executive vice-president of the european commission (schulz ), or fatih birol, head of the international energy agency (birol ) . there is one thing that must not be overlooked in this politico-economic competition: public funds are still short and must be used reasonably. otherwise ill-designed (green) subsidies can quickly turn into a part of the problem instead of being the solution. previous "cash for clunkers" programs warn as an example of a misguided recovery measure. these programs were introduced in many countries after the financial crisis and provided financial incentives to trade old, less fuel-efficient cars for new, more efficient ones. empirical analyses have shown very mixed results regarding both the economic and the environmental stimulus effects of these measures (grigolon et al. ; li et al. ; mian and sufi ) . at the beginning of every discussion about (green) recovery programmes, it is therefore important to develop transparent and sensible criteria based on which public aid should be allocated. after the initial bail-out programmes, public recovery programmes to stabilize the economy are now debated politically. certainly, this generates an unprecedented window of opportunity for structural transformation. moreover, the distribution of public aid may also justify committing beneficiaries to public interests to a certain extent. consequently, the currently available political degrees of freedom should be used to promote the transition of society towards sustainability. subsidies to branches like tourism, aviation and agriculture-which are particularly hit by the crisis and are lagging behind in terms of sustainability-should be paid conditional on meeting minimum environmental standards. new investments into long-lived, fossil-fuelled assets must be avoided. a recovery program cannot only be about re-establishing the status quo ante by assigning large public funds, possibly creating new barriers for sustainability transitions. in this respect, it makes sense to implement recovery programs that are in line with the objective of mitigating climate change-as called for by many at the moment. however, such green recovery programmes must not be arbitrary. green recovery programmes must go beyond green subsidies. first of all, it is also important to reduce unnecessary barriers for green investments, for example by revising legal constraints to the expansion of renewable energies like solar photovoltaics of wind power. moreover, any green recovery program can only effectively and efficiently spur decarbonization if it combines with a carbon price and the abolition of environmentally harmful subsidies. the direction of recovery must be crystal clear. otherwise green subsidies risk being ineffective and costly approaches to mitigating climate change (kalkuhl et al. ; palmer and burtraw ) , while imposing additional burdens on public budgets and reducing political degrees of freedom in the future. for subsidies to be economically justified, they need to meet clear criteria. for green recovery programs to succeed in the competition for public funds with other important policy fields (such as health or digitalization), they must help stabilize the economy. moreover, policy makers need to be aware that some of the currently observed economic problems might even resolve without any government aid. it can expected, for example, that global supply chains will resume and that people will catch up on purchasing durable goods like cars, at least partly. it is exactly (the maintenance of) environmental regulation that may help steer this consumption towards more sustainable modes. government interventions must take effect where permanent disruptions are looming. one example: innovative green business models may particularly be at risk if banks limit loans in the presence of the current uncertainties (lehmann and söderholm ) . in this case, government loans may provide direct assistance. in contrast, attempts to lower prices for goods and services-e.g. for cars (vat reduction, purchase premiums) or electricity (reduction of energy levies)-are rather inappropriate means to stabilize the economy. such measures fail to address the actual sources of insufficient investments or reduced purchasing power, and are therefore inefficient ways of spending public budgets. furthermore, it is unclear whether and to what extent such discounts will be passed through to final consumers by market prices (peltzman ) . green recovery programs should focus on government interventions that would also have been economically reasonable without the covid- crisis-for example, to correct market failures next to the co externality (bennear and stavins ; fischer and newell ; lehmann )-and that have the highest priority for climate policy. moreover, those measures should be implemented for which rational concepts have been drafted already and that can be realized promptly. positive examples of such "no-regret measures" can be found in the transport sector, for instance. this sector is severely lagging behind in terms of climate change mitigation, and economic rationales for public expenditures exist at least partly (briggs et al. ; low and astle ). in addition to that, numerous actors have already developed elaborated programmes of measures. those measures that can be implemented quickly, should now be launched-for instance to electrify the transport sector or to strengthen public transport. (green) recovery programmes must not only address the expenditure side. a currently still disregarded issue is the question how the required billions of euro could be raised. public expenditures for a green recovery program should at least partly be funded by polluters by implementing a carbon price and abandoning ecological harmful subsidies. such policies internalizing environmental costs would not be an extra sacrifice-but rather part of the solution both for revenue problems and for the redirection towards sustainability (for a review of the double-dividend hypothesis, see goulder ). the coronavirus crisis has opened up a window of opportunity for transformation. this should be used without getting off the regulatory track. green recovery programs must not be reduced to a mere competition for green subsidies. abandoning barriers to green investments and imposing a carbon price are equally important. where economically sensible, green subsidies should contribute to both stabilizing the economy and mitigating climate change. moreover, smart green recovery programs may contribute to raising revenues for the additionally necessary public expenditures. the world is currently facing the largest pandemic since spanish flu in . this has led to a lockdown policy on an unprecedented scale and measures of social distancing that are expected to continue. in france, as elsewhere, the "great lockdown" has disrupted food production chains through simultaneous shocks to demand and supply. populations have seen their food consumption habits be severely modified (e.g. closure of restaurants and markets). fearing food shortages, consumers have often stockpiled basic necessities, which has led them to actually provoke shortages, albeit temporarily. food production has also been disturbed, most notably by the reduction in the available workforce, whether domestic (due to the lockdown, illness or childcare) or foreign (temporary closure of borders). the issues at stakes are numerous, including deglobalization and environmental impacts, in both the long and short terms . the pandemic will lead to unprecedented uncertainties in food supply chains. in addition to the health crisis, a dramatic drop in worldwide gdp is anticipatedapproximately %-with a partial catch-up in , according to the international monetary fund (imf ). the reduction in income will soon impact food consumption. some authors are already pointing to an increase in social inequalities. the most fragile populations could slide into severe food insecurity, including in developed countries, as shown by deaton and deaton ( ) for canada and power et al. ( ) for the uk. however, the fao is rather reassuring about the total volume of food (cereal) at the global level. it also notes that the world price index has fallen in recent months. this does not necessarily mean that this decline is effective regardless of the type of agricultural production. in particular, fruits and vegetables could become more expensive. agriculture is by nature a risky activity, and the adoption of eco-environmental practices can only increase this risk. as a consequence, organic farmers, for instance, could be particularly affected by the combination of health and economic crises. the cost of organic production is therefore structurally higher, which could pose a problem in the event of an income shock. to add value to their production, many small farmers combine organic farming with short food supply chains. this makes it possible to regain a more comfortable margin than that allowed by mass distribution. however, here again, the health crisis has profoundly changed marketing channels. while supermarkets were stormed with shoppers at the beginning of the crisis, some short channels were closed, such as markets and restaurants, to preserve social distancing. this article therefore considers the consequences of covid- for sustainable agricultural practices, particularly for farmers who have chosen organic farming in short supply chains. to do so, we use the multi-level perspective (geels ) approach to conceptualize socio-economic transitions. the multi-level perspective (mlp) approach analyses transitions as mutation processes from one socio-technical regime to another under the pressure of macrolevel forces and the emergence of market niches that could provide the basis for a new regime (geels ) . we show here why local food supply chains are perceived by some farmers-especially the smallest-as a way to enhance the economic value of eco-friendly agricultural practices, such as organic farming. in france, the current dominant socio-technical regime based on conventional agriculture emerged gradually in the s. conventional agriculture diffuses slowly at first because of the high investment costs. however, the french state and farmers' unions support it as a means of increasing production and improving living and working conditions. farmers are then inclined to take on more debt and become dependent on their suppliers (phytosanitary products, seeds, tools, etc.), the food industry and supermarkets. as a result, this modernization of agriculture has been controversial from the outset, at least with regard to farmer autonomy and the country's food sovereignty (levidow et al. ). the early development of organic farming in france began in the s due to the effects of three negative effects of conventional agriculture, namely pollution, soil impoverishment and the lack of autonomy mentioned above. the organic label has become a marketing argument that is profitable for large farms, since they achieve significant scale effects on these standardized products. large scale organic farming is therefore less demanding and, by its nature, allows to benefit from the effects of scale. this is new competition for small farmers struggling to differentiate themselves. the organic label itself does not allow farmers to free themselves from the pressures upstream and downstream in the production chain. to differentiate themselves from large organic farms, some farmers are starting to sell in open-air markets again. at the same time, the idea that fair trade can concern north-north relations and not only north-south relations is beginning to emerge, which favours the development of community-supported agriculture. a new initiative is gradually emerging: collective farmers' shops. these initiatives began, for example, in the south of france in the mid- s and represent a more important restructuring of the farmers' market. in the medium term, the drop in income could increase the consumption of basic necessities (giffen goods) to the detriment of organic products. everything will depend on the elasticity of the demand for these products. until now, organic products have been extremely popular. it is possible that "industrial organic" will take market share from a "more artisanal organic" if consumers still want to consume organic products but cannot afford to spend a large part of their budget on them. farmers in short supply chains often have several distribution channels simultaneously, especially market gardening farmers. this allows them to be more responsive to demand. thus, the closure of farmers' markets and restaurants can be compensated for by farm shops, community-supported agriculture (csa), fixed point or home delivery, or collective farmers' shops. regarding hygiene measures, farmers are faced with two main types of strategies: receiving customers in the original locations or digitizing the process, including the more marginal case of automatic food dispensers (e.g. for eggs). selling in physical places must respect social distancing, which discourages consumption. however, conserving this method makes it possible to maintain the relational proximity between farmers and consumers. initially, this relational proximity is one of the main arguments in favour of short supply chains, as it is supposed to allow better traceability of products and to fight against social isolation (especially of the elderly). the other strategy, therefore, is to limit direct contact between human beings by means of computer tools. internet platforms already existed before the crisis and are expected to develop. some were public, such as those set up to supply school canteens. the pandemic has accelerated this process, and some regions have launched their own platforms. this solution is very time-consuming and seems difficult to sustain, unlike the others, which should have an impact in the long term. another practice has been reinforced: farm shops. during the lockdown, some consumers presumably had more time to cook and go to farms. others fled from supermarkets, considering that the products were handled by a large number of people and that there was too much traffic. it is difficult to predict whether these changes in behaviour will have a long-term impact. in addition, the pandemic has raised concerns about the reliability of international distribution channels. it is therefore possible that some policies may be sensitive to the has been growing at a very fast pace (see an indicative list of references in supplementary material ). however, little attention has been paid to the reliability of this type of epidemiological data to make statistical inferences. our initial aim was to produce a detailed statistical analysis of the relationship between weather conditions and the spread of covid- . this question has attracted significant attention from the media (e.g. ravilious ; clive cookson ) and the research community (e.g. araujo and naimi ; carleton et al. ; see a wider list in supplementary material ) due to the possibility that summer weather might slow the spread of the virus. after going through all the steps of such an analysis, we reached the unexpected conclusion that the limitations of the available covid- data are so severe that we would not be able to make any reliable statistical inference. this applies, for example, to the data provided by the john hopkins university (dong et al. ) and the data collated by xu et al. ( ) . this is a concerning and very important finding considering that such data are being widely used to make crucial policy decisions on a wide range of topics. since invalid causal inferences could be made with the publicly available covid- data, and then enter policy-making discourse, there is an urgent need to raise awareness among the scientific community and decision makers regarding the limitations of the information at their disposal. the elements discussed in this paper are also likely to be applicable to other epidemiological datasets obtained with insufficient testing and monitoring, either during exceptional epidemics or seasonal outbreaks. several challenges could undermine any causal statistical analysis of the influence of a potential determinant, such as the weather, on the spread of covid- . to start, confounding variables are likely to pose a significant problem: many factors (e.g. changes in policy or social interactions) are simultaneously influencing how the disease spreads. in addition, significant challenges come from the limitations of the covid- case count data itself. firstly, testing capacity has been a major issue in most countries. before march , very few countries had sufficient testing capacity. by april , highincome countries had significantly increased their testing capacity, but testing remained critically infrequent in most low-and middle-income countries. figure , panel a, illustrates the effect that insufficient testing capacity has on the number of confirmed cases. it distinguishes between three phases of limited (i), intermediate (ii) and widespread (iii) testing. in phases i and ii, there is a risk that the number of confirmed cases depends more on the number of tests available than on the actual number of people who have covid- , questioning the validity of any analysis relying too heavily on these data. moreover, there have been numerous concerns regarding the accuracy of the covid- tests performed so far (ai et al. ; apostolopoulos and tzani ; hu ; hall et al. ) . figure , panel b , illustrates the effects of both false-negative and false-positive test results on the number of confirmed cases. false-negative results would imply that the number of confirmed covid- cases is underestimated. false-positive results would imply that people who do not have covid- are included in the number of confirmed covid- cases. concerns regarding test accuracy create an additional problem of measurement that might affect statistical analyses. the two above-mentioned challenges are inherent in all current datasets of covid- confirmed case count and mortality. in addition, specific datasets may have imperfect geographical or time coverage. to look at the impact of the weather on the spread of covid- , we initially used a well-established approach, similar to the ones used previously to look at the impact of the weather on other diseases (e.g. deschenes and enrico ; gasparrini et al. ) (see details in supplementary material ). however, the fundamental measurement issues associated with the covid- case count data cannot be corrected by statistical techniques, as we outline in the following. the main problem is that the weather could be influencing the number of tests carried out and the segment of the population tested. for example, other respiratory diseases are often similar to covid- in their symptoms (e.g. who ) and are more common during cold weather (e.g. deschenes and enrico ; gasparrini et al. ) , which could influence the number of tests performed on people displaying symptoms of respiratory infection. therefore, even if the model correctly identified the impact of the weather on covid- case counts, it cannot distinguish between the impact of the weather on the spread of the disease and its impact on testing. table provides a non-exhaustive list of elements that could undermine any analysis of the impact of the weather on the spread of covid- using data on confirmed cases. the evidence suggests that the weather may correlate with the number of tests conducted and who gets tested. we have not been able to find any specific covid- -related evidence that the weather could impact test accuracy (e.g. the weather affecting the nasopharyngeal or oropharyngeal swabs used in the pcr analysis), even though this could be possible. other points of concern include: the fact that there may be indirect effects of weather conditions on other factors that could have an impact on the spread of covid- (such as social interactions or air pollution); the heterogeneity of impacts across populations and subgroups within a population; and the fact that some people may have travelled and therefore been infected in a different place from where the cases are reported. we ran our model (as detailed in supplementary material ) and provide results and robustness checks in supplementary material . the model would technically suggest a negative correlation (e.g. colder days would be associated with more confirmed covid- cases, and hotter days with fewer cases). yet, these results could be highly misleading since these estimates are likely to be substantially biased because of the aforementioned reasons. figure , panel a, provides an illustration of how we could have obtained a negative correlation even if temperature had no impact or a positive impact on the spread of covid- in our sample. the total number of estimated cases is given by the size of the circles as a function of temperature (x-axis). the circles in green correspond to the effects we are interested in-those that explain the influence of temperature on the spread of covid- . if temperature has no effect on the spread of covid- , then the green circles should be the same size at low and high temperatures. the pink circles represent the possible effect of temperature on testing (as reported in table ) under the illustrative assumption that high temperatures reduce testing frequency. in this case, the overall result is a negative correlation between temperature and confirmed covid- cases, even if temperature has no effect on the spread of the disease. in practice, we naturally do not know the direction of the bias caused by the effect of temperature on testing when using standard statistical methods. there is also no way for us to evaluate the contribution of each of these effects (green table non-exhaustive list of reasons why weather conditions could affect the number of covid- tests carried out and who gets tested potential reason potential implication unrelated respiratory diseases are weather sensitive (e.g. deschenes and enrico ; gasparrini et al. ) and can be confused with covid- (e.g. ai et al. ; chen et al. ) more patients with symptoms of unrelated respiratory diseases could be tested during cold weather the prevalence of other weather-sensitive respiratory diseases might make false-positive results more likely, especially if only radiographic imaging is used, since it is possible to confuse these diseases for covid- (e.g. ai et al. ; chen et al. ) the incidence of other pathologies (e.g. cardiovascular diseases) is influenced by the weather (e.g. deschenes and enrico ; gasparrini et al. ) hospital capacity and the workload of medical staff and testing structures are affected by weather conditions, with potential implications on the number of tests conducted at-risk individuals suffering from unrelated conditions are more likely to be tested for covid- , even if they only have mild symptoms for covid- people may be more inclined to seek medical attention depending on the weather (e.g. norris et al. ) due to weather conditions, people may or may not decide to seek medical attention, affecting the number of patients going to the hospital with covid- , and the workload of medical staff or pink) in our estimate. we arrive at the final size of the circles and cannot be sure if the association that we are interested in is either negative, null or positive. figure , panel b, focuses on the risk that effects could be different across different samples. the circles in blue capture other underlying factors that are influenced by temperature (such as acclimatization or the level of social interactions in the population), as well as other socioeconomic factors (such as the demographic characteristics of a population). these factors could be radically different in different regions but may also evolve over time (e.g. between winter and summer seasons). there are strong reasons to be concerned with the scenario illustrated in figure , panel b. in our sample, for example, we only have data from the start of the pandemic until end of april ; some countries (e.g. china) may be over-represented in the dataset; and the average daily temperature is relatively low at . °c. furthermore, many countries have implemented a stringent containment policy during the period covered by the sample. containment policies may have heightened (or lowered) the sensitivity of the spread of the disease to the weather because social interactions are limited. we are not able to observe how for example, sars-cov- is more infecƟous; the weather affects people's immune systems; people-to-people transmission is higher. for example, changes in the number of people with respiratory diseases other than covid- , affecƟng the number of tests performed. for example, social interacƟons evolve; or samples are for different countries. example temperature (a) esƟmates are likely to be biased because the weather influences data collecƟon (b) esƟmates could vary between samples fig. effects potentially captured by our estimate. the size of the circles represents the estimated number of cases at different temperatures. these are examples that do not correspond to actual data. in these examples, we assume no correlation between temperature and the effects in green (see legend), a negative correlation with the effects in pink (example ) and a positive correlation with those in blue. (color figure online) the impact of the weather on covid- might change at different gradients of social interaction. finally, our estimate is based on small, observed changes in temperatures, and not on radical increases or reductions in temperatures. the spread of covid- may respond differently to large variations in temperature, e.g. by °c or °c across seasons, making seasonal predictions even more unreliable. strong precautions need to be taken before using covid- case count datasets for inference. the results of our model using existing covid- data would seemingly imply a negative association between temperature and confirmed covid- cases. any projection of covid- cases with such estimates could conclude that, during the upcoming months of june to september , southern hemisphere countries would be exposed to higher risks of covid- spread and northern hemisphere countries to lower risks. these types of unsubstantiated results could be used as a misinformed justification for an early relaxation of effective social distancing measures in the northern hemisphere. these findings have equally strong implications for statistical analyses focusing on other questions that rely on covid- confirmed case count and/or mortality count data. even though the exact nature of the effects may change, such studies are also at risk of capturing the effect that their parameters of interest have on tests and test results. for example, studies interested in the effect of containment policies may have to consider that these policies substantially affect testing because they change the awareness of the disease in the population, political demands for more testing or the risk of contracting other respiratory diseases. other studies may also produce estimates that are very specific to the current circumstances in the development of the pandemic and are, therefore, not suitable to use for forecasts of what could happen in the coming months. in the medium term, more reliable data need to be gathered, for example through experimental studies that randomly test a sample of the population for covid- . in the short term, we are in a situation of fundamental uncertainty about how different factors affect or are affected by the widespread societal changes we see with the covid- pandemic. therefore, scientists, policy makers, journalists and the general public need to be very cautious when discussing how the spread of covid- correlates with the weather or any other factor. in the long term, this paper suggests that more attention should be given to how epidemiological data is recorded and used during exceptional epidemics and seasonal outbreaks, since insufficient testing and monitoring can undermine essential statistical analyses. this article calls for the complementary use of different methods for data collection, such as random testing in samples of the population. sars to novel coronavirus-old lessons and new lessons global rise in human infectious disease outbreaks carbon emissions come roaring back: will the economy, too? 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( ) consulted on th april use of radiographic features in covid- diagnosis: challenges and perspectives an empirical investigation into factors affecting patient cancellations and no-shows at outpatient clinics cohen is the first author. he had the original idea, wrote most of the paper and the code to produce the econometric analysis. he also coordinated the team. jani ensured the material was consistent with the epidemiological evidence. li produced the required climate data for the statistical analysis. lu helped on literature review, on coding the econometric analysis and on producing the tables. schwarz helped on data coding and matching and created the projections. all authors contributed to the text. data and materials availability all data and software are publicly available at https :// githu b.com/morit zpsch warz/covid - -weath er-oxfor d. key: cord- -hp ao i authors: narajewski, michal; ziel, florian title: changes in electricity demand pattern in europe due to covid- shutdowns date: - - journal: nan doi: nan sha: doc_id: cord_uid: hp ao i the article covers electricity demand shift effects due to covid- shutdowns in various european countries. we utilize high-dimensional regression techniques to exploit the structural breaks in demand profiles due to the shutdowns. we discuss the findings with respect to coronavirus pandemic progress and regulatory measures of the considered countries. the ongoing coronavirus pandemic in and especially the preventive measures to reduce the covid- disease changed drastically the patterns of our behaviour. many countries in europe and in the world introduced multiple levels of restrictions: companies sent their office employees to work from home, schools and universities closed, many factories limited or stopped their production, curfews and similar stay-at-home orders. all these factors impact the energy demand by decreasing the overall level and changing its behaviour. in this paper, we analyse the change in electricity demand pattern in selected european countries caused by the covid- shutdowns. for the analysis we consider the five most populated countries of the european union: germany, france, italy, spain and poland. the spread of the coronavirus as well as the undertaken coronavirus measures are on multiple levels in these countries in spring . also, the demand shifts are different for each of the countries what is depicted in figure . in europe, the pandemic started in italy and this is also reflected in the electricity load change in figure . a very high rise of the number of infected people in the beginning of the outbreak resulted in a very strict lock-down in the whole country (flaxman et al., ; saglietto et al., ) . thus, we focus particularly on the electricity demand of italy. the time of this analysis its progress differs significantly -from a very similar in spain to much lower in poland. in the next section, we present the data used for the analysis of the electricity demand. then, the utilized methodology and the model are discussed. the fourth section consists of the results which are presented and analysed separately for italy and for the other countries. the last section concludes the paper. the data utilized in purpose of this exercise was downloaded from the publicly available entso-e ( ) transparency platform. we use the actual total load data of all mentioned countries, and they span the data range from january to april . a small part of the data is presented in figure . it shows the electricity demand in electricity demand is usually observed. therefore, in order to recognize whether the change in the load is shutdown-, season-, or weather-driven we need a sophisticated demand model to disentangle the reduction effects. for exploiting the structural changes in the electricity demand due to the shutdown we apply a high-dimensional time series change-point models to the electricity log-load of each country. as baseline for the analysis of the structural changes we consider a model that is very similar to the load forecasting model of ziel and liu ( ) that was successfully applied in the framework of the global energy forecasting competition for electricity load forecasting. we refer for technical details to the aforementioned paper. however, here we want to describe the relevant model properties that are important to understand and interpret the results. for the analysis we consider a baseline model that assumes no structural changes in the data. then, this model is augmented by change-point components. first, we describe briefly the baseline model, to proceed with the change-point part. the baseline model contains mainly two types of components i) pattern-based time-varying coefficients and ii) autoregressive effects. the time-varying coefficients vary mainly seasonally and capture daily, weekly and annual effects. for the annual effects we distinguish between calendar-based effects (e.g. an effect that occurs every specific calendar date, e.g. christmas on december) and effects that are driven from the meteorological cycle with a periodicity of . days. the latter contains rather smooth changes as the meteorological impact changes smoothly over the year. further, the model contains interactions between the seasonal components, especially the daily cycle may change over the year. next to date-based calendar effects we also include other calendar effects. most notably holiday effects from public holiday that have a varying date, e.g. easter monday. the intercept of the considered model changes with all the mentioned time-varying components. the autoregressive components contain historical load data from the last hour up to the last weeks. however, we only let the most recent information to vary over time with selected time-varying components, but keep the remaining autoregressive terms constant. the autoregressive components absorb a lot of information from the past, indirectly also the information from typical external regressors like temperature. here, we want to remark that we double-checked that the additional information of temperature in our model is negligible. in simple words: if we are at pm today and want to predict the load in hour for today, i.e. at pm, the temperature (forecast) for pm does not help a lot to improve the load forecast as the temperature information is hidden in the most recent demand observation at pm, see e.g. haben et al. ( ) for similar findings. given the baseline model, we augment the time-varying intercept of the model by changepoint components that allow for different types of structural breaks. this is: i) a permanent change in the load level, ii) a permanent change in the load level for the daily profile (e.g. a load reduction for only certain hours of the day), iii) a permanent change in the load level for the weekly profile (e.g. a load reduction for only certain hours of the week). these structural breaks are implemented using dummies for relevant time sets. we restrict the space of possible changes to all observations after march which is before the coronavirus spread widely in europe and issued the covid- crisis in europe. we estimate the model using lasso which is tuned by the bayesian information criterion (bic). to analyse the results adequately, we estimate the model and then simulate from the estimated model times for the time range from march onwards. this allows us to get other plausible paths of the effect. we regard the mean of the mentioned trajectories as the profile under the shutdown. we also simulate from the estimated model where we set all change-point effects to . this allows us to mimic a load situation without the covid- shutdowns. again, the corresponding average describes the profile that we want to compare. another interesting aspect is that the structural change due to the shutdown of the nonnecessary commercial business is quite smooth and requires a couple of days to settle at the corresponding load level. this suggests that after the mentioned shutdown some businesses were still running for a few days prior closing. first, let us note the overall decrease of the load in the shutdown scenario. an interesting effect is the flattened morning peak (around am - am). this is most probably a result of many people working from home or not working at all and thus lesser utilization of production capacities, office building and electrified public transport etc. interestingly, the evening peak in demand is preserved and currently it is clearly the most electricity consuming part of every day in the week. this is reasonable as because of the lockdown, more people are cooking at home or using electricity-based entertainment. furthermore, the difference in electricity demand between saturday and sunday shrank heavily due to the shutdowns. another interesting feature is that we see shifts of the morning load peak within the day. this is best visible on sundays: usually at am the load level would increase by about . gwh (≈ % of the night load) from the night level. during the shutdown the increase starts later, at am we still remain at the night level load. a plausible explanation would be a 'getting up late'-effect. so the italians tend to sleep longer during the lockdown period. figure presents the electricity demand in the other considered countries: germany, france, spain and poland. the overall pattern of the rising deviation between the shutdown and no-shutdown models is similar to the one in italy, but respectively delayed. however, the level of the deviation differs among the states, what was already depicted in figure . interestingly, in france we observe an impact of the shutdowns before they went live. the reason may be that the limitations were announced accordingly earlier and the residents and companies of france may have started changing their public activity earlier, following the other countries' recommendations. furthermore, even before the national lock-down all big events, football matches etc. were being cancelled. however, there might be interactions with the export of electricity (esp. to italy) and temperature effects. concerning the latter, the period from march to april was relatively cold in europe, and france has a high temperature dependency in the electricity demand due to large electric heating capacities. figure shows the comparison of weekly demand over hours of the day between the shutdown and no-shutdown scenarios for germany, france, spain and poland. again, the plots are based on the week from march to april . similarly as in the italian case, we observe an overall demand decrease for every country. let us note that except of the level change, the weekly demand pattern remained almost the same in germany. on the other hand, in france, spain and poland the flattening of the morning peak and preserving the evening peak are present similarly as in italy. this can be also explained by lesser activity in the morning connected to the professional life and remained or even higher activity in the evening due to entertainment. the 'getting up late'-pattern is also visible in all the considered countries. still, it is most distinct in the mediterranean countries: france and spain. the shutdowns introduced due to the covid- pandemic have impacted significantly both the level of the electricity demand in europe and its weekly pattern. the revocation transparency platform report : estimating the number of infections and the impact of non-pharmaceutical interventions on covid- in european countries short term load forecasting and the effect of temperature at the low voltage level covid- in europe: the italian lesson lasso estimation for gefcom probabilistic electric load forecasting key: cord- -iska pt authors: sharma, pravesh; ebbert, jon o; rosedahl, jordan k; philpot, lindsey m title: changes in substance use among young adults during a respiratory disease pandemic date: - - journal: sage open med doi: . / sha: doc_id: cord_uid: iska pt background: news articles, commentaries, and opinion articles have suggested that ongoing social distancing measures coupled with economic challenges during covid- may worsen stress, affective state, and substance use across the globe. we sought to advance our understanding of the differences between individuals who change their substance use patterns during a public health crisis and those who do not. methods: cross-sectional survey of young adults ( – years of age) assessing respondent characteristics and vaping, tobacco, alcohol, and/or marijuana use. we calculated prevalence estimates, prevalence changes, and prevalence ratios with associated % confidence intervals and looked for differences with the chi-square test. results: of the total sample, . % (n = / ) young adults reported vaping or using tobacco, alcohol, and/or marijuana. among the respondents reporting use, . % reported a change in their use patterns. among respondents reporting changes in substance use patterns during the pandemic (n = ), . % reported an increase in alcohol use, . % reported a decrease in vaping product use, and . % reported a decrease in tobacco product use due to covid- . substance use changed significantly for respondents with increasing degree of loneliness (continuous loneliness score: prevalence ratio = . , % confidence interval = . – . ), anxiety (prevalence ratio = . , % confidence interval = . – . ), and depression (prevalence ratio = . , % confidence interval = . – . ). conclusion: self-reported substance use among young adults was observed to change during a pandemic, and the degree of loneliness appears to impact these changes. innovative strategies are needed to address loneliness, anxiety, depression, and substance use during global health crises that impact social contact. strain function as independent and interactive risk factors for substance use. , scientists have observed changes in the use of substances during large scale, traumatic events such as increased cigarette and alcohol use in adults following hurricane katrina compared to pre-hurricane prevalence. similarly, it was observed that utilization of cigarettes, alcohol, or cannabis increased among new york city residents, especially who experienced symptoms of post-traumatic stress disorder and depression, following the september terrorist attacks. however, studies tracking substance use in young adults during an ongoing major respiratory threat and large-scale traumatic events are lacking. this study assessed the self-reported changes in substance use among a young adult population during an ongoing respiratory disease pandemic with imposed social distancing. we assessed differences between individuals who reported changes in substance use and those who did not in domains of demographic characteristics, self-reported anxiety, depression, loneliness, and substance use and direction of change. we developed a survey tool (survey tool is provided as supplemental appendix) to understand the prevalence of substance use within a young adult population in a large, mixed urban/rural midwestern setting. patients were included in this study if they had been seen at one of our outpatient practice settings over the prior . months, were between the ages of and years as of january , and had a documented email address. the patients included were seen for any indication including, but not limited to, substance use. we did not have any specific exclusion criteria. based on recent analysis of national health interview survey (nhis) data, the percentage of persons aged - years, who reported using e-cigarettes at least once during their lifetime and now used "every day" or "some days," was . %. since the original intent of this study was to characterize electronic vaping product (evp) use in an outpatient setting, we wanted to have a meaningful sample size such as of subjects, therefore, we deployed the survey to patients. an electronic survey tool was deployed via qualtrics ® survey software (provo, ut). mayo survey research center, who deployed the survey, has the proper arrangements both internally (with our institutional review board (irb) and in compliance with hipaa) and with qualtrics ® to be used for clinical research. in the month of march , due to covid- , local and state government officials implemented stay-at-home guidelines and encouraged social distancing between individuals. the orders were further extended to the month of april and may. our survey was administered in april during the time of this stay-athome order. all survey responses were anonymous to the research team. the study was approved by irb at mayo clinic through expedited review procedures and determined that the study constitutes minimal risk. due to the timing of our survey launch with the covid- pandemic, we included one additional question to assess "do you think your use of electronic vaping products, tobacco products, alcohol, or marijuana (any of these) changed (increased or decreased) since coronavirus (covid- ) outbreak?" if individuals answered "yes," we then provided subsequent questions to assess the directionality of change of each product type as: "which of the following products and to what direction this change happened?" with the response option of "increase / decrease" for each substance type. our survey instrument also included demographic information (age, gender, educational status) and requested each respondent to answer "have you ever been told by a healthcare provider that you have [anxiety; depression]?" these factors were used in the present analysis. we assessed the presence of loneliness through the validated three-item university of california, los angeles (ucla) loneliness scale. we present both continuous loneliness scores (range, - ) as a sum of individually answered questions, as well as a dichotomized loneliness variable where the continuous loneliness score was greater or equal to , similar to other reports. the higher ucla loneliness scale scores indicate greater degrees of loneliness. we focused our analyses on the differences between individuals who changed substance use patterns and those who did not. we present frequencies and simple proportions (%), and due to the cross-sectional nature of our study, we have calculated prevalence estimates, prevalence changes, and prevalence ratios (prs) with associated % confidence intervals (cis). analyses were done via chi-square test unless test assumptions were unmet, whereby fisher's exact test was performed. we used statistical analysis software (sas) version . (cary, north carolina), and employed a p < . for significance for statistical testing and considered our pr significant if they did not span the null. our overall survey had a response rate of . % ( patients of ), . % ( patients of ) of whom reported vaping or using marijuana, tobacco, and/or alcohol during the covid- pandemic. of the individuals using substances who reported current use of one of our substances of interest, reported a change in their substance use patterns ( . %; / ; table ). overall, . % ( patients of ) of the population used substances of interest and changed use patterns. compared to individuals not reporting a change in substance use, individuals reporting a change in substance use were younger (p = . ), had more self-reported anxiety (p = . ), had more self-reported among participants reporting substance use change, we observed the largest reported change in directionality among those who used alcohol ( . % increase). participants reported decreased usage among inhaled substances (vaping product . % decrease; tobacco product . % decrease), and a split in reported changes by marijuana using participants ( . % increase, . % decrease). when analyzing within substance type groups (electronic vaping, marijuana, tobacco product, alcohol), we did not observe significant differences by report of loneliness, anxiety, or depression for changes in substance use direction (increased vs decreased usage). we observed that, overall, less than one in five respondents used substances of interest and changed their use patterns during the respiratory disease pandemic. more than % of respondents reported vaping or using marijuana, tobacco, and/or alcohol. we observed that . % of respondents who used substances of interest reported a change in their substance use patterns during the covid- pandemic, with the highest proportion of respondents reporting a change in the amount of inhaled substances (decrease) and alcohol (increase) they used. we also observed that the proportion of respondents reporting a change in their substance use patterns differed by age, self-reported anxiety and depression, and degrees of loneliness. we observed that respondents who reported a change in their substance use patterns reported decreased use of inhaled substances. since covid- is a respiratory illness, and media coverage has disseminated information about the severe manifestation of this virus on the lungs, especially among those who smoke and vape, young adults may be exhibiting increased caution about using combustible forms of substances. this notion is supported by rohde and colleagues who surveyed adult tobacco smokers and found that respiratory harm health consequence was a major reason for vaping discouragement. conversely, we observed reported increased use of alcohol. studies have shown that young adults often indulge in drinking behavior not only for celebration but also to cope with aversive mood states, which prolongs or worsens negative affective states and further drinking. the increase in alcohol use by young adults could be related to an attempt to alleviate negative affective states triggered by social strain, as we observed change in substance use tied to increasing degree of loneliness in our population. in a recent study, it was projected that prolonged or intermittent social distancing measures need to be practiced until to prevent the resurgence of covid- . if social distancing is to continue until or we are at risk of future pandemics then nontraditional socialization opportunities for people with poor emotional health and loneliness to mitigate social strain are essential. social isolation may also affect patient's access to treatment, therefore, multidisciplinary and integrated treatment delivery approaches utilizing digital platform and technology are required to better serve patients with poor mental health. our study has limitations including a reliance on selfreported data and potential for recall bias and we did not assess amount of change. we suffered from low response rate ( . %), lower than other surveys of the same population. research has shown that young adults may be less likely to participate in research studies, and studies of self-reported risky behaviors such as substance use can struggle to enroll. our study is limited because apart from the loneliness scale, no other survey components were validated. our study also had several strengths, including the timing of our study during the covid- pandemic, and the use of a validated measure of loneliness. we need to advance our understanding of how best to support individuals with anxiety, depression, and loneliness during times of social distancing measures. our data emphasize the importance of advancing our understanding of how substance use changes with pandemics and what infrastructure could be built leveraging technology to provide assessment and support to patients with mental health concerns and substance use. severe acute respiratory syndrome coronavirus (sars-cov- ) and coronavirus disease- (covid- ): the epidemic and the challenges the covid- pandemic in the us: a clinical update a bold response to the covid- pandemic: medical students, national service, and public health chronic stress, drug use, and vulnerability to addiction relationship between loneliness, psychiatric disorders and physical health ? a review on the psychological aspects of loneliness incidence of drug problems in young adults exposed to trauma and posttraumatic stress disorder: do early life experiences and predispositions matter alcohol and cigarette use and misuse among hurricane katrina survivors: psychosocial risk and protective factors sustained increased consumption of cigarettes, alcohol, and marijuana among manhattan residents after notes from the field: use of electronic cigarettes and any tobacco product among middle and high school students: united states a short scale for measuring loneliness in large surveys: results from two population-based studies loneliness in primary care patients: a prevalence study e-cigarette health harm awareness and discouragement: implications for health communication a motivational perspective on risky behaviors: the role of personality and affect regulatory processes projecting the transmission dynamics of sars-cov- through the post-pandemic period social relationship quality among patients with chronic pain: a population-based sample surveying adolescents enrolled in a regional health care delivery organization: mail and phone follow-up: what works at what cost all authors have contributed substantially to the manuscript. the author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. ethical approval for this study was obtained from mayo clinic institutional review board (approval number/msr #: - -h ). the author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: this study was funded by the office of mayo clinic health system research. the irb reviewer approved waiver of the requirement for the investigator to obtain a signed consent form in accordance with cfr . as justified by the investigator. pravesh sharma https://orcid.org/ - - - lindsey m philpot https://orcid.org/ - - - supplemental material for this article is available online. key: cord- -mhqe xjz authors: küchenhoff, h.; guenther, f.; höhle, m.; bender, a. title: analysis of the early covid- epidemic curve in germany by regression models with change points date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: mhqe xjz we analyze the covid- epidemic curve from march to end of april in germany. we use statistical models to estimate the number of cases with disease onset on a given day and use back-projection techniques to obtain the number of new infections per day. the respective time series are analyzed by a poisson trend regression model with change points. the change points are estimated directly from the data without further assumptions. we carry out the analysis for the whole of germany and the federal state of bavaria, where we have more detailed data. both analyses show a major change between march th and th for the time series of infections: from a strong increase to a stagnation or a slight decrease. another change was found between march th and march st, where the decline intensified. these two major changes can be related to different governmental measures. on march, th, chancellor merkel appealed for social distancing in a press conference with the robert koch institute (rki) and a ban on major events with more than visitors (march th) was issued. the other change point at the end of march could be related to the shutdown in germany. our results differ from those by other authors as we take into account the reporting delay, which turned out to be time dependent and therefore changes the structure of the epidemic curve compared to the curve of newly reported cases the first phase of the covid- pandemic in germany was managed relatively successful in comparison to other countries in europe. therefore, it is worth taking a closer look at the course of the pandemic in germany, which has already led to controversial discussions. this particularly concerns the important question about the effectiveness of various control measures. there are several publications using data from different countries on the effects of control measures, see, e.g., [ ] , [ ] and [ ] . as [ ] point out, many of such studies are undermined by unreliable data on incidence. many papers use data provided from the johns hopkins university (jhu) [ ] . these data are based on cumulative registered cases in different countries, which induces several problems, particularly the fact that not all cases are reported and that there is delay between the day of infection and the reporting day. furthermore, the systems of reporting vary between countries, which makes comparisons between countries difficult. therefore we focus on the analysis of the epidemic curve in bavaria and germany. the availability of case based data for bavaria and detailed data for disease onset for germany is essential for our analysis. in a recent paper on germany by [ ] , the authors use a complex bayesian modeling approach based on the daily registrations in the jhu data. an important claim by [ ] is that the lock-down-like measures on march rd were necessary to stop exponential growth, however, this result contradicts for example results by the german rki [ ] . furthermore, these approaches were critically questioned by [ ] and [ ] , where the latter emphasized the importance of taking into account the delay by reporting and incubation time, when analyzing the possible effect of non pharmaceutical interventions. we follow this line of argument and use a statistical model to estimate daily numbers of infected and of persons with disease onset on a certain day. for bavaria, we use detailed case-based data, while a modeling approach is utilized for german data. we analyze the respective epidemic curves using a segmented regression model with change points. the paper is organized as follows. in section , we present the data and the the strategy of estimating the relevant daily counts. then the segmented regression model, which is the basis for further analyses, is presented. in section , we present the results followed by a discussion in section . the latter is not always known: partly because it could not be determined and partly because the case did not (yet) have any symptoms at the time of entry into the data base. a procedure for imputation of missing values regarding the disease onset has been developed by [ ] , using a flexible generalized additive model for location, scale and shape (gamlss; [ ] ), assuming a weibulldistribution for time t d > between disease onset and reporting date. we estimate the delay time distribution from data with disease onset and impute missing disease onsets based on this model. for the german data, no individual case data were available, so instead we used estimated disease onset data provided by the robert koch institute (rki), see [ ] and [ ] . the method used by the rki is similar to our approach applied to bavarian data [ ] . to interpret the course of the epidemic and possible effects of interventions, case based data on time of infection is essential. however, as such data is generally not available, one simple approach is to shift the curve to the past by the average incubation period. the average incubation period for covid- is about five days [ ] . a more sophisticated approach is to use the incubation period distribution as part of an inverse convolution, also known as backpropagation, in order to estimate the number of infections per day from the time series of disease onsets [ , ] . we assume a log-normal distribution for the incubation time with a median of . . days and a % percentile at . days [ ] . these are the same values as used by [ ] . for our calculation, we use the back-projection procedure implemented in the r package surveillance [ ] . to analyze the temporal course of the infection we use the following poisson regression model with over-dispersion and change points (see [ ] , [ ] ): where e(y t ) is the expected number of new reported cases at time t, k is the number of change points, and x + = max(x, ) is the positive part of x. the change points are used to partition the epidemic curve y t into k + phases. these are characterized by different growth parameters. in the phase before the first change point cp the growth is characterized by the parameter β , in the nd phase between cp and cp by β = β + γ . the next change is then at time cp . in the rd phase between cp and cp the growth parameter is given by β = β + γ + γ . this applies accordingly until the last phase after cp k . the quantities exp(β j ), j = , . . . , k + can be interpreted as daily growth factors. since model ( ) is a generalized linear model given the change points, the parameters of the model (including the change points) can be estimated by minimizing the quasi likelihood function for the poisson model. due to the estimation of the change points the numerical optimization problem is not straight forward. for the estimation of the model we use the r-package segmented, see [ ] . the starting values are estimated by discrete optimization using all possible integer suitable combinations of change points. the number of change points k is varied step-wise up to a maximum of k = . it is examined whether the increase of the number of break points leads to a relevant improvement of the model fitting (over-dispersion parameter). models with more than change points since they are hardly interpretable and the danger of overfitting is high. we apply the segmented regression model to time series of the estimated daily numbers of infections for bavaria and germany. since the back propagation algorithm yields an estimate for the expected values of the number of daily infections and does so by inducing a smoothing effect, as a sensitivity analysis for the location of the breakpoints, we also apply the model to the time series of the daily number of disease onsets. when comparing the results, it should be taken into account that the onset of the disease is on average days after infection. in figure , the three different time series of daily cases (reported, disease onset and estimated infection date) are presented. the delay between the three time series for bavaria and germany is evident. furthermore, the curves do not just differ by a constant delay, but there is some change in structure of the curves. the curve relating to the date of infection is clearly smoothed due to the back projection procedure (cf. section . ) and has a clear maximum both for bavaria and germany. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october , . ; for the bavarian data on disease onset, the model with k = change points gives the best result with an estimate of the over-dispersion parameter of . , i.e., the variance of y t is . times higher than the value of var(y t ) = e(y t ) otherwise expected under the assumption of the poisson regression model. the over-dispersion for a model with k = change points is substantially higher ( . ), which suggests that the model with four change points should be preferred. table , left panel. the model delivers five phases starting with a steep increase, which is slowed down in the second phase. in the third phase starting at th- th of april, the increase is stopped and there is decrease in number of disease onsets in the fourth phase, which is accelerated in the fifth phase. the poisson model for the infection date gives a substantially better fit than the disease onset model. the model with four change points has an underdispersion by the the factor . compared to . for the model with three change points. therefore, we use the model with three change points to avoid overfitting. the result can be seen in figure (right panel) and in table (right panel). the phases are similar to those for the disease onset, but the change from increasing curve (phase and ) to a decreasing phase (phase ) is direct without a plateau in between. taking the mean incubation period of five days into account, we combine the results for the two models for bavaria, which implies four phases of the epidemic: st phase there is a substantial increase in new infections in both models. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october , . ; https://doi.org/ . / . . . doi: medrxiv preprint the results for the german data are presented in figure and in table . for the disease onset model, the overdispersion is substantially lower for the model with four change points than with three change points. however, the overdipersion of the model with four change points is rather high ( . ) and the confidence intervals for the change points are rather big, especially for the first part of the time series where two change points are estimated. while the distinction in the first three phases is unclear, there is a clear turning point between march th and march th. the fifth phase with a further slowdown starts at the end of april. as can be seen from figure there are further estimated change points in both models, which have wide confidence intervals and do not fit well together in the models. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october , . ; https://doi.org/ . / . . . doi: medrxiv preprint the present analysis is a retrospective, exploratory analysis of the german and bavarian covid- reporting data during mar-apr . the analysis does not include cases that have not been recorded. if the proportion of undetected cases changes over time, this can distort the curve and thus the determination of the change points. therefore, additional data on daily deaths and hospital admissions and the number of tests performed should be considered. furthermore, it is possible to estimate the proportion of undetected cases with the help of representative studies such as the one currently conducted in munich, see [ ] . our analysis is based to a considerable extent on imputed data, see [ ] , which is a results of missing data w.r.t. the disease onset. since changes in behavior do not occur abruptly, the assumption of change points is also problematic in itself. therefore, the interpretation of change points should always be done in conjunction with a direct observation of the epidemic curve. our analysis is based on the onset of the disease (more precisely: the onset of symptoms) and a back projection to the date of infections, and therefore, despite its limitations, is better suited to describe the course of the epidemic than the more common analysis of daily or cumulative reported case numbers. in the analysis of the bavarian and the german data in different settings, the main result is the change point, where the exponential growth was stopped and is clearly visible between march th and th. the timing of this change point coincides to the implementation of the first control measures: the partial ban of mass events with more than people. furthermore, in a press conference on march th chancellor merkel and the president of the rki appealed to self-enforced social distancing (https://www.bundesgesund heitsministerium.de/en/coronavirus/chronologie-coronavirus.html. furthermore, the extended media coverage from bergamo, italy, as well as the voluntary transition to home-office work could be related to this essential change in the course of the pandemic. in bavaria and in germany, the change point at the end of march of infection date is apparent. this change point is associated with different measures taken in march (closing of schools and stores on march th and the shutdown including contact ban on march st). since there were many measures administered simultaneously, it is not possible to attribute individual measures to the development of the epidemic curve. the claim by [ ] , that the shutdown on march st was necessary to stop the growth of the epidemic is not supported by our analysis. there is a change . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october , . point in the epidemic curve after that date, but the major change from an exponential growth to a decrease was before the shutdown. the difference in results can be explained by the different data bases used for the respective analyses. while [ ] use data based on daily registered cases, in our analysis, data on disease onset are included. as can be seen from figure and from the results of our data analysis, the delay distribution of the time between disease onset and reporting day changed over time. using this information is a crucial difference between our analysis and that of [ ] . in a recent technical addendum [ ] the authors re-fit their model on more appropriate data. these analysis -in our opinion -clearly show that the effective reproduction number decreased earlier than in their initial analysis, however, they attribute the decrease to a sir model peculiarity, where a linear decrease in the contact rate can lead to the incidence curve dropping despite r(t) > . the above discussions illustrate how complex the interpretation of even simple sir models is and the question is, if such sir modeling is not too simple to really allow for questions to be answered model based (no age structure, no time varying reporting delay, no incubation delay). in contrast, our approach is more data driven with a minimum of modeling assumptions and without the need to include strong prior information about the change points. directly using a segmented curve with exponential growth (decline) is in line with common models of infectious diseases in its early stages, where the limitation of the spread by immune persons plays no role. the problem of using complex models with many parameters for the evaluation of governmental measures has also been highlighted by [ ] . our approach is similar to that of [ ] . however, using change point analysis for variables derived from daily new infections appears problematic, since assumptions modeling the reproduction number r(t) or the cumulative numbers are questionable. more specifically, the use of the time-varying reproduction number r(t), a standard measure to describe the course of an epidemic is challenging, as different definitions have been proposed in the literature that also imply different interpretations (see [ , ] ). however, the analysis of r(t) as a relative measure can be useful, when one wants to analyze data from different countries with non comparable reporting systems, see [ ] . we prefer the direct use of a poisson regression model with a more plausible assumptions about the error terms instead of using ols for logarithmic case numbers. furthermore, we apply a direct maximum likelihood estimation of the change points of the segmented regression model. for the interpretation of the model based on disease onsets, we also use a simple difference of five days to take the incubation time into account. our results are similar to that of [ ] . however, the claim that there is no evidence for the effect of governmental measures is not supported by our analysis. our result is in line with that of [ ] , where a stop of exponential growth in great britain has been before the shutdown. furthermore, the effect of governmental measures as whole is clearly documented in the literature, see, e.g., [ ] and [ ] .our result on a possible effect of the ban of mass events is also in line with the results of [ ] . . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october , . ; the temporal connection between the change points in our analysis and various control measures should be interpreted as an association, rather than a direct causal relationship. in the end many other explanations exists and from a simple time series analysis it is not possible to say to what extent the population already had changed their behavior voluntarily, as for example observed in mobility data [ ] , and in what way the measures contributed to this. more speculative alternative explanations would include the possibility of a seasonal effect on coronavirus activity (e.g. related to temperature) or changes in test capacity or the case detection ratio. however, given the re-emergence of the epidemic in the fall of at high test capacity and at relatively high temperatures shows that contact behavior is the major explanatory factor for virus activity. nevertheless, any analysis of observational time series data including only a limited amount of explanatory factors has to be interpreted with care and with respect to the many uncertainties which remain regarding covid- [ ] . despite the limitations of the approach, we argue that it is advantageous and important to directly interpret the epidemic curve and the absolute number of cases, rather than indirect measures like the r(t). furthermore, the reproduction rate does not contain information about how many people are currently affected, or whether the infected persons belong to risk groups. the course of the time-varying reproduction number calculated by us for bavaria fits well with the change point analysis [ ] . a value of r(t) > corresponds to a rate of increase > , noting that the time delays in the interpretation of r(t) must be kept in mind. it should be noted, that the presented analysis is retrospective. control measures have to be decided based on a completely different level of information than what the retrospectively established epidemic curve suggests. the simple observation of the course of the reported case numbers by reporting date is also problematic because this course is strongly influenced by the reporting behavior and the methods and capacities of the test laboratories. typically, substantially fewer cases are reported at weekends than during the week. therefore, the estimation [ ] is an important step to estimate the better interpretable curve of new cases, but is limited by assumptions and limitations itself, that need to be considered when interpreting the results. since the impact of the measures also depends on how they are implemented by the population (compliance), the results cannot be directly transferred to the future. nevertheless, it remains a remarkable result that the clear turning point of the early covid- infection data in germany is associated with non drastic measures (no shutdown) and strong appeals by politicians. all data used for the analyses and all code to reproduce the models, figures and tables in the manuscript are openly and freely available from https://gi thub.com/adibender/covid -changepoint-analysis-germany-bavaria. all . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted october , . ; analyses were performed using the r programming language [ ] . figures were created using r package ggplot [ ] . estimating the effects of non-pharmaceutical interventions on covid- in europe physical distancing interventions and incidence of coronavirus disease : natural experiment in countries the temporal association of introducing and lifting non-pharmaceutical interventions with the time-varying reproduction number (r) of sars-cov- : a modelling study across countries. the lancet infectious diseases lockdown-type measures look effective against covid- but evidence is undermined by unreliable data on incidence an interactive web-based dashboard to track covid- in real time. the lancet infectious diseases inferring change points in the spread of covid- reveals the effectiveness of interventions the limits of estimating covid- intervention effects using bayesian models a phenomenological approach to assessing the effectiveness of covid- related nonpharmaceutical interventions in germany nowcasting the covid- pandemic in bavaria. medrxiv flexible regression and smoothing: using gamlss in r. the r series schätzung der aktuellen entwicklung der sars-cov- -epidemie in deutschland -nowcasting the incubation period of coronavirus disease (covid- ) from publicly reported confirmed cases: estimation and application a method of non-parametric backprojection and its application to aids data associations of age and sex on clinical outcome and incubation period of shiga toxin-producing escherichia coli o :h infections monitoring count time series in r: aberration detection in public health surveillance estimating regression models with unknown breakpoints modelling covid- outbreak: segmented regression to assess lockdown effectiveness segmented: an r package to fit regression models with broken-line relationships. r news protocol of a population-based prospective covid- cohort study munich model-based and model-free characterization of epidemic outbreaks -technical notes on dehning the limits of estimating covid- intervention effects using bayesian models a new framework and software to estimate time-varying reproduction numbers during epidemics association of public health interventions with the epidemiology of the covid- outbreak in wuhan, china did covid- infections decline before uk lockdown ? estimating the impact of mobility patterns on covid- infection rates in european countries covid- 's known unknowns r: a language and environment for statistical computing elegant graphics for data analysis we would like to thank katharina katz and manfred wildner from the bavarian state office for health and food safety (lgl) for providing the data and for useful discussions. we also thank nadja sauter for help with visualizations. key: cord- -wuug jjv authors: ahlers-schmidt, carolyn r.; hervey, ashley m.; neil, tara; kuhlmann, stephanie; kuhlmann, zachary title: concerns of women regarding pregnancy and childbirth during the covid- pandemic date: - - journal: patient educ couns doi: . /j.pec. . . sha: doc_id: cord_uid: wuug jjv objective: better understand knowledge, attitudes and practices of pregnant women and mothers of infants around coronavirus disease (covid- ). methods: a -item electronic survey was distributed to pregnant and postpartum women (infants < months) who were > years, english-speaking and enrolled in prenatal programs. data is summarized using central tendency, frequencies and nonparametric statistics. results: of ( % response rate) participants, . % reported negative changes in mental status measures (e.g. stress, anxious thoughts, changes in sleep patterns). all reported risk-reduction behavior changes (e.g. handwashing/use of sanitizer, social distancing). significant changes were reported in employment and financial status due to the pandemic. increases in alcohol consumption among postpartum women were also reported. few reported changes in prenatal, infant or postpartum healthcare access. conclusion: this study provides initial insight into the knowledge, attitudes and practices of pregnant and postpartum women during the covid- pandemic. this study is limited as participants represent a single midwest community and social desirability response bias may have impacted responses. however, results may inform future interventions to support pregnant women and mothers of infants during pandemics. practice implications: providers should consider the impact of such events on mental status, access to resources and changes in behaviors. first identified in wuhan, china, the coronavirus disease (covid- ) caused by severe acute respiratory syndrome coronavirus (sars-cov- ), is novel enough that minimal information is known regarding impact on pregnant women and infants [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the cdc recommends pregnant women protect themselves from exposure to covid- [ ] as early data suggests negative outcomes for infants of covid- -infected women may include preterm birth, fetal distress, premature labor, respiratory distress and perinatal death [ ] [ ] [ ] [ ] . with overwhelming misinformation regarding covid- [ ] and females expressing significantly higher levels of stress, anxiety and depression [ ] , it is crucial to assess the impact on perinatal women. the purpose of this study is to better understand knowledge, attitudes and practices of perinatal women regarding covid- . this observational cohort study utilized a convenience sample of pregnant women or mothers of infants < months enrolled in programs serving women at high-risk for poor birth outcomes (e.g. low income, uninsured). participants were > years, residing in sedgwick county and english speaking. a brief study overview and the research electronic data capture (redcap) [ ] survey link and informed consent information were emailed on april , . two reminders were sent at -week intervals. the -item survey included demographics, pandemic-related behaviors [ ] , pregnancy, infant and self-care, access to healthcare, mental health, and financial stability [ ] of potential participants, ( %) completed the survey; ( . %) were pregnant and ( . %) postpartum. no significant demographic differences were identified between pregnant and postpartum respondents; therefore, data is combined (table ) . participants reported knowing a lot (n= ; . %) or a little about covid- (n= ; . %), with few knowing nothing/being unsure (n= ; . %). most reported being very concerned (n= ; . %) or somewhat concerned (n= ; . %), with a minority not very/not at all concerned about covid- (n= ; . %). three ( . %) had been tested for covid- . significant reductions in financial status and employment were reported during the pandemic (table ). unmet needs (n= ; . %) included lack of essential items (e.g. diapers) due to low stock in stores or finances, reduced social support and inability to access lactation, therapy and other support services. few participants experienced changes in healthcare access (table ) . for those who did, it was at the physician's request prenatally, but the woman's preference for infant and postpartum appointments. participants reported changes in mental status related to the covid- pandemic (n= , . %), including increased stress (n= ; . %), increased anxious thoughts (n= ; . %), changes in sleep patterns (n= ; . %), reduced motivation (n= ; . %), increased fearful thoughts (n= ; . %), changes in appetite (n= ; . %), racing thoughts (n= ; . %), difficulty in focus and concentration (n= ; . %), depressed mood (n= ; . %) and increased tearfulness (n= ; . %). most reported no change (n= ; . %) or increases (n= ; . %) in social support. however, . % (n= ) reported decreases in support from family, friends, coworkers, and support services. many (n= ; . %) described self-care strategies such as focusing on things they can control, daily physical activity (e.g., walking, yoga), cleaning and organizing, meditation, reaching out to family and friends on the phone and limiting exposure to the news. all (n= ; %) participants reported behavior change due to the covid- pandemic, including frequent hand washing/use of sanitizer (n= ; . %), reducing time in places with > people (n= ; . %), reducing contact with people outside of own household (n= ; . %), avoiding contact j o u r n a l p r e -p r o o f with others who had symptoms (n= ; . %), avoiding touching eyes, nose or mouth (n= ; . %), and purchasing or making a face mask (n= ; . %). additional behaviors are in table . the majority reported following the state and county stay-at-home orders (enacted / / and / / respectively) all (n= ; . %) or most of the time (n= ; . %), and fewer some of the time or not at all (n= ; . %). of those currently pregnant or recently delivered, half expressed concerns about their partner's ability to attend the delivery (n= ; . %). some changed their birth plan (n= ; . %), reducing to one support person due to hospital limitations, requesting induction, or reducing elective procedures. other pregnancy changes included canceled events (e.g. baby showers, parent courses) and not allowing visitors after infant is home. changes in infant sleep practices were reported with . % (n= ) following or planning to follow the american academy of pediatrics safe sleep guidelines (alone, on the back, in a clutter-free crib) [ ] prior to the pandemic compared to % (n= ) at time of survey (p= . ). increased unsafe practices were related to bedsharing. for most, breastfeeding behavior/plans did not change ( . % (n= ) breastfeeding; . % (n= ) not breastfeeding). however, two ( . %) not planning to breastfeed decided to, six ( . %) who planned to breastfeed decided not to and eight ( . %) were unsure. other infant care behavior changes included limiting contact with infant, scheduling appointments to avoid sick patients and taking precautions such as hand washing and protective barriers when leaving the house. most (n= ; . %) reported no change in family planning strategies. of those who reported a change (n= ; . %), reasons included missed appointments delaying receipt of contraception and deciding to postpone subsequent pregnancies. due to covid- women also reported decreased likelihood of talking to doctor about medications and mental health concerns. if a covid- vaccination became available, . % would be interested in receiving it (n= ); . % (n= ) would not and . % (n= ) were unsure. concerns included side effects or sickness (n= ; . %), cost (n= ; . %), allergy to vaccines (n= ; . %) and perception it is unnecessary (n= ; . %). this exploratory study identified behavior changes of perinatal women specifically attributed to the covid- pandemic. surveys were collected while cases of covid- were increasing daily [ ] . the state and county stay-at-home orders expired may , [ ] but were in effect for most of the data j o u r n a l p r e -p r o o f collection. positive behaviors, such as adherence to the stay-at-home orders and following cdc risk reduction recommendations [ ] , were reported by most participants. in addition, smaller numbers reported positive impacts such as increased physical activity and decreased consumption of unhealthy foods or use of substances. for most, no changes in frequency of healthcare visits were reported. some participants reported negative behaviors attributed to the pandemic, including decreased physical activity or increased unhealthy behaviors; increased alcohol use was significantly higher postpartum. a few reported engaging in bedsharing due to the pandemic, which increases risk of sleeprelated infant death [ ] . some of these behavior changes may be a direct result of increased stress and anxiety due to the pandemic. in general, up to one quarter ( %) of women experience psychological distress during pregnancy which can lead to poor birth outcomes [ , ] . stressful events also have significant deleterious effects on birth outcomes [ ] . in the current study, most participants ( . %) reported changes in mental status, such as increased stress and anxious thoughts. when a physician struggles with anxiety related to pregnancy during the covid- pandemic [ ] , it is unsurprising survey respondents also reported such feelings. the continuity of clinical care reported presents an opportunity for providers to assess mental status, as well as unmet needs during the pandemic. it also offers opportunity to reinforce risk reduction behaviors (e.g. infant safe sleep) and areas within the women's control (e.g. handwashing, limiting exposure, individual stress reduction activities). providers should consider addressing social support concerns as nearly a quarter of participants reported decreases due to the pandemic. social support may provide a buffering affect against preterm birth for women with high stress [ ] and lack of support has been linked to increased levels of antenatal anxiety and depression [ ] . as such, helping women identify strategies to maintain social support systems is of utmost importance. this study is limited as participants represent a single midwest community and were enrolled in perinatal programs, so may not reflect women not engaged in services. self-report data may reflect social desirability response bias. behavior change was assessed specifically as a result of the covid- pandemic and due to lack of time-frame-specific information it was not possible to compare breastfeeding and sleep practice data to those normally observed in the postpartum period. j o u r n a l p r e -p r o o f j o u r n a l p r e -p r o o f coronavirus pandemic and worries during pregnancy; a letter to editor coronavirus disease (covid- ) and pregnancy: responding to a rapidly evolving situation emergency caesarean delivery in a patient with confirmed coronavirus disease under spinal anaesthesia a case of novel coronavirus in a pregnant woman with preterm delivery a case report of neonatal covid- infection in china experience of clinical management for pregnant women and newborns with novel coronavirus pneumonia in tongji hospital management strategies of neonatal jaundice during the coronavirus disease outbreak coronavirus disease (covid- ): pregnancy and breastfeeding outcome of coronavirus spectrum infections (sars, mers, covid - ) during pregnancy: a systematic review and meta-analysis maternal and perinatal outcomes with covid- : a systematic review of pregnancies clinical analysis of neonates born to mothers with -ncov pneumonia middle east respiratory syndrome coronavirus (mers-cov) and covid- infection during pregnancy coronavirus: the spread of misinformation immediate psychological responses and associated factors during the initial stage of the coronavirus disease (covid- ) epidemic among the general population in china research electronic data capture (redcap): a metadata-driven methodology and workflow process for providing translational research informatics support national immunization surveys engagement with perinatal mental health services: a cross-sectional questionnaire survey task force on sudden infant death syndrome, sids and other sleep-related infant deaths: updated recommendations for a safe infant sleep environment sedgwick county health department, sedgwick county covid- dashboard kansas' statewide stay-at-home order actually only lasts until may , fox related infant deaths: evidence base for updated recommendations for a safe infant sleeping environment mindfulness-based cognitive therapy for psychological distress in pregnancy: study protocol for a randomized controlled trial mindfulness-based interventions during pregnancy: a systematic review and meta-analysis. mindfulness (ny) change in birth outcomes among infants born to latina mothers after a major immigration raid facing a pandemic while pregnant identifying the women at risk of antenatal anxiety and depression: a systematic review key: cord- -x sfwqnk authors: butler, colin d.; higgs, kerryn; mcfarlane, rosemary anne title: environmental health, planetary boundaries and limits to growth date: - - journal: encyclopedia of environmental health doi: . /b - - - - . - sha: doc_id: cord_uid: x sfwqnk published almost years ago, the limits to growth remains relevant to contemporary environmental health, though, paradoxically, this relevance is scarcely recognized. the seminal ideas it presented provide a useful background, as do the later planetary boundaries analyses, with which to consider key issues in contemporary environmental health. to be more than reactive, it is necessary to understand the complexity and interactions of integrated environmental health risks, including the possibility of significant global population decline within the current century. this contribution provides an overview to the limits to growth, linking it especially to the “planetary boundaries” of climate change, biodiversity loss and novel entities (including artificial substances and genetically modified organisms). the gradual increase in the amount of primary energy required to generate useable energy is also argued to be an under-recognized contributing factor to the decline in real wages growth for much of the world’s population since then, although this aspect may be improving. these elements have positive and negative health effects, which we discuss. only ones that allowed civilization to continue without crisis. if the human economy maintained business as usual, the team found, it would collide with the physical realities of a finite planet by the second half of the st century, triggering social collapse. initially, the book received a positive response and some of the recommendations were adopted by several countries. canadian prime minister pierre trudeau and us president jimmy carter each commissioned studies of the impact of physical limits on the global future and their national prospects. although these studies examined the outlook only as far as , their conclusions to that date confirmed those of the original ltg study. right from the start, however, economists were critical, even abusive, creating a negative impression that persists in some quarters, todayddespite the rapidly accumulating evidence of the basic robustness of the ltg projections and assumptions. robert gillette, who attended the ltg launch for the journal science, noted that the "assumption of inevitable economic growth" represents "the very foundation" of economics. any "limit" to growth challenged this foundation. it is unsurprizing that most economists attacked the ideas vigorously, an assault that illustrates the conflict between the core assumptions of economists and those of the physical sciences. economics adopts a standard model where production and consumption exist in a circular flow, without a natural context. it is a world of business and individual, producer and consumer, labor and goods. the physical world, which supplies resources and provides a site where wastes can be discharged, is not seen as essential and does not affect the equations, though, occasionally, the concept of "externalities" (which can be negative or positive) is mentioned. ecological economists reject the argument that human activity is independent of nature, which they consider a conceit. instead, nature is accepted as the indispensable foundation of human activities. physics really matters; questions of depletion and pollution are inescapable. several researchers have compared the mit projections with what has actually happened since, establishing that the correlation between the standard run and real world trends over the intervening years is extremely close. one of these researchers, graham turner, compared the standard run's modeled trajectory with years of historical data (see fig. ). he concluded that the data for - approximated the standard run of the ltg model, although the figure shows a slight but favorable divergence for the trajectory of non-renewable resources, such as fossil fuels, phosphate and concentrated, rich sources of ores. systems ecologists charles hall and john day also compared the standard run with actual data to . despite the common perception that the ltg work had failed, the model's performance was not invalidated, unlike models made by economists which are rarely, if ever, accurate over such a long time span. in , jørgen randers, part of the original ltg team, using an updated model, compared the ltg projections with real world data up to . he found that real world outcomes have approximated the second ltg scenario, the "standard run with extra resources," or "pollution crisis." based on observed data further processed by turner (up to ) trent modelled by the ltg study ( - ) (ltg) peak health? c : population declines due to increasing cases of regional overload meadows et al. for - and updated by turner with data to . it also introduces the concept of "peak health." this is the point at which human population well-being reaches its zenith, if the ltg model (of decline this century) proves reliable. although the timing is imprecise, peak health will precede the exact moment of maximum population. peak health and unwanted population decline are not inevitable; even today they could be postponed, perhaps indefinitely, by enlightened policies and technological breakthroughs. many resource analysts have identified declining "energy return on energy investment" as a key to understanding the slowing of the rate of improvement of living standards for most people in high-income countries. in turn, these indicators reflect the decline in easily accessible fossil fuels. this subject will be returned to. it is important to understand that the broad trends described in the figure and used in the ltg model do not capture the entire world. no model can. these trends were chosen because they captured many aspects of the material world, both human and natural, including feedback loops and potential crises that might threaten the human economy and thus wellbeing. indicators of these trends were chosen for a number of reasons, including researchers' attempts to choose representative indicators that would faithfully reflect the trends, the need for parsimony and, especially back in , the difficulty of finding accurate data. the decline in population that is modeled is a logical consequence of the decline in resources per capita, whether non-renewable, or as food, industrial output and services. if these inputs decline, the modelers assume, so will human population, which depends on them. the concept of the ecological footprint is among the most important developments in thinking related to the ltg. devised in the s by william rees and mathis wackernagel, this measure enables ecological impacts (individual, national or global) to be quantified and compared. on one hand, it estimates the ecological assets required to produce the resources consumed by any discrete population; this includes food and fiber plants, livestock and fish, timber and other forest products, space for urban infrastructure and whatever "sinks" are needed to absorb the waste produced, especially carbon dioxide emissions. the unit of measurement adopted is the area of biologically productive land and water, usually expressed in hectares. on the other hand, the ecological footprint also estimates the productivity of a country's actual ecological assets (cropland, grazing land, forest land, fishing grounds, and built-up land). researchers using the ecological footprint methodology calculated that, while the world's biocapacity averages . ha. per capita, high-income "developed" countries greatly exceed this average. examples include the united states ( . ha. per capita) and the united kingdom ( . ha. per capita). the us, which has far more productive land available than the united kingdom, appropriates . times its own biocapacity through imports, and the united kingdom almost three times. many island nations and arid countries such as saudi arabia exceed their biocapacity by a factor of more than ten. the ecological footprint has the strengths and weaknesses of any aggregate indicator: the concepts and units are easy to understand by policy-makers and the public, but it does not encompass all aspects of human environmental impact (methane, e.g., is not integrated). this matrix needs to be used in conjunction with other indicators. another related development is the framework called "planetary boundaries," devised by a large team led by johan rockström (see following section). although the environmental health literature has long identified links between health and indicators used in the ltg model, such as food, services, and pollution, there has been little recognition among the health community, including within public health, of the possibility of a reduction in population this century. such reductions, as mentioned above, are forecast by most ltg scenarios, including the standard run (see fig. ). such a reduction, were it to occur, will have impacts on public health. there are a few exceptions to this generalization. in , the human ecologist frederick sargent, in an article in the american journal of public health, warned that human "interventions in and manipulations of the processes of the planetary life supportsystem (ecosystem) have produced a set of complex problems" (page ). in , the visionary socialist economist barbara ward co-authored "only one earth" with the microbiologist, pioneering "earth physician" and pulitzer prize winner, rené dubos. this book stated in part "the charge to the [ stockholm] conference was clearly to define what should be done to maintain the earth as a place suitable for human life not only now, but also for future generations" (emphasis added) (page xiii). health is implicit in this statement, as is sustainable development. in mcmichael, who was influenced by dubos, echoed sargent's term, writing in the foreword to his influential book planetary overload, that "the most serious potential consequence of global environmental change is the erosion of earth's life-support systems. yet, curiously, the nature of this threat to the health and survival of the world's living speciesdincluding our owndhas received little attention" (page xiii). a quarter of a century later, little has changed. although keynote talks by mcmichael and john last at the conference of the international epidemiological association warned of the dangers to global public health of global environmental change, there has been barely any recognition or follow up, at the broad integrated dimension. ltg receives little recognition now. a literature search for the term "limits to growth" in association with "health" reveals little other than work involving the authors of this contribution and their close collaborators. this is unfortunate. the persistence of single-issue approaches to address complex problems retards our ability to act effectively. this is the case where there is singular focus on climate change. the impact on human health and wellbeing is modified by many interacting factorsdincluding population, global demand and availability of resources and services and the waste and pollution we generate. other than noting the exponential growth of carbon dioxide in the atmosphere, ltg did not address climate change as such, but it modeled these factors as part of a complex system. although infrequently, the issues of resource depletion and population pressure have featured in some medical curricula, from at least the s, preceding publication of the ltg by over a decade. pioneering writers and speakers have included colin bertram, peter parsons, and roger short. john guillebaud, the world's first clinician professor of family planning and reproductive health, has repeatedly spoken on the issue of both consumption and population, to diverse audiences including medical practitioners, nurses, medical students and others, since . in , a special issue of the american journal of public health focussed on peak oil, an important aspect of ltg. a scattering of other articles in the health literature have mentioned peak oil, but ltg is far more than peak oil. it is also far more than global warming. the reasons for the general failure of the public health literature to engage with ltg are complex but include an incorrect belief that ltg was discredited, over-specialization within the public health community, political suppression of the core ideas, and a lack of funders. the issue has had very few champions. this lack of engagement is not from lack of evidence. the term planetary boundaries (pbs) was first published in . these boundaries were defined, initially, as referring to nine earth system processes (see table ), each of which had been, is and will be modified by human actions. the first planetary boundary paper (published in in ecology and society, subtitled "exploring the safe operating space for humanity") explicitly acknowledges its debt to the ltg framework. a "safe operating space" implies the existence of multi-dimensional limits, just as does the word "boundaries". nonetheless, the links between pbs and the ltg are mostly implicit. although the focus of the pb work is on identifying the criteria for a "safe operating space" for humanity, rather than that of other living species, the concept acknowledges that humans depend on the diversity of life on earth. the first pb argued to be outside the safe operating space is biological diversity. analogous to nine bodily systems (renal, hepatic, neurological and so on) the large multidisciplinary team ( co-authors) that was responsible for the first pb articles argued that these earth system processes can still provide useful services, even if functioning outside their optimal range. however, pushed too far, even one aberrant bodily function can cause death, and just one extremely disturbed earth system process might trigger catastrophic consequences for humanity. perhaps, for example, a precipitous loss of insects could disrupt pollination, food supply, and the survival of birds and other vertebrates. in turn, loss of birds might trigger additional crop losses, as their biological control of insects and other pests (i.e., of pests that do survive) is lost. another example is the reduced complexity of the microbiome of those who live in cities and other modified environments. this in turn has been hypothesized to be a causal role in the emergence of auto-immune diseases such as type diabetes. further, just as with bodily processes, earth system components are linked to, interact with, and are influenced by common causes. humans may live for decades with chronic illness, but die quickly if multi-organ failure develops. so too, earth system processes interact, but exceeding multiple planetary boundaries, either simultaneously or in close proximity, risks precipitating a steep decline towards a civilization-crippling condition. other writers have also commented on the similarities between the earth and the human system, including james lovelock, the originator of the gaia hypothesis and an early user of the term "planetary medicine." table shows links between planetary boundaries, ltg and human health. the exact extent to which we are breaching planetary boundaries is still being explored. the team's paper argued that two problems are already extremely dangerous (red zone) and two others are well on the way (amber zone), also noting that the designated boundaries are inter-related and most have overlapping implications. the team considers loss of biosphere integrity as the most critical problem. rates of extinction are reckoned to be at least times the background rate, possibly as much as , times. populations of vertebrate species declined by more than half between and and the biomass of wild mammals is now only about % of the total, which is dominated by humans and livestock. that remaining % is under siege, including for substances such as rhinoceros horns and pangolin scales, which have alleged therapeutic benefit, even though chiefly constituted of keratin, just as are fingernails. biological diversity of lower order organisms (within soils, among pollinators, and in the species traditionally utilized for plant-origin food, resources and medicines) are similarly in decline. also under deliberate attack are forests, especially species with valuable timber, or growing on land that can be used for crops, including oil palm. many forests are also at unintended risk, due to roads and global warming, each of which also exacerbates the risk of fire. global warming is also likely to further hasten biodiversity loss. in some cases biodiversity decline and climate change, acting together or independently, also promote the survival of new pests. infestations of tree borers, able to survive warmer winters in large numbers, render trees, forests and their associated animal life more vulnerable to disease and fire. humans, a form of life, depend on the fabric of other life on earth for their survivaldfor food, clean air and water, and numerous other ecosystem "services" (see glossary and below), as well as for novel substances, including drugs. at some level of bio-alteration the reduction in ecosystem services will reduce in a non-linear way that could cascade in a manner harmful to all civilization. smaller scaled examples include the collapse of regional fisheries or the decimation of regional harvests by novel diseases or pests, such as in ireland in the late s. the khapra beetle, a pest from south asia that has evolved insecticide resistance threatens significant (up to %) loss of rice, post-harvest, in some regions. devastating drought in the "dry corridor" of guatemala, el salvador and honduras has been blamed for contributing to the influx of migrants attempting to enter the united states between and the present ( ). for rockström, steffen and colleagues the second most pressing danger is the radical disruption of the biogeochemical cycles, particularly nitrogen and phosphorous. in nature, most nitrogen was inert in the atmosphere, though some was mobilized by bacteria and leguminous plants. applied as fertilizer, nitrogen has greatly expanded food production, but is now cascading through our rivers, groundwater and continental shelves, initiating algal blooms and dead zones. in the case of phosphorous, the other widely dispersed fertilizer, there is an added dangerdphosphate rock is a resource in decline, with grim implications for future agriculture, especially where populations will lack the financial capacity to import it, as prices rise. land-system change is argued to be in the "amber zone," close to crossing the boundary into extreme danger, if it has not already crossed it. millions of hectares of vegetation are still being cleared every year and wetlands continue to be drained. stocks of "blue carbon," stored in plants and trees associated with water, such as kelp and mangroves, are also under threat. land-system changes enable more food, fiber and other financially valued products to be grown, but amplify the harm to several pbs: biological integrity, climate and biogeochemical cycles. oil palm plantations are displacing tropical forests in asia, africa and, increasingly, latin america, where clearing already provides cattle pasture, soybean and sugar cane. such plantations involve the death of vast numbers of individual animals and the annihilation of immense tracts of tropical forest. this boundary is underpinned by the declining remainder of tropical, temperate and boreal forests. these forests have a major role in land surfaceclimate coupling. in addition, agricultural land-system change may ultimately result in land degradation giving rise to erosion, loss of topsoil, sedimentation of waterways and degradation of coastal zones. in dryland regions, degradation is referred to as desertification. large areas are affected. the united nations organization considers that billion people are at risk of desertification globally, half of whom live in africa where they face major challenges to water and food security. increasing urbanization also drives land-system change, typically in areas of high agricultural productivity. vast urban regions impact surface energy (through the "heat island" effect), alter hydrological and biochemical cycles, net primary productivity and biological diversity. they are also major foci of pollutants. as humanity becomes predominantly urbanized it is with these land systems that most of us have most intimate contact. also in the amber zone is climate change. remaining below the c target, which is thought to provide a reasonable chance of avoiding catastrophic climate change, necessitates technologies which do not yet exist for extracting carbon from the atmosphere. most nascent carbon reducing technologies require considerable energy, although new forms of cement may soon be feasible and environmental health, planetary boundaries and limits to growth affordable on a large scale. research since suggests that the c target may need to be adjusted downwards to provide a reasonable chance of avoiding calamitous warming, in which case climate change may already belong to the red zone. even if the commitments made at the paris conference of the parties are all honored, it currently seems likely to many analysts that global temperatures will be c hotter than pre-industrial times by and nearly c higher by . these estimates depend on a number of variables: whether nations will adopt more ambitious pledges in the near term; whether technologies will emerge that can, at a low energetic cost, suck carbon back out of the atmosphere; whether unknown tipping points will be crossed, forcing a temperature surge. if these variables prove unfavorable, the aspirational . c maximum target may be reached by the early s. there is no guarantee that the damage can be held to approximately c, in particular due to the risk of amplifying feedbacks such as the release of carbon dioxide and methane from the arctic, and/or the drying and burning of the amazon forest. the capacity of the ocean to absorb co is also declining. that will slow the rate of ocean acidification, but increase atmospheric heat trapping. even if temperature rise and rainfall intensity can be contained, crop yields will decline and many places will become unliveable due to excessive heat and humidity or coastal inundation. glaciers that act as a bank to store water and in some cases whose melt supplies electricity to billions in asia and south america will shrink, coral reefs and many other species will disappear, and significantdeven catastrophicdsea level rise will result. in greenland and along the entire coast of west antarctica ice shelves are already retreating or collapsing as warm seawater intrudes underneath, grounding lines retreat, and the glaciers behind them accelerate in their march to the sea. climate scientist james hansen and many glaciologists warn that the disintegration of the polar icesheets involves non-linear processes, and the timing, though still unknown, may be far quicker than assumed, and may include rapid, even unstoppable collapse of ice cliffs in series in parts of antarctica and greenland. the impact upon human wellbeing resulting from stress on biological diversity will be compounded by climate change and the fragmentation of society. for example, a complex economic and social fabric enables the importation of food and other resources to an increasing number of regions, some of which have been in this vulnerable situation for decades. such mechanisms are fragile. today, five countries are recognized as afflicted by famine: yemen, somalia, south sudan, n.e. nigeria and two regions of the democratic republic of the congo (kasai and tanganyika). in the long run, if climate change and other aspects of adverse ecological change intensify, then it is also possible that regions that are current net food exporters will also experience famine; if this evolves then conditions in food-importing regions will inevitably deteriorate. alongside these four major crises, the researchers also identify the threat from various forms of pollution. most of these are discussed elsewhere in this encyclopedia. however, we briefly discuss novel entities. novel entities is a recently introduced term, first identified as a planetary boundary by the pb team in , evolving from chemical pollution in the earlier pb publications. the pb team defines novel entities as "forms of existing substances, and modified life forms that have the potential for unwanted geophysical and/or biological effects." most novel entities have been generated in the anthropocene, the human-dominated era, defined roughly as the time since the start of the widespread combustion of fossil fuels, in the th century. they include synthetic molecules such as chlorinated fluorocarbons (cfcs), ddt, dieldrin and other organochlorines used as biocides and compounds used in industry such as polyvinyl chloride. cfcs, by harming the stratospheric ozone layer, clearly impinge on an earth system function (and thus indirectly on human environmental health); the destruction of the stratospheric ozone layer causes uv light to reach the earth's surface to a greater extent than prior to the widespread use of cfcs, leading to the potential for an increased incidence of skin cancer, ocular problems and immunosuppression. here, however, we focus mainly on the biological effects of novel entities. novel entities are not confined to new chemical compounds, as the pb authors note. genetically altered organisms can be conceptualized as novel entities, as are nanoparticles (such as in sunscreens and cosmetics), and blue light from computer and phone screens. humans are also exposed to numerous other emerging environmental hazards, especially since world war ii, and to human-generated ionizing radiation (x-rays were once routinely used to help fit shoes). possible health risks of nonionizing radiation, such as from mobile phones, are discussed briefly below, as are novel behaviors, foods and other novel environments. a lancet commission report estimates that , compounds have been synthesized since , with perhaps widely disseminated in the global environment. although some are regulated, and a few have been banned, the pace of their introduction greatly exceeds that of epidemiological investigation and legal constraint. for example, the international agency for research into cancer (iarc), which is closely affiliated with the world health organization (who) has recently concluded that the widely applied herbicide glyphosate (commercially known as "round up") may be carcinogenic. these findings have been resisted by some companies and their agents and supporters. thousands of studies of novel entities have found or suggested that many are carcinogenic, while others act as endocrine disruptors or harm health in other ways. some have been linked with massive ecosystem disruption, including colony collapse disorder (of bees) and "insectageddon." the lancet commission on pollution reported that fewer than half of the most widely dispersed chemicals have undergone any testing for safety or toxicity. interactions between such chemicals have received even less examination. the immunological and allergenic effects of most novel entities are also barely explored, and could contribute to the changing pattern of allergic diseases, auto-immune conditions and autism. while some novel entities have been regulated (e.g., x-rays) and banned (such as the "dirty dozen," including the organochlorine dieldrin, which was, as a rare exception, strongly linked with breast cancer), hundreds or thousands of others are released annually onto the market. in both industrial and rural societies, almost the entire population has been exposed to hundreds of chemicals whose concentrations can be measured in tissue samples, while for thousands more, no test exists. there is little support from policy decision makers around the world for precautionary approaches to many potential risks. for example, there are concerns that mobile phones can cause brain tissue to warm up, if the receiver is held close to the ear. however, there are also concerns about the effects of non-ionizing radiation on brain tissue, and claims of an increased risk of malignant brain tumors in heavy users of mobile phones. cardiac and neurological disorders are also plausible consequences of the rapidly increasing use of wireless devices, including smart meters. infrasound from wind turbines is another novel entity. such sounds disturb the sleep of many people who live close to them, and there may also be other harmful effects including vertigo, as well as chronic diseases worsened by chronic poor sleep. such concerns have often been dismissed as "nocebic" (i.e., through apprehension and negative thoughts) as high quality evidence for health impacts is lacking. the precautionary principle would place the onus on industry to prove safety. novel behaviors, foods, organisms and environments are also emerging in the anthropocene. examples include reduced weight bearing exercise in childhood and adolescence (leading to a higher risk of early-onset osteoporosis), increased screen watching and the partial replacement of tangible, local friends and acquaintances for virtual social networks. novel diets include the widespread consumption of sweetened drinks, a known factor in obesity and harmful to health, while the greater variety of foods out of season, especially of fruit, is beneficial. there are also novel microbial and parasitic environments and novel microbiomes, each of which is likely to be associated with health benefits and risks. for example, humans and livestock farming provide opportunity for the amplification and spread of genes that convey antibiotic resistance. these genes are favored wherever antibiotics are used by humans or fed to livestock to promote growth and limit infectious disease. antibiotic resistance genes have been shown to spread to environmental microbes in soil and water systems, to wildlife and to human and livestock pathogens. identified mechanisms for this transfer include air-borne transport of particulate matter and direct and indirect contact with waste products. the augmented "wild" population of antibiotic resistant genes is an added risk to human health and has poorly understood implications for other environmental microbial systems. novel or increased contact with mammalian wildlife creates further potential for interspecies transfer of pathogens, particularly viruses. this is discussed below (in biodiversity and health). since the s, there has been increasing recognition of ways that anthropogenic emissions of greenhouse gases (manifest in phenomena including global warming, weather wilding, jetstream oscillations, sea level rise and ocean acidification) is likely to impact human health, both positively (e.g., fewer cold waves in some areas) and negatively. there are numerous mechanisms for this. one that is perhaps most obvious is an intensification of extreme weather events, including heatwaves, droughts, flooding, and major storms including cyclones, typhoons and hurricanes. such events can have complex and delayed effects, such as from the savage hurricanes that flooded and devastated houston, texas and the us territory of puerto rico, as well as other regions. there is also speculation that the frequency, severity and locations of tornadoes may be affected. very intense flooding events, where weather systems remain almost stationary, have generated the neologism "rainbomb." changes in vector-borne diseases, food security, and sea level rise have long been forecast to occur due to global warming. global warming is already affecting migration, conflict and mental health, and these effects are likely to intensify. over the longer timescale, of decades to centuries, adverse effects are forecast to exceed benefits, perhaps by orders of magnitude, especially if the ice sheets in greenland and antarctica continue to melt. there are many ways health effects related to climate change can be categorized, such as through changes in temperature and humidity, vector ecology, water quality, water and food supply impacts, severe weather effects, air pollution, allergens, and migration, conflict and related mental health implications. a simpler classification has three main classes, conceptualized as "direct" (e.g., heatwaves), "indirect" (e.g., changes in vector ecology) and a third category, causally more displaced, with the potential for the largest burden of disease, through means such as large-scale conflict, migration and famine. in this classification, effects on mental health are regarded as "cross-cutting." dislocation from one's home due to a storm surge or a prolonged blackout (some parts of puerto rico lacked power for months following hurricane irma) can lead to depression and even suicide. such stress is also likely to exacerbate domestic violence, especially if associated with increased economic insecurity. increased rates of post-traumatic stress and anxiety are also likely in survivors. even worse than the mental trauma of a single extreme weather event are the health consequences, including to mental health, from conflict, famine and forced migration. of course, such "tertiary" effects have multi-dimensional causes, from ancient rivalries to recent and emerging contests over scarce resources, often aggravated by "youth bulges" and brutal repression. all writers on these "tertiary" topics, publishing in the academic literature, recognize the complexity of this issue, and frequently try to convey this by using the term "risk multiplier" to indicate how changes in climate can worsen (or in some cases reduce) the co-factorial causal contributors to conflict. that is, climate change is conceptualized as similar to a catalyst or enzyme. famines, wars and migration can all occur without climate change, but in some cases climate change can make these phenomena much worse. in some cases, such as sea level rise, climate change can be conceived as by far the dominant factor. however, even for vulnerable lowlying pacific islands, co-factors such as high population growth have contributed to vulnerability and the risk of migration, for example by depleting fresh water lenses, leading to the salinization of garden soil. many important diseases, including parasitic, vector-borne and zoonotic diseases are associated with invertebrates such as ticks, mosquitoes and blackflies, or higher order vertebrates. ticks transmit diseases such as lyme disease, mosquitoes transmit many illnesses such as malaria and yellow fever, while black flies transmit river blindness (onchocerciasis). the distribution of these insects and animals are shaped, not only by climate but by many other aspects of their ecology. often, the identification of the precise attribution to climate change is elusive and possibly fruitless. less intuitively, the epidemiology of many vector-borne diseases, including malaria, dengue fever and zika virus is also influenced by the ambient temperature in another way, by determining the growth rate of the parasite or virus within the cold-blooded vector. more rapid growth of these pathogens (i.e., in slightly warmer vectors) can, in some cases, lead to additional cycles of transmission, leading to explosive increases in cases. another way to think of these organisms is that their numbers and disease potential exist within a window or "sleeve" of climate and ecological suitability. it would be wrong to think that a warmer or wetter climate will inevitably increase the burden of these infectious diseases. as temperatures rise, insect populations may too, but only to a point. beyond that point, vector populations may in fact decline. similarly, excessive rain may reduce vector habitat (e.g., flushing the population away), as may unusually prolonged droughts (drying out the habitat). however, the epidemiology of vector-borne diseases is also influenced by human factors, such as insecticides (including impregnated bednets) and molluscicides, and by treatments such as vaccines (e.g., for yellow fever) and antimalarial drugs such as quinine. the impact of biodiversity loss on human health is being realized slowly. the dimensions of biodiversity (the diversity of genes, species and ecosystems) are not experienced or understood by most individuals, or policy makers, and challenge health researchers. as such, the impacts are dispersed across multiple scales of biodiversity and multiple dimensions of health and wellbeing much of which is discussed elsewhere in this contribution. the pb team has examined the genetic diversity within and between species and ecosystems and its functional role within a global system, separately. they conclude that the loss of genetic diversity has exceeded a safe limit, with uncertainty remaining over how this impacts the function of ecosystems. the alarming rate and extent of loss of genetic biological diversity has been discussed earlier. the loss of genetic diversity undermines the resilience of ecosystems. under relentless ecological change, biological diversity is replaced by ecosystems dominated by fewer, highly adaptive species, inadvertently or purposefully promoted by human activities. these include domestic species, pests and wild synanthropes, humans and the novel entities described above. genetic diversity is also the source of pharmaceutical discovery as well as the storehouse of traditional medicines. most naturederived pharmaceuticals come from plants; some come from traditional medicine practice but much is the product of systematic searching, modification and trial. nature produces an inspirational variety and complexity of molecules to further manipulate. the diverse origins of the pharmaceutical armory against hiv aids includes betulinic acid, derived from the bark of the tree betula pubescens; bevirimat, extracted from a chinese herb syzygium claviflorum; and ganoderic acid b, isolated from the fruiting bodies and spores of the fungi ganoderma lucidum. such a utilitarian appreciation likewise extends to livelihoods dependent on different aspects of biodiversity. for some, particularly vulnerable groups and those in remote locations, survival is dependent on the ability to harvest freely (or illegally) from the natural environment. rich biological diversity is often helpful for the resilience of ecosystems functions, sometimes called "services." in the early s, the millennium ecosystem assessment, a global collaboration of over scientists, grouped these into four kinds, which they called supporting, provisioning, regulating and cultural. food production is classified as a provisioning service. for such a service, biological diversity is required for (supporting or underpinning) soil health, pest control (a regulating service), pollination and the genetics of livestock and crops. other products of provisioning services include clean water, bio-fuels and crop residues used to provide energy. other regulating services include carbon sequestration, climate regulation and disaster risk reduction. nutrient recycling is another example of a supporting ecosystem service. a sacred grove or an iconic species of deep significance to the beholder illustrate cultural services. disease-regulation as an ecosystem service is contested, but some diseases, such as lyme disease, are more prevalent in diminished and simplified ecosystems. the net effect of deforestation often favors mosquitoes that serve as vectors of human diseases including previously obscure pathogens such as zika and chikungunya viruses, or encourages urbanization or farm-foraging by fruit bat hosts of henipah viruses nipah and hendra. there is also a complex relationship between ecological change and malaria, which is, by far, the most important mosquito-transmitted disease. as discussed above, climate change also impacts these vector borne diseases. changes (driven by reductions) in biodiversity have increased zoonotic infectious disease risk especially due to intensive animal husbandry. livestock, which now dominate global vertebrate biomass, and intensive production, create the opportunity for viral amplification and mutations resulting in new and previously unrecognized animal diseases and zoonoses. these include h n (avian flu, via chickens), h n (swine flu, via chickens and pigs), possibly sudden acute respiratory syndrome (sars, via farmed civet cats and racoon dogs), nipah virus in malaysia (via pigs) and middle east respiratory syndrome (mers, via camels). novel or increased contact with mammalian wildlife creates further potential for interspecies transfer of pathogens, particularly viruses. such contact is facilitated by accelerated land-use change and wildlife harvesting and sometimes aided by domestic animals acting as amplification hosts. disease may transmit directly to humans or indirectly through domestic animals. many opportunities for viruses to jump between species may be required before a significant disease emerges. tropical areas of high biodiversity and under human pressure are considered "hotspots" for such diseases. novel zoonoses of wildlife origin such as hiv aids, ebola and sars corona virus have been the subject of strong interest in the late th century and early st century. the examples above have resulted in pandemics. many other smaller viral "spillover" events have occurred with localized impact only. natural or wild areas also reduce stress, depression and anxiety in those who visit them. this effect appears to be dependent on cultural and socioeconomic characteristics of the visitor and has deeper, religious dimensions for many indigenous people. as discussed earlier, the human microbiome links us to the external world. personal microbiodiversity is enriched by environmental and dietary diversity and, through mechanisms of immune regulation and the gut-brain axis, has a significant impact on physical and mental health. the benefits of experiencing biodiversity within natural settings appear to be physiological as well as psychological. however, the living environs of most people is one of reduced biodiversity, and for many most time is spent indoors. it is the capacity of earth's natural systems, the aggregate of species and ecosystem biodiversity, to provide resilience despite changing environmental conditions that should be of the most fundamental concern to health and well-being. the biosphere must absorb our wastes, including carbon emissions; buffer coastlines from extreme weather events; provide clean air, water, a moderate climate, and the renewable resources humanity seeks to consume. it is therefore of great concern that the global ecological footprint network (see "the ecological footprint" section) estimates that % of [global] biocapacity is consumed per annum. today, many determinants of health and wellbeing, including effective health services and their inputs, such as consumables and pharmaceuticals, are dependent on abundant and affordable energy. millions of people living in poverty, especially in sub-saharan africa and south asia, suffer multi-system consequences of air pollution, both indoor and outdoor, from smoke generated by their own household and by other households. in many locations, this is aggravated by the burning of fossil fuels such as coal and oil. many people, particularly women and children, undertake daily laborious effort to obtain fuel and water. access to electrical power, and even gas for cooking would bring significant improvements in health to the . billion people living without electricity. probably the least understood aspect of limits to growth is the concept and importance of declining energy return on energy investment (eroei). the number given for eroei is the ratio of useful energy obtained versus primary energy expended (see box ). the major energy carriers in use today are fossil fuels, especially coal, oil and gas. these are used not only for transport, heating and electrical power (over % globally), but also as a chemical stock to manufacture plastics and to make fertilizer. but, just as in the past when our ancestors bred, trained, housed and fed donkeys and draft horses for assistance with laborious tasks, fossil fuel needs to be wrested from the environment, whether drilled for, dug by hand or removed by robotic shovels. these processes themselves take energy. in addition, energy released or captured from these sources needs to be distributed and the infrastructure to do that needs to be maintained. coal needs to be transported and burned, with some of its energy captured through combustion. electrical energy needs to be distributed and regulated irrespective of its source (including solar, wind, hydro and tidal). to manufacture wind turbines or solar panels requires energy, as does the mining infrastructure described above. life-cycle assessment allows a full quantification of the energy invested in any form of energy extracted, which is clearly significant. in the heyday of fossil fuels, oil and coal were easy to extract, and their eroei was highdsome analysts report an average eroei of over in the late th century. in contrast, a review published in in nature communications found that, globally, up until , solar panels may have yielded no energy beyond that required for their manufacture and installation. in other words, under the least optimistic scenario, solar panels, cumulatively, have been a sink for energy, rather than a source until very recently. more encouragingly, the eroei for solar appears to have increased considerably in the last decade, perhaps to or , especially in locations with high insolation, such as in the tropics. the climate footprint of solar is much lower than of coal and will continue to decline, especially as the efficiency of panels increases and the electricity they generate is used to manufacture additional ones. the eroei for wind is widely agreed to be even higher than for solar, so these two sources have promise as major substitutes for fossil fuel energy, even though researchers still debate whether renewables will yield energy abundant enough to fuel the current consumption-oriented economy. in addition, ugo bardi and sgouris sgouridis argue that the window for a successful transition is narrow: a very large investment of available energy is required, while still maintaining adequate energy for ongoing services. moreover, the world's economic system may fail to allocate the necessary resources in the necessary timeframe (by in this analysis). bardi and sgouridis are skeptical that market forces can effect this transition. they calculated that, as of , capital investment is only about one tenth of what is required. energy investment is also inadequate. though not impossible, any transition to renewables will be challenging and requires a substantially greater rate of energy and capital investment than is currently allocated. there is a widespread understanding that fossil fuels have been crucial to the human colonization and domination of the biosphere. the importance of energy is explicit in the work of many environmental writers, and implicit in the military actions of many great powers, who have frequently acted with violence or duplicity to acquire or maintain energy resources, from the middle east to the timor sea. without fossil fuel, modern civilization could not have evolved in the way it did, whether to create highways, intensive agriculture, skyscrapers or the space age. although, in the middle ages, the harnessing of water power for work from milling grain to sawing wood ("sawmilling") was widespread, such industry was necessarily confined to suitable riversides. ancient mariners crossed straits and sometimes oceans, powered by oars and blown by the wind, but the scale of maritime trade was miniscule compared with that made possible by steam, oil and nuclear-powered vessels. while this dependence on energy is well-known, though rarely highlighted in economic histories, the fact that eroei is steadily declining is rarely mentioned in mainstream media or outside of specialist journals; it is claimed that fracking and shale oil now negate peak oil, though insufficient attention is paid to the instability of an industry reliant on proliferation of drilling sites and rising costs. the decline of eroei may be disconcerting to a public ill-prepared for the future austerity which such a decline implies. such consequences would not only affect health services, but the myriad other processes necessary for health which rely on affordable energy, including agriculture. concern about the impact of global ecological change on health is growing. so too is an understanding of the need for multi-sector collaborations. few groups are yet addressing the deeper issues of pbs. however, many once disparate groups are converging as they seek to improve equity in health with a focus on global problems of biodiversity decline, environmental degradation and climate change. "planetary health," promoted by the prestigious medical journal the lancet, is currently prominent. others include eco-health, one health and the in vivo planetary health group. predicting future human health (or survival) under the status quo is difficult. ecological systems typically demonstrate nonlinear responses to perturbations and it is likely that current consumption patterns will precipitate dramatic shifts in biodiversity, ecological function and health-supporting services. impacts of environmental change are disproportionately experienced by poor and rural communities. advocacy and action to prevent these health risks is an essential role for those concerned with public health. this entry has reviewed the issue of limits to growth, its more modern formulation as planetary boundaries and the relevance of both concepts to global population health. it has used these frameworks to classify and extend some environmental health risks. these include novel entities and behaviors, and global risks including climate change, biodiversity loss, land-system change and biogeochemical cycles. these risks are escalating and we recognize the shortfall in assessing the health risk of new pollutants, entities and behaviors. when considered together with economic and population growth, and with ever-increasing resource and energy use, these environmental health risks may foreshadow significant population decline. by using the ltg and pb frameworks in this contribution, society can frame preventative measures on the scale at which this is required. without urgent change, future global population health, and survival, is imperiled. environmental quality in a growing economy. resources for the future health, population, limits and the decline of nature climate change, food security and population health in the anthropocene a further critique of growth economics encyclical letter laudato si of the holy father francis on care for our common home collision course: endless growth on a finite planet the limits to growth great transition: the promise and lure of the times ahead union of concerned scientists, . world scientists' warning to humanity the interaction of human population, food production, and biodiversity protection is global collapse imminent? re-assessment of net energy production and greenhouse gas emissions avoidance after years of photovoltaics development come on! capitalism, short-termism, population and the destruction of the planet understanding social-ecological systems fischer: a critical look at food security strategies key: cord- -tnsubtjr authors: baztan, juan; vanderlinden, jean-paul; jaffrès, lionel; jorgensen, bethany; zhu, zhiwei title: facing climate injustices: community trust-building for climate services through arts and sciences narrative co-production date: - - journal: clim risk manag doi: . /j.crm. . sha: doc_id: cord_uid: tnsubtjr the goal of this paper is to analyze how and with what results place-based climate service co-production may be enacted within a community for whom climate change is not a locally salient concern. aiming to initiate a climate-centered dialogue, a hybrid team of scientists and artists collected local narratives within the kerourien neighbourhood, in the city of brest in brittany, france. kerourien is a place known for its stigmatizing crime, poverty, marginalization and state of disrepair. social work is higher on the agenda than climate action. the team thus acknowledged that local narratives might not make much mention of climate change, and recognized part of the work might be to shift awareness to the actual or potential, current or future, connections between everyday non-climate concerns and climate issues. such a shift called for a practical intervention, centered on local culture. the narrative collection process was dovetailed with preparing the neighbourhood’s th anniversary celebration and establishing a series of art performances to celebrate the neighbourhood and its residents. non-climate and quasi-climate stories were collected, documented, and turned into art forms. the elements of climate service co-production in this process are twofold. first, they point to the ways in which non-climate change related local concerns may be mapped out in relation to climate change adaptation, showing how non-climate change concerns call for climate information. secondly, they show how the co-production of climate services may go beyond the provision of climate information by generating procedural benefits such as local empowerment – thus generating capacities that may be mobilized to face climate change. we conclude by stressing that “place-based climate service co-production for action” may require questioning the nature of the “services” rendered, questioning the nature of “place,” and questioning what “action” entails. we offer leads for addressing these questions in ways that help realise empowerment and greater social justice. today, there is some irony for us to work on a paper tackling issues of priorities, justice, and the interconnectedness of climate issues with most issues of social justice. as we are writing these words, we are locked-down, contributing, through isolation, to a fight against a very disruptive world scale pandemic. we have somehow imperfectly shifted our priorities. many of us observe that covid- has shifted all sorts of priorities. some of us long for a world a world where priorities will be, at long last, straightened-out -thanks to the lockdown and the realization that some things can grind to a halt if really needed (latour ) . some see current events not so much as a crisis, but as a necessary, and painful, wake-up call leading to an evolution in our ways of inhabiting the world. if there is lesson to be learned from covid- , it is that priorities are contextual and short-term crises, mutations, may blind us from more pressing challenges (the intertwining of climate change and pandemics takes many twists and turns, see for instance ord ) . we have known for a long time that pandemics and climate change may be connected, and climate justice may very well be one of these connections. this paper contributes to the growing body of the literature on climate service co-production; coproduction understood here as a normative practice that consists in "the deliberate collaboration of different people to achieve a common goal" (bremer and meisch , pg. ) . we chose to analyze a climate service co-production situation where potential climate information users were not necessarily aware of the importance of climate change in their daily lives. we developed an intervention allowing for the analysis of how and with what results place-based climate service coproduction may be enacted within a community whose locally salient concerns do not include climate change. while envisioning local climate change adaptation action, one may have to face the fact that for some communities climate change might not be high on the local agenda. the issue is not necessarily that such communities negate the salience of climate change and its impacts. some communities may have other challenges that are more pressing and not obviously climate related: marginalization, poverty, resource depletion, crime, sense of powerlessness, health, housing, education, cultural erosion. some communities are facing short-term challenges for which all their energy is mustered -other stressors do matter (e.g., ahmed et al. , mccubbin et al. ). yet these communities are and will be facing climate change and its impacts. it is quite likely they are already in a position where climate change adaptation may be necessary. they may thus need climate science attuned to their specific challenges, however they are not yet part of the visible "demand side" for climate services such as climate information. climate science will have to deploy itself in accordance with their priorities, which are not climatecentered. these situations raise questions about the legitimacy of climate action in the face of social and economic hardship not directly connected to climate challenges. who has a say in priority setting? can an issue seen as salient from outside a community be somehow made salient for those within the community? by whom, and under what conditions, is exogenous priority-setting acceptable? concern about exogenous priority setting and its associated marginalization led our team of scientists and artists to engage in fieldwork in the kerourien neighborhood, the results of which we present in this paper. we share a story of seemingly competing priorities, external and internal. through climate service co-production, which we see as an avatar of transdisciplinary research, we conducted a real-life experiment where scientists and artists focused on developing approaches for place-based climate service co-production by reaching out to a community that has other, more pressing concerns than climate change. the kerourien neighborhood, in the city of brest located on the coast of the brittany region of france, is better known for its stigmas (e.g., high unemployment rate, state of disrepair, drug-related violence) than for its awareness of climate issues. yet climate change is the symptom of broader issues, and is intimately connected to social issues (see krauss ) and it seemed to the research team that kerourien's challenges deserved to be explored through the lens of climate change. we first present a summary of the literature we drew from to design our on-site intervention, including: climate change adaptation and local contexts, art and science approaches to climate intervention, and climate service co-production as an empowering transdisciplinary practice. we then describe the cocliserv project and the kerourien community. the results section focuses on the process in which we participated, its outputs in terms of approach, procedural and substantive benefits, and entry point for place-based climate information design. we focus on locating climate change within apparently non-climate related narratives and on the ubiquity, yet invisibility, of climate change within kerourien. we further discuss the procedural and substantive benefits of the work conducted. local context, climate services, and art and science co-production "climate science usability is a function both of the context of potential use and of the process of scientific knowledge production itself" (dilling and lemos ) . it is now widely documented that climate change adaptation is dependent upon local contexts and there are no one-size-fits-all solutions (ipcc ). culture (adger et al. ) , local knowledge (naess ) , local institutions (berman et al. ) , the intertwining of local and global political forces and history (kennedy et al. ) , are examples of dimensions that define the ability of a community to face climate change. the provision of meaningful and usable climate information at the local level, therefore, entails some sort of tailoring. there are now case studies illustrating the mismatch between climate science and local interests (e.g., baztan et al. ) providing information does not necessarily mean local users will be able to make use of it. climate science may need to be linked more directly to local communities, to their particular capacities and contexts of vulnerability (ipcc , vaughan et al. . as bremer et al. ( ) put it, "scholars and practitioners have been ill-equipped to describe the contexts where climate services are introduced, or interpret how context can shape the way they develop" (pg. ). co-production has been proposed as one approach for addressing the challenge of turning climate science into a usable climate service. not only does knowledge co-production allow for better usability and implementation, it has many increasingly documented benefits such as social learning, empowerment, and inter-and intra-community trust building. for the purpose of this research we used, as starting point, vaughan and dessai's ( ) definition of climate services: "the aim of climate services is to provide people and organizations with timely, tailored climate-related knowledge and information that they can use to reduce climate-related losses and enhance benefits, including the protection of lives, livelihoods, and property" (pg. ). when considering this definition, we focused on providing people with climate tailored knowledge, "people" understood as members of local communities. the central challenge we wished to address was that of "tailoring" climate knowledge and information for communities at the margins, who might not be very aware of climate issues. such tailoring of climate knowledge is closely associated with the ability to establish iteration (dilling and lemos ) and dialogue (vaughan and dessai ) between scientists and non-scientists in the course of knowledge production and use. climate knowledge co-production, the "deliberate collaboration of different people to achieve a common goal" (bremer and meisch , pg. ) has been proposed for quite some time (e.g., lemos and morehouse ) to address challenges of initiating and maintaining such reiterations and dialogues. but what if this dialogue needs to be established on grounds other than those of climate change? bremer and meisch ( ) conducted an extensive mapping of the literature on climate change research co-production. they identify a series of eight "conceptual lenses" and call for a "selfreflexive transparency when using co-production concepts" (pg. ) to address the concepts' polysemy. within their framework, the work we conducted lies at the juncture of several objectives associated with these lenses: we want to integrate non-scientists as co-investigators (extended lens); we wish to sustain interactions between climate science providers and users (iterative interaction lens); we pursue a goal of empowering local experience, and thus of local knowledge (empowerment lens); we recognize the need to facilitate social learning about climate issues (social learning lens); and we are embedded in a culturally-rooted goal to improve public service through the joint engagement of government agencies and citizens in the production of new knowledge (public services lens). all these objectives are associated with acknowledging the current uneven distribution of access to, and benefits from, climate services development (vaughan and dessai ) . for instance, harjanne ( ) surveyed institutions related to climate services to identify how they justify the need for climate services (as a departure from climate science), and identified the following rationales: the global and widespread nature of the climate challenge; specific industry needs; socio-economic value; technological potential; and deficient supply and demand. there seems to be very little room left for marginalized communities. in the work presented here, we envision the co-production of climate services, not because we perceive co-production as a "value in itself" (voorberg et al. ) , but because we see co-production as a means to create and nurture sustained interaction with marginalized communities while contributing to their empowerment. we wish to explore means for correcting the inequitable distribution of climate change knowledge for action. this uneven distribution in part reflects the fact that not all communities are equal and some are facing such immediate challenges that climate change may be invisible to them. over the course of the research presented here, this called for working to shift awareness to the actual or potential, current or future, connections between everyday non-climate concerns and climate issues. such a shift required a practical intervention centered on local culture. we chose to work hand-in-hand with artists to conduct such an intervention, as art is well-identified as an approach to make visible the "invisible or almost-visible" phenomenon of climate change (knebusch ) . art is also identified as facilitating access to narratives in general, and climate narratives in particular (roosen et al. ). art-based intervention has been proposed in the context of climate change for quite some time (lippard , volpe ). this is not without pitfalls such as instrumentalising art, and reproducing dominant categories and codes through art (miles ) . experiments have shown that through public participation and activism, art may be empowering, and may shift attention to issues that question dominant paradigms (sommer and klockner ) . several dimensions have been identified for collaborations between art and science: new understandings and capacities within and across the arts and sciences involved (gabrys and yussof ) ; catalysing explorations of the scientific context and critical re-imaginings of research practices (rödder ) ; helping to engage multiple senses and emphasizing social interaction within research practices; aiding participating researchers in thinking creatively (jacobson et al. ) ; redesigning social relations to natural systems (armstrong and leimbach ); rearticulating politics and knowledge (latour ) ; offering more effective approaches to engaging multiple publics in climate-compatible behaviour change; and engaging explicitly with the underresearched issue of the role of place attachment and local, situated knowledge in mediating the influence of climate change communication (burke et al. ) . capitalizing on these observations, we developed the working hypothesis that iterative art and science approaches have the potential for instigating and sustaining community dialogue through efforts to co-produce climate services. we saw art as essential for making the concept of climate services more meaningful in a specific place, and focused on narratives as an entry point for coconstruction. artists are ideally placed to challenge existing narratives and to provoke the exploration of new narratives. the intermeshing of nature, culture, emotions and reason around weatherscapes implies that approaching local climate through science could be put into perspective by a more intuitive approach seeking to represent the world as perceived by the senses. such an approach has been achieved by including the arts in social dynamics together with scientific research and innovation (bilodeau, ) . we envision the integration of art and science not in terms of using the arts to communicate scientific findings, but rather in terms of gaining access to elements that are generally excluded from scientific inquiry to convey a more complete picture of the challenges at hand. developing a strong connection between art and science enables the rearticulation of the scientific description of the world (latour, ) . we proposed to test if an art and science based 'recomposing' of the world (latour, ) allows for locally clarifying ambiguous concepts such as climate change and associated "services". our investigation entailed examining the potential of art 'gestures' (citton, ) and the 'practices of everyday life' (de certeau, ) to facilitate cultural translation between different fields of knowledge and the associated diversity of priorities. the results presented here are from a broader project entitled "co-development of place-based climate services for action" (cocliserv, http://cocliserv.cearc.fr), implemented in parallel at five sites: jade bay in germany (krauss ) , bergen in norway (bremer et al. currently under review) , dordrecht in the netherlands (marschütz et al. ) , the gulf of morbihan (da cunha et al. under review), and the kerourien neighborhood in brest, france. these sites follow a shared methodological design starting with narratives as an entry point (krauss ) and extending to incremental normative scenario design (vanderlinden ) . these steps are transversely associated with: (a) identifying available climate information and potential gaps to be addressed now or in the future, (b) developing a suite of representational tools and participatory approaches (e.g., outreach material, art and science procedures, participatory metadata scheme developments and mapping, citizen science), and (c) creating a knowledge quality assessment protocol tailored to the needs of the project. the narrative dimension of the project is seen simultaneously as a "first step" and a step that yields results in and of itself. more details on the cocliserv narratives can be found in the "narratives of change" work, led by werner krauss (krauss , krauss and . one of the central challenges in designing the cocliserv project related to generating a sufficiently broad and diverse set of ground-truthing sites to assess how the approach would fare in different settings as it was being developed and tested. within the participating communities, the kerourien site -the focus of the experiment we present here-was unique. in kerourien, climate change is not a visible concern; residents of kerourien face other daily struggles that take priority and the research team had to adapt to their situation. the peri-urban neighborhood of kerourien is in the port city of brest (brittany, france). it is located in a coastal area with low population density on the western edge of france. kerourien is a minute walk from where the bay of brest connects to the mer d'iroise, part of the atlantic ocean system. it is technically coastal, however its residents are not seafarers or fishers, they live in a suburban housing development similar to many found throughout france. climate change and its effects have been studied in the brest area for decades. we know today, as shown for example in l'hévéder et al. ( ), that temperature plays a fundamental role in ocean circulation; stratification; chemical and biogeochemical processes such as degradation, dissolution, precipitation; and in controlling the spatial distribution, metabolic rates and life cycles of marine flora and fauna (e.g., bissinger et al. , chen , helmuth et al. , philippart et al , thomas et al. in l'hévéder et al. ). kerourien's context is that of an oceanic region with sea temperature sensitives to global change, this area is a biogeographic boundary zone and in recent years warm water species have become much more common (southward et al. ) . ecological problems related to sea surface temperature change also include alterations in nutrient delivery from land to sea; arrival of invasive species and changes in host-pathogen relationships and biological interactions (poloczanska et al. ). these observations are combined with coastal erosion and extreme weather events such as droughts, exceptional storms, and heat waves, as experienced in . the - city climate plan for brest pays particularly close attention to mitigation, and thus to energy production and consumption, concluding that: (i) housing (heating, hot water, and all uses of electricity) is responsible for % of energy consumption in the brest metropolitan area and % of greenhouse gas emissions. the energy used in the brest metropolitan area is mainly of fossil origin (gas: % and oil: %), the net quantity stored annually in the brest metropolitan area is estimated to be , tonnes of co , i.e., around % of its annual emissions. while adaptation planning is a legal requirement, mitigation seems to remain higher on the city's list of priorities. kerourien has residents ( census), and the neighbourhood is mostly organized around post-war housing projects. it is a priority area for the city, which implies a well-identified social vulnerability including demographics such as: . % single-parent families, with % of its residents under the age of , and more than nationalities represented. kerourien is one of the most diverse areas in the city and faces the most challenges in terms of urbanization, migration, and disempowerment (fig ) . thirty-two percent of its inhabitants are unemployed versus % in the rest of the city, with an apex of % for youth between the ages of - versus % for the rest of the city. the schooling rate for youth ages - is % versus % for the rest of the city. this is kerourien's current context. from the neighbourhood's beginning there have been efforts for improvement conditioned by its unbalanced social structure. currently five formal social work organizations conduct on-going efforts here, but with high unemployment and growing imbalances, the question often arises: "who helps whom"? key observations . on the process of linking a local event and our climate services centered inquiry the initial step of our co-production work focused on collecting narratives. this was an ad hoc process, which evolved along the way. we present our approach and associated observations as the first part of our results, describing the organisational stages and observations of procedures and materials ( table ) . as a starting point, we formed a working group with four partners who had participated in preparing the project, including: the maquis, the centre social couleurs quartier, the theatre du grain, and the cearc research center. the five initial meetings focused on identifying the means to simultaneously mobilize the neighbourhood around locally salient issues while giving access to potential conversations about climate-related concerns. the upcoming th anniversary of the neighbourhood was chosen as an opportunity to achieve this goal. a list of stakeholders was established by the four core partners with the provision that snowballing was possible if a category had been overlooked. accordingly, over the course of events we incorporated additional partners, but to a limited extent and very progressively. the initial stakeholder list comprised eight groups and institutions. after limited snowballing, it expanded to members (table ) these stakeholders were invited to a foundational meeting to identify shared objectives within the context of organizing kerourien's th anniversary celebration. the goal that generated the most widespread support was to dispell the myth that "in kerouien only bad things happen". the anniversary celebration was thus named les belles histoires de kerourien (kerourien's beautiful stories). the group of stakeholders formally became the local coordination committee for the th anniversary. at that point, the aim of climate service co-production seemed remote. however the intention, climate-services wise, was to gather narratives in order to explore the linkages between explicit concerns and climate issues, which seemed mostly invisible in kerourien. working on les belles histoires with and for kerourien residents seemed to be a challenging and promising opportunity. we held monthly meetings of the -member local coordinating committee. these occurred under the leadership of its rapidly established executive board, composed of four members of the initial core working group. in the course of these meetings, stories were progressively identified. four important developments were observed: . members of the group acknowledged the need to increase the involvement of community members; . systematic archive research became necessary because, according to those around the table, the stories being told needed anchoring in dated, identifiable events that those present had not necessarily witnessed but that were still part of the memories of the neighbourhood; . an interview protocol was devised to simultaneously involve more members of the local community and gather stories in the words of those most affected; . practical matters were delegated to implementation committees tasked with managing specific practical concerns for the th anniversary celebration, such as: welcoming/ticketing; hiking, cycling, scooters; canteen/kitchen organizing; reports, video editing, projections ( in total). these were powerful recruitment tools, and many community members participated. these developments were important on two levels. first, the local coordinating committee turned to the a priori climate-centred transdisciplinary team to mobilise their expertise in terms of public participation and research. second, it allowed for a more systemised approach to recruitment and collecting local and non-local narratives. a transdisciplinary research design was then adopted to prepare the th anniversary celebration and its associated art form. this included systematising the identification of archive materials, their sorting, key-wording, storage, and subsequent use. these archives contained materials such as: personal photographs, drawings, memorabilia, media excerpts, and recorded songs collected through on-site participant observation. gathering the archive had two effects. first, it allowed us to put kerourien's stigma in perspective both as the product of historical and political processes, and as the product of the amplification of anecdotal events. second, compiling the archive also lent a sense of materiality to the process. a semi-structured interview protocol was designed and the associated interview framework was developed. both were developed with input from the local coordination committee and members of the local community. the interview protocol contained the following questions:  where were you born?  what was the path you took that brought you to kerourien?  can you tell us about the first time you came to kerourien? how did you feel?  tell us about three events in kerourien that have been important to you (personally).  how do you feel when you look at kerourien today?  can you describe three dreams you have for kerourien in (in years)?  what would it take to make them come true?  do you have anything you would like to add? the interview process led to the collection of narratives that stressed the need for les belles histoires. interviewees were proud to be part of the neighbourhood and expressed a deep wish to shift the stigma and be seen by the city as the decent people they recognise themselves to be: "what i like is the moments when you help people and they thank you. help with a stroller, or the shopping bags of an elderly lady, hold a door. small moments like that and people thank you. a thank you. not much, but attention, a gesture, a coffee, a smile." "we have lots of things to do. and in kerourien we think, we act, we do a lot of things for people to gather. faced with the disorder of the world, here kerourien is bubbling with ideas. how, in the face of global reality, we invent something here to rebuild, to find meaning, with the people of the neighbourhood. many people do not give up. continue tirelessly to fight, to resist. day by day. right here. now." at that point, our experiment in climate service co-production had taken quite a turn. first, narratives were being collected with a level of robustness that would make them usable in a science-centred process. second, the team of climate social-scientists and artists were putting their skills to work to serve the local community in realms that may seem far removed from climate issues, but that allowed for a fascinating exercise in trust building and, as a result, quite extensive data collection. papers, objects, newspaper clippings, bits and pieces of local narratives, were made physically available. this led to another important development: obtaining two rooms and bathrooms fully dedicated to those preparing the th anniversary celebration. this locally anchored what had otherwise been a "placeless" exercise. stories, archives, drawings, timelines all could be pinned on the walls. interviews were conducted in the les belles histoires room. involvement continued growing, with community members "just passing through" to "take a look," and thereafter staying around, becoming contributors to the anniversary celebration preparations and the archive and narratives corpus we were compiling (table ) . for the climate service co-production work, it meant a wealth of stories were made available. ongoing dialogue with and participation from the artists meant that a central art form was taking shape. this acted as a filter, prioritizing local issues as they appeared in the stories being told. a script and a scenography emerged, encapsulating the stories being told in kerourien. table : summary description of the data collected through the joint process of preparing kerourien's th anniversary and collecting narratives as part of the climate service co-production process. this process (table ) led to material and procedural benefits. on the material front, robust data were collected. these were used for creating an art form and for laying the foundations for the climate service co-production process. on the procedural front, the neighbourhood was highly involved and, more importantly, trust had been built and mobilised for collective action. start of the project's implementation in kerourien / setting up the working group / these four partners have a well-established working relationship: trust between them is well established. issues of symbolic hierarchies, associated with status (artists are not as regularly employed as social workers or scientists) initial meetings and identification of a modus operandi / decision taken to contribute proactively the neighbourhood's th anniversary celebration. the anniversary was to take place in june , then delayed to october . content and program are still a total blank slate. identification and invitation of all potential stakeholders / foundational meeting of the local coordination group. the initial working group is turned into the local coordination group executive board / proactively implementing the local / / meeting facilitation was organized around the following aim: identifying a common goal for all present, around which everyone could be mobilized. of the members, are residents of the neighbourhood and act as initial proxies for the coordination groups' decisions. resident population. monthly meetings of the local coordination group / discussing the planning and programming of the th anniversary. from / to / all decisions taken collectively. when the group deemed it necessary, votes were organized. this was the first step to spread the word and be publicly explicit with the intentions and partnerships. tasking a specific group with conducting interviews. / - / obtaining fully dedicated facilities ( rooms and bathrooms). agreement with the city as landlord. moving the date of the anniversary from june to october. the decision was taken to document the process more systematically. establishing thematic working groups / specialized working groups are created. these working groups are open to anyone willing to contribute to organizing the th anniversary. the public presentation of the "beautiful stories" process. / / at h, h and h to be sure all residents have the chance to come if they wish. formal closure of the social centre after arson. / to / / the perpetrators are identified but the ongoing efforts of trust-building are damaged as well as the front door of the social centre itself. weekly meetings / discussing the process and detailed programming of the th anniversary. from / for some of the working groups. some meetings are working-group focused, some are plenary with all partners. poster proposed by the graphic artist, discussed at the coordination level and the final version made available to spread the word. a new iconic image for the neighbourhood. final programme available. efforts increase progressively going from weekly to daily. - / the number of people engaged grows day by day, the thematic working groups structure is key. kerourien's beautiful stories. a popular success with a strong public and mass media impact. "beautiful stories" review meeting. / people express that kerourien is whole again, community members have a renewed sense of sharing common narratives. what did the climate service co-production component of the process learn from the exercise up to now? first, we witnessed progressive involvement from community members. by embedding ourselves in a process focused on highly salient non-climate related issues we gained access to community members and to issues that would not have been accessible otherwise, including a diversity of narratives. more importantly, and this came a bit as a surprise, the transdisciplinary abilities of the research team were called upon to help achieve non-science centered goals. an initially asymmetrical situation became a situation of more balanced cross-fertilization. somehow our respective specialized lines of action became blurred and from this trust and capacity for action grew. this situation created a central question: how do we manage the different natures of the various processes at hand -celebrating kerourien, preparing an art form, and identifying how local issues relate to climate issues within the context of climate service co-production? as stated above in section . , our working hypothesis was that all issues are connected with climate issues, and these connections deserve to be made explicit. this hypothesis led us to commit to maintaining the blurring of the lines as artists working with and for social scientists when it came to the climate dimension; and social scientists working with and for artists when it came to preparing the art form, rooting it in local issues and giving the identified and collected stories back to the community. the process described here between artists, social scientists, and community members led to the joint identification of a series of three narratives seen as fundamental for the local community. these are available in detail, including interview quotes and the final script of the play, in the supplementary materials. we summarize these materials here, highlighting the most important characteristics. the narrative identification and explication process was conducted through a systematic analysis (iterative thematic analysis), conducted by the social scientists. the creative writing and scenography was conducted by the artists. these phases included on-going interaction with community members, and thus evolved under their constant scrutiny, including during the first ( figure ) and following days of the play. here we present the narratives in the order of their "discovery". a first narrative that stood out was that of the kerourien neighbourhood, its origin, and the trajectory of its first residents. schematically this narrative reads like this: when this narrative was identified, the experiment in climate service co-production could have taken a wrong turn. we could have had the illusion that access to the "place" had been gained. however the place, as a stabilized geographical and administrative space, located in brest, france, turned out to be a misconception. kerourien is made of many places, associated with the diversity of it inhabitants. does that mean this story should have been dispelled as useless? certainly not, it gives a wealth of information. for example, there once was agriculture, and the weather matters when discussing construction work. yet this is not kerourien's only story. other stories matter, and we did gain access to those as well. the second important story of kerourien reads as follows (see supplementary materials for details and interview excerpts): . we, the newcomers, settled in kerourien after leaving home (from abroad) in order to find a better life (and yes we did find it, at first). . since the s kerourien's population has been in flux (and individuals carry their sometimes heavy personal histories, and their traditions, seen as source of positive diversity). . integration within a diverse mix is proactively pursued (not always easily, xenophobia exists, as well as clear challenges associated with highly intercultural settings). promises that were made, mostly of vertical social mobility, are unkept). . there is nostalgia for elsewhere and anger toward "here." (kerourien encapsulates the love-hate relationship that any migrant community may have with its new country). box : "the first generation laid low. the second generation crashed. the third is restless." -kerourien's recent-present narrated as a multicultural entity. once this narrative was identified and clarified, we stumbled on something fundamental in terms of climate services development. kerourien is, for its population, just one of the places that matters. when envisioning brest's humid climate, it is compared with a place of birth, which is, for many, the place where they long to return upon retirement. if we are about to co-produce place-based climate services, we must prepare ourselves to expand our geographical horizons beyond brittany. further conversations indicated that these narratives of kerourien's history and the histories of its "newcomers" were connected though other avenues beyond simply being from kerourien. both "newcomers" and those "from the barracks" share life trajectories of adversity, proactive fights against dominant forces, and the need to contribute to a sense of justice. these characteristics are connected to the third dominant narrative we identified. . the harshness of the life in brittany, the war, and post-war reconstruction, created in brest strong unions, a strong civil society, ready to fight for its rights (even more so in low income neighbourhoods such as the barracks first, and kerourien thereafter). . the fight for social justice led, in the s and early- s, to genuine progress that reinforced kerourien residents' conviction in favor of social justice and the need to fight. neighborhood now, during the presidential election when the far-right candidate made it to the second round (people registered en masse as voters in order to have their voices heard). . today in kerourien community members continue walking the same streets while their context becomes increasingly fragile, they still want to take charge, but feel increasingly unable to shape their world. box : "i have a say" -kerourien narrated as a place fighting for justice. at this stage, the experiment in climate service co-production had access to a unique and locally validated dataset of narratives. these encapsulated local priorities, concerns, and, to a much smaller extent, their interaction with weather and climate. the narratives we collected show that kerourien residents are, indeed, stressed by their daily economic constraints along with societal challenges related to gender, racism and well-being. they do not seem to show much interest in kerourien's weather and climate. furthermore, when envisioning a relationship to the nexus of place, weather, and climate, the narratives seem to indicate that for the so called "newcomers", weather and climate has mostly been experienced in places other than kerourien. newcomers may have experienced weather and climate, but in diverse ways, associated with their diverse origins. moving from the comores, entails seafaring, flying, and having lived in intense relationship with the indian ocean, its power and associated weather patterns. this experience is not easy to relate to the experience of those migrating from mali who have experienced rainfall deficits at the margin of the sahara desert. the relatively mild and humid climate of kerourien may be seen as a relief for some, and as nuisance for others. the limited time some have spent in kerourien may simply not allow for much of a relationship with the local weather to exist. in order to further our inquiry, we tried to identify traces, even faint ones, of weather or climate information. this led us to reorganize the corpus (table ) in terms of weather and climate issues. three ways of grouping the associated narrations emerged from this reanalysis: climate/weather information sources; manifestation of climate or weather in one of the dominant narratives; and reference made to places where climate issues may be radically different than in kerourien and that are seen as central to the life experiences of the narrators. residents of the kerourien neighbourhood have access to mass media and their coverage of weather (mostly through weather forecasts, or coverage of extreme events, mainly storms) and climate issues. they also have access to information sources produced by authorities (such as information booklets about brest 's climate action plan) or by social action ngos (such as information booklets developed by an ngo centred on fighting for housing rights and covering the paris conference of parties in ). they thus have access to the dominant techno-scientific discourse on climate change. very little is available on their everyday concerns, on the places they hold dear, be it kerourien or their hometown in syria, mali, morocco or haiti. very little is even said about climate change impacts on water quality in the bay of brest. one striking finding from our analysis of the collected narratives is the inability of dominant climate change discourse to significantly influence local narratives. not that weather and climate are absent, they simply take another form. we identified several traces of weather and/or climate in the narratives we collected. first, living conditions in brest are very connected to the omnipresence of rain, drizzle, and water in a way that resonates with the situation in bergen (bremer et al. under review) . there is "mud" in the barracks, construction sites are dangerous because of "mudslides" and "slippery conditions." rain is associated with gray skies. coming from warmer climes: "it's not the cold that bothers me the most. it's the gray. i don't like fall. it's too wet, too gray". nowadays the weather in kerourien, for its residents, is relative to elsewhere, places that, thanks to the filters of nostalgia, are characterized by milder conditions, blue skies, easy living. the traces of weather and climate we found are not often from "over here". for many now, kerourien is not necessarily associated with their future. in the future, climate and weather encounters will occur elsewhere. the framing of the weather in kerourien per se follows the enthusiasm-to-regret cycle of the "the first generation laid low. the second generation crashed. the third is restless" narrative : "when i arrived there was snow. i was coming out of the oven. from a hot climate up to °c in february. and i was going into the refrigerator. from hot there to cold here. i was happy to see the kids playing with the snow." the climate of the emotions guides the judgement of the external climate, that of rain, cold and grey skies, shaping a "sense of climate." when the future is envisioned in kerourien, we interpret it through a climate lens as a future that will be the product of the fight between forces of progress and reactionary forces, with kerourien on the side of progress: we observed that, while rarely present in explicit terms, weather and climate are part of the background of kerourien's narratives. from here, we turned to connecting to climate change issues through these stories. these stories, their material, ethical, and emotional dimensions have been recognized as important by community members. for the climate service co-production team, they constituted an escape route away from the dominant technocratic climate change discourse that is currently a non-starter for kerourien residents. by engaging in the process of jointly creating an art form and gathering data while preparing an event with local significance, we managed develop trust and establish mutually beneficial relationships between kerourien community members and the transdisciplinary research team, even leading to the co-production of climate services. vincent et al. ( ) stress that climate services are more than information alone. they are about producing long-term relationships and trust. in kerourien the situation was slightly different, as we were not in a position to engage in a relationship by talking about climate change (see krauss for another instance of such a situation). our challenge stopped at simply engaging in a relationship, let alone one built on climate concerns. we were in a situation where we wanted to "prime the pump" of a co-production process. trust-building in our case study was a condition for co-producing climate services, and a product of our process. this intervention primed not only the co-production of climate services, but also a desirable positive feedback loop. another important observation relates to mutual learning, in the sense of learning to do things together (see perron , vincent et al. ) . one key element that stood out in this experiment was the way in which the transdisciplinary and scientific skills of the research team were put to work for non-scientific purposes, namely the preparation of a neighbourhood's anniversary celebration. learning and trust-building were deeply intertwined for this. as our case shows, those on the science-side of co-producing climate services may render other services to the communities with whom they engage. through this observation we see that co-producing climate services may actually be more about transdisciplinary science than about climate science. this suggests climate service co-production is following the example of sustainability science (see mauser et al. , polk but with a narrower, and much more local focus. finally, we collectively identified a limited set of narratives encapsulating what kerourien residents had to say about themselves. in the words of some of the participants, this process, and the associated les belles histoires, did put the kerourien community back on track to becoming whole again. these stories constitute what the residents all share, at least for the time being. having this renewed common ground is making them stronger than before our co-production intervention. a stronger community will be able fare better in the face of climate change. for our purpose of coproducing climate services, a stronger community will be a stronger partner, more reliable, and more aware of its actual needs. not only did we develop trust with the kerourien community, we all became stronger in the process, stronger for engaging in substantive work, stronger for taking stock of the substantive effects of our intervention. climate services are often assessed against their usability. our research demonstrates this is not sufficient; the procedural benefits of co-producing climate services should be assessed as well. context dependency will make such evaluation particularly challenging. this should not prevent climate service funders from taking stock of the wider social benefits the actions they support bring to co-producing partners. . getting the priorities straight: adopting local values and contributing to justice. the initial step of our co-production process allowed us to anchor our actions in local stories and relate directly to our partner community and its values. this allowed us to free the co-production team from dominant (and technocratic) climate change and adaptation discourses. rather than adopting the pervading culture represented in the climate literature available to the community, we adopted narratives associated with everyday life, hardships, the joys and pain of migration, and engagement for greater justice. through the lens of priority setting, climate service co-production has much to learn from participatory research and participatory planning. for instance, one aspect we did not address explicitly in this experiment in co-production was that of gender. in the realm of participatory research there many analyses showing that one should be explicit about gender and other identity dynamics at play -the "whos voices? whose choices?" questions that need to be answered (cornwall ) . in the case of climate service co-production, the dominant discourse may totally blind co-producers with its technocratic, mainstream scientific stance; it seems too often to consider gender, race, class, and other social categories, as not necessarily part of what deserves attention. within the realm of climate change, our results point to "the importance of (re)politicizing coproduction by allowing for pluralism and for the contestation of knowledge" (turnhout et al , pg. ). as krauss ( ) writes, "a focus on narratives shifts the attention from the impact of climate on society to the myriad of entanglements between human and non-human actors in a changing climate" (pg. ). this shift in focus will allow us to ground further steps of climate service co-production in the priorities of those most vulnerable to the vagaries of the world. . extending the geographical boundaries of empowerment and the fight for justice: revisiting the concept of place finally, paying attention to local stories and the role of weather and climate within these stories led us to the realization that locally place-based climate service co-production may actually entail working with multiple locations and associated issues. co-production challenged our routines (krauss ) . it pushed us to reconceptualize "place" as extending beyond the circumscribed location where our co-production partners were living at the time. this opened up a rich perspective in terms of climate service co-production. in the course of our work, place became a relational concept, the definition of which belonged to the members of the co-producing community -what mattered what their sense of place (see stedman ) . sense of place is an integrative concept (saarinen et al. ) , and it carries both the characteristics of the environment and of the individual or group perceiving it. sense of place connects with place attachment, and others have shown, as we observed, how memory is critical for migrant populations' relationships to places (rishbeth and powell ) . by adopting this extended concept of place, the co-production team had to accept that knowledge transcends national boundaries, and that time-scales may relate to individual trajectories of past, present, and hoped-for futures. huot and liberté-rudman ( ) , analyzing the interplay of occupation, place, and identity, propose that individuals perform their identity in relation to place and occupation. this resonates with our results and the dynamic nature of the judgement individuals expressed of the place where they live and of the (now imagined) place they once left, and to which they long to return. the status of place shifts through time, as a manifestation of changes in context, occupation and identity. place-based co-produced climate services in such situations need to be reinvented in order to offer information that is dynamic, reconfigurable, and multi-layered. this is another central challenge for the climate service co-production research agenda. what did we learn by reaching out to a community for whom climate issues were not central? we learned that things are not what they seem; at least when one is "co-producing placebased climate services for action." "places" have multiple meanings, are not bounded, and depend very much on personal experience and mobility. the conceptualization of climate services, and the potential for co-production, depends on the reference discourse one mobilizes. climate services may generate many things: from the reproduction of a dominant liberal narrative emphasizing individual responsibility to a unique opportunity to contribute to justice and the redistribution of power. and "action" in this case entailed being mobilized to co-produce an anniversary celebration and the corresponding theater play, along with a stronger partner community as a result. if we return to our original line of questioning, "how and with what results might place-based climate service co-production be enacted within a community for whom climate change is not a locally salient concern?", we have some answers. in order to engage with a community in a climate service co-production exercise despite the community's seeming lack of interest in climate issues, we took the long way around. we engaged in relationship-building and making our skills available for many purposes while progressively connecting with the community, its concerns, interests, and finding intersections with climate issues. we were transparent in terms of our interest in climate concerns, yet we kept that agenda on the back burner while accepting to be mobilized ourselves around the community's more pressing issues. with what results? from the development of a trust-based relationship with the community to the building of a stronger community, this experiment allowed us to question the dominant paradigm that may be crippling for climate service co-production: the assumption that climate and weather are evident parts of the fabric of everyday life; that it is not always necessary to be explicitly focused on climate to be able to co-produce climate services. these three points lead us to question the definition of climate services we used at the onset of our work and as a starting point for this paper. paraphrasing vaughan and dessai we are inclined to propose a working definition of climate services that will need further ground-truthing: the aims of climate services are to provide people and organizations with timely, tailored climate-related knowledge and information that they can use to reduce climate-related losses and enhance benefits, including the protection of lives, livelihoods, and property, as well any process designed to reinforce the ability to: these conclusions seem to indicate that it might be worth considering integrating climate service coproduction into all community development activities. preparing for a changing climate and preparing the requisite knowledge base should be part of the everyday routines of local development, especially in neighbourhoods with a long list of seemingly more urgent concerns. they might be the hardest hit by climate change. cultural dimensions of climate change impacts and 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priorities to advance climate services what can climate services learn from theory and practice of co-production art and climate change: contemporary artists respond to global crisis a systematic review of co-creation and co-production: embarking on the social innovation journey we thank the reviewers and the guest editor for the care taken in the process of reviewing our manuscript. this paper was made in the course of the cocliserv project figure : kerourien's regional context (a); detailed map of the neighborhood (b) with the location of two representative sites: c : the water-tower, c : the builders' bar in , with cows grazing. key: cord- -n lttt authors: balsari, satchit; dresser, caleb; leaning, jennifer title: climate change, migration, and civil strife date: - - journal: curr environ health rep doi: . /s - - - sha: doc_id: cord_uid: n lttt purpose of review: in this article, we examine the intersection of human migration and climate change. growing evidence that changing environmental and climate conditions are triggers for displacement, whether voluntary or forced, adds a powerful argument for profound anticipatory engagement. recent findings: climate change is expected to displace vast populations from rural to urban areas, and when life in the urban centers becomes untenable, many will continue their onward migration elsewhere (wennersten and robbins ; rigaud et al. ). it is now accepted that the changing climate will be a threat multiplier, will exacerbate the need or decision to migrate, and will disproportionately affect large already vulnerable sections of humanity. worst-case scenario models that assume business-as-usual approaches to climate change predict that nearly one-third of the global population will live in extremely hot (uninhabitable) climates, currently found in less than % of the earth’s surface mainly in the sahara. summary: we find that the post–world war ii regime designed to receive european migrants has failed to address population movement in the latter half of the twentieth century fueled by economic want, globalization, opening (and then closing) borders, civil strife, and war. key stakeholders are in favor of using existing instruments to support a series of local, regional, and international arrangements to protect environmental migrants, most of whom will not cross international borders. the proposal for a dedicated un agency and a new convention has largely come from academia and ngos. migration is now recognized not only as a consequence of instability but as an adaptation strategy to the changing climate. migration must be anticipated as a certainty, and thereby planned for and supported. people have always been on the move in search of a better life and often simply for survival. population movements, both voluntary and involuntary, have been of interest to those in power and especially so in the last years, coinciding with the period of great explorations, wars of extensive scale, accelerated population growth, and increased control of political borders. the international legal regime created in the years after world war ii, when at least million refugees were stranded in europe and uncounted millions rendered homeless in africa and asia, established rules and regulations relating to definitions and options for people who had left one country for any reason and desired entry into another. these rules have been used to establish the legitimacy of an individual's claim to be a refugee. in the decades since the end of wwii, these rules have proved insufficient to capture the needs and motives of desperate people on the move [• • , ] . migration has now become the reality for nearly one billion people. on a seasonal basis, the numbers are larger, although on smaller temporal and spatial scales. in this paper, we use movement and migration interchangeably to mean displacement, whether voluntary or forced, temporary or permanent, and across spatial scales. diverse political, economic, and environmental causes have triggered large migrations in the past five decades (see table ). a surge in human population creating rising resource demands, an explosion in industry and technology with heavy reliance on fossil fuels, and many different kinds of wars and ethnopolitical conflicts have brought us to this point: nearly one billion people live away from their places of birth, among whom about million (or . % of the global population) have crossed international borders. those on the move include people seeking work, collective groups of people fleeing war and oppression, environmental and climate refugees, and many who fit several of these categories [• • , - ] . in the first half of the twentieth century, migration into wealthy countries of the north and west was driven by major wars and the collapse of countries. in the s, however, the influence of environmental degradation and then climatechange-induced distress began to emerge in the scientific and policy discourse [ ] . this phenomenon is not new-periodic shifts in environmental conditions or sheer over-population of fragile ecosystems have prompted migration for centuries. advances in remote-sensing and surveillance capabilities that have tracked population displacements in recent years have generated strong empirical evidence that migration is a key survival mechanism in the face of extreme weather events, including floods and droughts; that most climate-related migration takes place across short distances within countries or across contiguous borders; and that households which lack migration options are inherently more vulnerable and less adaptable to the impacts of climatic variability [ ] . the drivers of migration are complex and there is now growing recognition that climate change is a threat multiplier, meaning that while it may not directly cause conflict, it can significantly exacerbate the conditions that lead to conflict, destitution and displacement [ ] . until recently, large migrations usually resulted in the eventual integration of people into the social and economic lives of communities and states in their path. the processes were complicated and much hardship and suffering were often experienced by the first generation who made the move. in recent decades, however, the international and regional responses to migration have become much less welcoming and non-porous, requiring large swathes of people to undertake perilous and often fatal journeys by road or by sea to find a route into some country that might allow them entry. popular discourse has often reduced the complexity underpinning the decision to migrate to a tussle over resources between those native to the land and those that have arrived from elsewhere. over the course of this century, it is expected that heat and irregular rainfall will render many agricultural lands infertile and contribute to land degradation, the expansion of deserts, food insecurity, and permanent alteration of many regional economic systems [•• ] . there is an extensive history of how prolonged episodes of severe drought in many regions of the world have caused hardship for pastoralists and farming populations whose lives and livelihoods depend upon periodic rains. in general, several years without rainfall are required before these populations decide they must leave [ ] . they sell assets to try to survive until the rains come and then over time their animals, and table notable international migrations in the past five decades (nonexhaustive list), reconstructed from "migration waves," national geographic, august when fully impoverished, they then abandon home [ , ] . where people choose to go depends upon location and custom, and their ability to leave; movements may be to urban areas, coastal regions, or adjacent lands which remain fertile. these paths out inevitably lead to social collisions with other populations, and again, consequences vary considerably. the world community has had more experience with periodic drought in sparsely settled rural areas than in dense cities where, in general, wealth and infrastructure have buffered urban populations from the direct effects of decreased rainfall. with accelerating climate change, however, the intensity and duration of droughts are likely to increase, impacting urban water supply [•• ] ; urban heatwaves will also become more frequent [ ] . as greenhouse gas concentrations continue to rise, regional warming will intensify [• • , ] ; models suggest that alterations in atmospheric dynamics may lead to large changes in precipitation, particularly in subtropical and mid-latitude regions. [ ] [ ] [ ] . the ipcc report forecasts that, by the late twenty-first century, intense heat and dryness will affect countries extending across north africa into egypt and sudan and northeast to saudi arabia, the levant including syria, iraq, and iran [ ] , to parts of india and china in asia, and across the southern united states and mexico [ ] [ ] [ ] . intense regional heat may render some areas uninhabitable [• ] . a model suggests that business-as-usual approaches to climate mitigation and population growth may expose one in three humans in this belt to high temperatures currently found only on . % of the earth's surface [ ] . these environmental conditions raise fundamental concerns about the viability of agriculture in these important regions, as they are seen now to be forcing millions of farmers, pastoralists, and smallholders into increasingly precarious livelihoods and worsening food insecurity [• • , ] . in iran, models suggest that soil may be increasingly infertile by mid-century, contributing to increased dust storms and falling agricultural output [ ] [ ] [ ] . in wealthier settings, such as australia, high transition costs may occur as commercial farmers seek to control additional resources and invest in more efficient systems to maintain agricultural output [ ] . an additional risk factor may be the habitation of regions that will become more desirable as a result of climate change and may become the object of resource competition with adjacent states or populations [ ] . dependence on water from transboundary rivers that upstream political entities may seek to control also creates inherent vulnerability and opportunities for conflict [ ] . other impacts of climate change will impose similar migration choices. evidence of accelerating sea-level rise due to climate change is now extensive. mainstream estimates range from . to . m by the end of the twenty-first century [ , ] , and higher rates are possible if ice sheet and permafrost melting occur faster than anticipated [ ] . displacement of populations whose dwellings are inundated is the most obvious concern and is already occurring in some low-lying islands and river deltas. displacement from these areas will be a widespread issue within a few decades [ ] [ ] [ ] . in addition, repetitive instances of saltwater intrusion from sea-level rise and storm surges will render well water non-potable and farmland infertile, causing livelihoods stress even for people whose dwellings are not directly affected by rising waters [ ] [ ] [ ] . storms will increase in severity and frequency as a result of climate change [ ] [ ] [ ] . high winds, hail, and heavy rainfall can directly damage crops and can lead to extensive surface flooding, landslides, and structural damage; in the case of tropical cyclones, these may also be accompanied by deadly coastal storm surges causing millions of dollars in damage [ ] [ ] [ ] . the resulting physical and emotional trauma, in addition to damage to individual livelihoods, businesses, and entire sectors of the economy can be substantial and, in some cases, accelerate emigration of affected persons or even lead to abandonment of heavily damaged locations [ ] [ ] [ ] . in the arctic, melting permafrost and sea ice are fundamentally altering transportation systems and rendering some subsistence-based lifestyles unsustainable, as maritime resources are harmed by warming, acidifying oceans [ , [ ] [ ] [ ] [ ] . wildfire has become a new hazard in the region, threatening settlements in the tundra and boreal forest and in some cases permanently altering the ecosystem [ ] . this threat is not limited to the far north; inhabitants of some regions in california may soon be unable to purchase fire insurance, suggesting significant actuarial concern about the longterm habitability of certain fire-prone locations [ ] . current economic and political systems ensure that these slow-moving but inevitable changes will force millions to move for reasons not envisioned in the legal regimes instituted in the s and s [ ] . how migrants are received in host communities however has profound and long-lasting implications on the future of both. migration affects most aspects of human social interaction and in itself imposes environmental change when large influxes of refugees lead to increased demands on ecosystem services as well as on space, food, and shelter [ ] . in the face of inevitable large-scale migration expected within and from the weak economies of latin america, africa, the middle east, and south asia, the current xenophobic approach to keeping out migrants ensures devastating and destabilizing consequences for humans and the planet. a full review of specific vulnerable populations in the face of climate change projections has been extensively explored by other authors [ , ] . here, we focus on four regions at the confluence of climate-related hazards noted above: the african sahel, the middle east and north africa (mena), the "dry corridor" in central america, and south asia, as together they host the world's most vulnerable billions. . the sahel: migration and conflict related to climate change and resulting agricultural and ecological problems are increasingly prominent issues in the sahel, a semi-arid region stretching across africa from ethiopia to senegal. smallholder rainfed agriculture and herding are becoming increasingly difficult due to unpredictable availability of water and in some locations expansion of the sahara desert [ , , ] . these stressors and competition for available resources may have contributed to conflicts in nigeria [ ] , uganda [ ] , sudan [ ] , and kenya [ ] , although the relative causal contributions of climate change, governance, population pressures, and preexisting sectarian divisions to outbreaks of organized violence remain a subject of intense debate [ , , ] . many people facing unpredictable weather, food insecurity, and in some cases violence have left their land for urban centers in african nations, where conflict with existing populations and discord over space and essentials may occur [ ] [ ] [ ] . others have followed longer, more dangerous migration routes across the sahara and the mediterranean in hopes of reaching europe. these routes are characterized by conflict with populations and governments along migration routes and substantial hazards during transport across desert and ocean [ , , ] . even after arrival in host countries, migrants remain a source of intense political tension and experience a variety of forms of discrimination and violence [ , ] . . the mena region: climate models for the rcp . (business-as-usual emissions trajectory) in the middle east and north africa estimate that by there will be at least a days a year (and by , over days) when temperatures will cross into the th percentile for the region, with the hottest temperatures exceeding °f [ , • ] . the same models suggest that in the same time frame ( ) the heat stress on the soil in many areas would desiccate organic life to as much as inches deep, precluding crop growth even if adequate irrigation were provided. although there is some disagreement, the preponderance of the literature suggests that climate change has contributed to civil strife in syria [ ] [ ] [ ] . there, a prolonged drought in the east and northeast linked to climate change forced pastoralists and farmers to abandon their land, sell their animals, and trek to the cities in the western region of syria. in - , it is estimated that approximately one million people migrated to these urban centers, including the seat of the assad government in damascus [ , ] . underlying political grievances in these urban areas, already stretched for resources by . syrian government in the spring of led rapidly to an outright civil war [ ] . that war, now in its tenth year, has killed approximately , people, created . million refugees and at its peak - million internally displaced persons. the ongoing ramifications of this conflict are undermining stability in much of the middle east as well as europe. . central america: in central america, another pattern of climate change, drought, livelihood collapse, discord, and distress migration leading to conflict has occurred [ ] . since , the "dry corridor" in nicaragua, honduras, el salvador, and guatemala has experienced a series of devastating multi-year droughts [ ] . this series is consistent with the predicted effects of climate change in the region, and there is now substantial evidence linking drought and agricultural hardship to the effects of climate change [• • , , ] . according to the food and agricultural organization (fao) of the united nations, crop losses in the region from to range from to % and left . million people in need of humanitarian assistance [ ] . these losses have had a profound effect on the viability of agricultural livelihoods, with the majority of households resorting to crisis coping methods such as selling agricultural tools for food. even so, as of the last quarter of , more than % of the population in the dry corridor are without sufficient income to purchase necessary food [ ] . facing uncertainty about future rainfall, many have chosen to leave, with emigration from the region increasing by % between and [ , , , • ] . the majority of people moved overland along this "dry" corridor to seek better prospects northwards. this trek has resulted in increasing conflict between migrants and authorities in transit nations and along the southern border of the usa. this border, historically the final destination and hopeful push-off point in the usa for many of those attempting to escape the worsening conditions to the south [ ] , has for the last five years now been effectively closed [ ] . . south asia: the densely populated gangetic plains in south asia also fall in the zone that will experience high temperatures as a result of climate change in the coming decades. a model suggested that extremes of wetbulb temperature in south asia, under the representative concentration pathway . (rcp . ) scenario, are expected to approach or exceed the critical temperature of °c ( °f), considered an upper limit of human survivability [ ] . in addition, bangladesh will face the consequences of rising sea levels and coastal flooding that is already imperiling millions of coastal dwellers. the south asia region has already experienced severe heat waves in recent years, including the fifth deadliest heat wave in recorded history killing people in pakistan in . per the world bank report, by , south asia could see - million climate migrants in scenarios under models of urgent climate mitigation, and up to million under a more pessimistic scenario [ ] . the population is highly dependent on the summer monsoons and rainfed agriculture, making it particularly vulnerable to temperature and rainfall changes. the region is likely to have the highest number of foodinsecure people by mid-century [ ] . the southern indian highlands between chennai and bangalore (containing many densely populated cities), parts of northwest india, and nepal may see in-migration, while bangladesh and the gangetic corridor from lahore to delhi will be hotspots for out-migration. coastal cities such as mumbai, dhaka, and chennai are likely to reel under the burden of continued rural to urban migration, until intensifying heat, and worsening flooding and coastal storm surges make life untenable for most of the millions who live at the economic margins of these megalopolises, compelling them to move again. models estimate that the number of people trying to live in the flood plains of south asia may rise from million in to at least million by , even as climate change renders these regions more hazardous [ ] . growing religious and ethnic intolerance in south asia does not bode well for the region when the overwhelming survival need for populations in this century will be more movement not less. rohingya refugees from myanmar are trapped in what is now the world's largest refugee camp in coastal cox's bazar in bangladesh, where they are denied formal refugee status, and remain stateless, unable to seek formal work, advance educational opportunities, or do much more than barely survive [ ] . in neighboring india, the government launched an attack on its minorities by re-defining citizenship, arresting dissenters, and beginning to build detention camps for muslims and unregistered populations in the northeast of the country [ , ] ; the border with neighboring bangladesh, a likely source of climate refugees, is now heavily militarized [ ] . india is currently the greatest exporter of migrants-nearly million-to the gulf states, the usa, the uk, and elsewhere. the experience of populations in the sahel, mena, dry corridor, and south asia, where the climatologic roots of their displacement may overlap considerably with conflicts arising from deep-seated political enmities, suggests patterns of mixed climate change and social crises now occurring in many regions. as they seek stability, livelihoods, and food in new locations, the implications of these movements for affected populations and host regions have become a topic of increasing concern in policy circles although few robust response strategies have emerged from these deliberations. these patterns are expected to intensify and occur more frequently as climate change becomes more extreme in the coming decades. a vexed question over the last years for social scientists and demographers has been whether uprooted populations contribute to intra or inter-group conflict during migration or in the host country. the expert consensus from the late s through early s was that societies could adapt, migration was not clearly caused by climate change, and there was little to no evidence for the influence of environmental stress (let alone climate change) on the incidence or persistence of armed conflict [ ] . shifts in these arguments began to emerge in the mid- s, as the effects of climate change became more marked [ ] , the number of resource wars increased with surges in forced migration [ ] , and research and policy analysis contributed to enhanced understanding of the pivotal negative roles played by internal social fragilities and incompetent or malicious state leaders in the setting of external economic and agricultural shocks [ , ] . at present, available evidence indicates that distress migration from climate-induced environmental crises does sometimes lead to intense social conflict and in certain instances contributes directly to armed conflict. there are however numerous mediating variables: the capacity of the host government and its economy to absorb the incoming population, the extent to which the incoming population is related politically or socially to another population threatening the state's internal order, and the underlying historical vulnerability of this society to civil war or armed conflict all play a role [ ] [ ] [ ] . these variables are crucial in all assessments of these interrelationships and the most pivotal of these is the fragility of the government and its recent conflict history. to put the empirical findings positively, a flexible and robust system of governance with a strong economy can respond to an influx of large numbers of people without deep social distress or conflict-provided sufficient foresight and resources are devoted to advance planning and mitigation. for decades, however, migration was not high on the global political agenda, and not imagined beyond the contours of movements of political refugees. the millennial developmental goals do not pay particular attention to migrants. this stance has changed rapidly in the first ten years of the twenty-first century, as evidence as well as the experience has accumulated to focus attention on the increasing numbers and kinds of distressed migrants moving within countries, within regions, and to europe and the usa. some are fleeing extreme weather events or slowonset but cumulatively dramatic environmental change. some are fleeing conflicts, exacerbated by climate change and resource tensions. a watershed moment in global political discourse around climate change and migrants occurred in - , when a series of international initiatives acknowledged not only the urgent need to address climate change but also the intersection of climate change and human mobility (see table ). in stark contrast to the mdgs, the sustainable development goals (sdgs) called for the "facilitation of orderly, safe, and responsible migration and mobility of people, including through implementation of planned and well-managed migration policies." the sendai framework for disaster risk reduction recognized climate change as a driver of human mobility. the world bank report and the atlas of environmental migration authored by the iom and others conclusively recognize the salience of "environmental migrants." there are to date no international conventions that explicitly address migration from environmental or climate-related causes, and no single un agency responsible for them. proposals for treaties and legal instruments focusing on climate refugees have largely originated from academia and ngos and are summarized in table . the iom does not favor a separate category of "climate refugees" or new treaties to address them, arguing that international attempts at controlling such flows will clash with current notions of national sovereignty [ ] . migration is now recognized not only as a failing consequence of myriad and intersecting determinants, including the failure to adapt to environmental changes, but also as a useful climate-adaptation strategy-if managed, planned, facilitated, and well prepared for in advance [ ] . most migration will continue to occur within countries, and will largely involve rural to urban population flows, mediated by a complex intersection of political, demographic, socioeconomic, and environmental drivers [ ] . while transient shocks like the nationwide lockdown in india amidst the covid- pandemic the conference also established a process for least developed countries (ldcs) and other interested developing countries to formulate and implement national adaptation plans (naps) to identify and address their medium and long-term adaptation needs. it proposed the idea of a climate risk insurance facility, and sought "ways to address rehabilitation from the impacts of such climate change-related events as sea-level rise [ ] ." the nansen initiative, [• • ] in response to cop , the nansen initiative, launched by switzerland and norway, was a state-led consultative process, which recognized that while most movement will take place within countries, there remains a significant protection gap (both legal and operational) for those who cross an international border. based on these consultations, the initiative published a consensus "agenda for the protection of cross-border displaced persons in the context of disasters and climate change," which was endorsed by states in [ ] . the protection agenda supports the integration of effective practices by states and (sub-) regional actors into their own normative frameworks, rather than calling for a new binding international convention on cross-border disaster displacement [ ] . the wim created a legitimate policy space to discuss and address the negative consequences of climate change if society's efforts to mitigate and adapt are not sufficient. it sought to implement approaches to address climate change associated loss and damage and recognized migration as an adaptation strategy. the paris agreement seeks to strengthen the global response to the threat of climate change by keeping a global temperature rise this century well below °c above pre-industrial levels. to strengthen the ability of countries to address the impacts of climate change, it seeks to facilitate appropriate financial flows, a new technology framework, and an enhanced capacity-building framework to align action by developing countries and the most vulnerable countries, with their own national objectives. to date, of the parties to the convention have ratified it. having notified intent, the usa can officially withdraw from the agreement on or after november , . many of the nationally determined contributions, as required by the paris agreement, from africa, asia pacific, and oceania refer to human mobility and its role as an adaptation strategy [ ] . sendai framework for disaster risk reduction, [•• ] the sendai framework adopted at the world conference on disaster risk reduction and endorsed by the unga in recognizes that population movements produce risk but can also serve as an adaptation strategy [ ] . the text however emphasizes disaster-related displacement and avoids explicit inclusion of mobility from conflict and violence, to gain member state consensus. agenda for humanity, [ ] the third of five principles, articulated in this political communique led by the un secretary general, as an output of the world humanitarian summit (whs) in istanbul, is titled "leave no one behind" and addresses displacement, migration, and statelessness. the platform on disaster displacement, [ ] the goal of the platform also launched at the whs is to follow-up on the protection agenda published by the nansen initiative. it explicitly recognizes the intersection of environment and climate change, and displacement. its four strategic priorities include addressing knowledge gaps, promoting identified effective practices, promoting policy coherence and mainstreaming of human mobility challenges, and promoting policy and normative development in gap areas [ ] . assembly and addresses migration due to environmental and/or climate change, as well as the environmental impacts of migration, large population movements, and the environmental sustainability aspects of migration. it notes that environmental factors drive both internal and international migration the global compact for safe, orderly and regular migration, [ ] the compact is an intergovernmental negotiated, non-binding, un global agreement initially adopted by countries that seeks to address concerns of state sovereignty and responsibility-sharing while upholding human rights and principles of non-discrimination as societies undergo demographic, economic, social, and environmental changes that may have implications for migration or result from it. the global compact for refugees, [ ] the compact seeks to invest in host communities to provide better education, healthcare access, and livelihood opportunities, moving away from contemporary dominant encampment policies [ ] . resulted in return migration to rural areas, we find that the current literature suggests overwhelming net rural to urban migration. the foresight commission report from the uk government cautioned against trying to prevent such migration (although it might lead to increased vulnerability of new urban migrants) because preventing them from leaving highly stressed or insecure environments could lead to graver outcomes for those trapped in rural areas with even less access to food and water [ ] . it recognized that the cities receiving these migrants were also particularly vulnerable to environmental change and without adequate preparation would also suffer the consequences of extreme heat and depleted water resources. now, almost a decade later, this scenario is already evident: rural populations moving into vulnerable urban cities, finding no sustenance are on the move again. a recent model developed jointly by the new york times magazine and propublica, and published in july , estimates that the number of migrants arriving at the us border from central america and mexico may rise to . million a year by , from about , a year in , in the absence of climate mitigation or adaptation strategies [ ] . the current international agreements are not remotely adequate to address ongoing and projected levels of human migration, regardless of how and how much climate change may influence it, nor are the decades-long and still current trajectories of industrialization, globalization, and development providing any signs of serious commitment to climate adaptation. realistic global and regional planning should therefore assume the worst-case scenarios under rcp . and begin immediately to prepare to make life tenable for the hundreds of millions that will urbanize and then attempt to move again as life becomes unendurable when the urban centers themselves exhaust resources and opportunities: the migration in the dry corridor heading toward the us border, or the perilous journeys over the mediterranean, are examples of migration patterns when the first stop of refuge is no longer a sustainable option. the wall-building, xenophobic, and insular strategy embraced by the usa, europe, china, and india to deal with the greatest challenge of our times is regressive, violent, and profoundly ignorant, in that it denies the core reality that for millennia, under threat, humans have moved to escape. it dismisses recent and strengthening scientific evidence that stability and security for hundreds of millions in latin america, africa, and asia will depend on the opportunity to relocate, even if seasonally, for work. facilitating visa, employment, and remittance arrangements can complement urgently needed climate mitigation strategies. water management in agricultural lands and in urban areas is likely to become critical to any adaptation plan. host communities, including the transition cities and towns we have examined and final destinations whether in-country or across borders, must start preparing now for the increased demand for food, water, shelter, services, and jobs that will arrive with these migrant populations. there is little indication table key legal instruments that have been proposed in the twentyfirst century mostly based on the premise that current options are not sufficient or are failing. the summaries here are based on [ ] draft convention on the international status of environmentally displaced persons, . [ ] university of limoges the principles underlying the convention include the principle of solidarity, common but differentiated responsibilities, effective protection, non-discrimination, and non-refoulement. the specific rights guaranteed to persons threatened by displacement include rights to information and participation, displacement, and the right to refuse displacement. the rights guaranteed to persons already displaced include those common to inter-state and internally displaced persons. the convention would include a world fund for the environmentally displaced that would provide financial and material assistance for the receipt and return of the environmentally displaced. proposal for a convention on climate change refugees, [ ] harvard university the authors argue for a new, independent convention that allows for the instrument to be creatively tailored to the complexity of the problem and to take a broad-based and integrated approach, on the basis that the problem of climate-induced migration "is sufficiently new and substantial to justify its own legal regime," instead of being forced within legal frameworks that were not designed to handle it. [ ] university of western australia the convention would assign rights and protections through a process of "request and determination" that would be based on scientific studies and the particular situation of the community. under the convention, displacement would be viewed as "a form of adaptation that creates particular vulnerabilities requiring protection as well as assistance through international cooperation." thus, the emphasis of this convention would be on the duty of a particular state to provide protection and humanitarian assistance to climate change displaced persons within its jurisdiction and to support governments, local communities, and agencies in fulfilling that duty. climate exile treaty, [ ] iit, madras, india the authors distinguish between "luxury" emissions referring to those associated with wasteful lifestyle choice and "survival" emissions that are associated with subsistence living. they invoke the "beneficiary pays" principle, which states that "countries that undertook and benefited from emissions activities are liable for the costs of combating negative externalities that resulted from them." that any of this preparatory action will happen: the pandemic has reaffirmed that the current global approach to solving intractable challenges does not embrace cooperation, mutual interest, and scientific rigor but instead retreats to adamant rejection of a future that is upon us. the post-wwii international regime sought to rescue, provide refuge, and rehabilitate millions rendered homeless by the wars and post-colonial independence struggles that followed. but in recent decades, large pulses of migration along the dry corridor in central america, the sahel, the middle east, and asia, some in the context of protracted wars, have culminated in a regime of reneged promises to protect, refusal to let the vulnerable in, and attempts at repatriation or worse, forced refoulement [ ] . these responses have followed the ascendancy of inwardlooking nation-states in the north and west who have observed explosive population growth elsewhere and interpreted this phenomenon as a threat to their own internal resources and stability. consequently, international borders have become increasingly tightly regulated and hostile to foreign entrants. complex visa arrangements are now a key driver of who can cross international borders, and under what conditions, inflicting with their harsh differentiating categories particularly punishing effects on women, children, and the elderly [ ] . the regressive treatment of refugees at the us-mexico border and at the gates of europe (with the exception of germany) is diametrically opposite to what is needed. weaker economies like turkey, jordan, lebanon, and even bangladesh have been kinder to the millions that have poured across their borders, albeit with wide variation in permitted integration. unless the most powerful governments around the world change course nowand they are showing little signs of doing so-over this century, the evidence shows, the impact of climate change on the hundreds of millions who will nevertheless move, on those in regions that will receive them, and on those who will not find any remedy through migration, will be impossible to bear. acknowledgments we thank abhishek bhatia for the assistance with editing the manuscript. dr. balsari and dr. dresser's research is supported, in part, by the living closer foundation consortium on climate and health. author's contribution the manuscript was jointly developed and written by sb, cd, and jl. all authors read and approved the final manuscript. conflict of interest the authors declare that they have no conflict of interest. open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link 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safety and dignity: addressing large movements of refugees and migrants: report of the secretary-general human rights of refugee and migrant women and girls need to be better protected. human rights comment-the council of europe commissioner for human rights publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord- - ljyk uw authors: el hamichi, sophia; gold, aaron; murray, timothy g.; graversen, veronica kon title: pandemics, climate change, and the eye date: - - journal: graefes arch clin exp ophthalmol doi: . /s - - - sha: doc_id: cord_uid: ljyk uw nan global climate change is primarily a sequel of human's impact on the planet. more than years ago, marsh recognized and praised the benefits of the interaction between man and nature. however, he was also the first to severely criticize their relationship, suggesting that further abuse by humans could result in the extinction of the species. this exploitation of earth's resources led nobel prize laureate paul crutzen to coin a new term: "anthropocene" or "the age of man" [ ] . humanity's disruptive behavior could have started with the industrial revolution in the mid-eighteenth century and has continued for the past three centuries. in , vitousek et al. estimated that - % of the world's land surface has been transformed or degraded by human activity [ ] . furthermore, a persistent increase in levels of fossil-fuels has released abundant greenhouse gases (ghg), contributing to a global crisis of air pollution. the energy imbalance as a result of pollution induces accumulation of heat with the subsequent warming of the planet. in fact, the intergovernmental panel on climate change (ipcc) calculated that the earth will warm by . °c during this century [ ] , causing a massive climate-induced change in the migration pattern of wildlife animals, bringing them into greater contact with humans. currently, the covid- pandemic has exposed our vulnerability. in a couple of weeks, it brought normal life to an almost complete halt. the primary aim of this review is to describe the impact of environment on the spread of zoonoses and how climate can influence the development of eye diseases. some of the possible outcomes from covid- will also be delineated, respectively. the climate system is interactive and evolves in time under the influence of several factors: little ice age is a period between and that is known for its colder temperatures, with an average drop of . °c. although most of the cooling may have been caused by a decrease in sunspot activity or a surge in volcanic eruptions, evidence suggests that it could be to some extent manmade [ ] . a vicious cycle had started: several plagues had claimed the lives of millions, leaving extensive land to reforest, lowering the levels of carbon dioxide (co ) with a subsequent decrease in temperature. on a global scale, extreme climate with freezing conditions increased harvest failures, famine, and malnutrition, resulting in prominent outbreaks of new and old epidemics [ ] . since the world has emitted-approximately-over . trillion tons of co [ ] , contributing to the increment of global temperatures over °c, with unprecedented changes since the mid-twentieth century. prosperity and advances in technology, the hallmarks of the second half of the century, are primary drivers of co emissions. therefore, the sustained human release of greenhouse gases is the main reason attributed to the rise in temperatures [ ] . to address climate change, the united nations (un) established the paris agreement, setting a target, limiting average warming to °c, urging the world to urgently reduce emissions. regarding health effects, several studies have estimated that global warming will induce deadly hurricanes by this article is part of the topical collection on perspectives on covid- intensified rainfall and stronger winds. furthermore, rising sea levels will acidify the ocean, and drought and fires will worsen pollution. such conditions have a lasting impact on sea temperatures and air quality, direly affecting ecosystems, and leading to waterborne and airborne diseases [ ] , aggravating chronic conditions and ultimately anticipating higher rates of premature death. an unstable climate may also threaten pathogen hosts, inducing relocation of microorganisms and hosts [ ] . these shifts have been suggested as the reason for emerging infectious diseases [ ] (fig. ) . in december of , dr. li wenliang, a chinese ophthalmologist, warned the world about the potential danger of a cluster of pneumonia cases in wuhan, china [ ] . the outbreak was later traced back to the seafood and wet animal wholesale market in wuhan. moreover, samples from the live animal section of the market were positive for the virus. a novel coronavirus was identified as the etiologic agent of this deadly disease. covid- rapidly proved to have human-tohuman transmission spreading through the world at a fast pace, jeopardizing the modern world. as of september , , covid- has infected more than million people in the usa, killing over , [ ] . however, these statistics might not be entirely accurate. several factors prevented the containment of the epidemic and enabled exponential growth of cases, revealing our country's flaws in the management of an outbreak of this magnitude. history has shown that the worst epidemics ravaged nearly entire civilizations, but the impact of an epidemic goes beyond the death toll, involving the global economic collapse and climate change. we can postulate that the aftermath of closing borders, bars, and schools, in addition to travel bans and shelter in place orders around the world, has brought some benefits to the planet. at least momentarily, mother earth seems to be healing. benefit: reduction of air pollutants nasa (national aeronautics and space administration) and esa (european space agency) have announced that china and italy are showing a cleaner air amid the quarantine. the dramatic drop-off of nitrogen dioxide (no ) pollutions have lifted the fog of densely populated cities around the world, in particular, los angeles and mumbai. in a similar manner, satellites from the copernicus atmosphere monitoring service measured a decline of - % in surface pm . over large parts of china in february [ ] . at large, carbon emissions have sharply fallen across continents. similarly, the united states energy information administration has reported fig. graphic depicting the cycle between climate change and zoonoses a predicted . % reduction in emissions during , as a ramification of the pandemic [ ] . a few months ago, the head of the un environment programme (unep) cautioned the world: "covid- is not a silver lining for the climate" [ ] . despite the favorable changes in carbon footprint, the mitigation might be temporary. patients and healthcare workers are now producing significant medical and hazardous waste. of more concern, americans have been hoarding supplies such as masks, gloves, and cleaning materials since the beginning of the epidemic. the proper disposal of these provisions is questionable. in wuhan, the first epicenter of the pandemic, hospitals' daily waste reached metric tons, a sixfold increment compared with the amount before the epidemic started [ ] . thus far, hospitals in the usa have not been burdened this way; however, it is uncertain how much additional waste will be produced as a result of the outbreak. the intriguing chain reaction of global warming in ocular health is ominous. the spectrum of eye diseases can be categorized by exposure to specific environmental factors, and their severity appears to be directly linked to the duration of that exposure. most atmospheric ozone is concentrated in the stratosphere. with a program launched in , nasa has continuously monitored the status of the ozone layer, partaking in the discovery of their depletion in the early s [ ] . the main function of the ozone layer is to protect life on earth by absorbing harmful ultraviolet light (uv). among the radiation reaching earth's surface, % belongs to the medium-wavelength band ( - nm) or uvb; and the long-wavelength band ( - nm) or uva accounts for the remaining %. the eye is one of the two organs susceptible to solar irradiance; hence radiation from direct sunlight and sky scattering and reflection from clouds, ground, and other surfaces have deleterious consequences [ ] ; and strong epidemiological evidence associates them with the development of photochemical damage to ocular tissues [ ] . the photochemical injury is predominantly due to photooxidative damage where the creation of reactive oxygen species plays a central role. the length of exposure, the wavelength of uv rays, and tissue irradiance determine the severity of the lesion: acute phototoxic lesions are seen on the ocular surface as photokeratitis and conjunctivitis and the retina as solar retinopathy. chronic exposure to solar energy may induce damage to the eyelids: keratoacanthoma, actinic keratosis, and neoplasias; conjunctiva: pterygium, pinguecula, metaplasia, or carcinoma of the conjunctiva; cornea: climatic droplet keratopathy (labrador), keratoconus, endothelial cell damage, and dry eye; lens: cataract and early presbyopia; and trabecular meshwork: glaucoma. regarding the retina, studies have failed to conclusively support the relationship of uv light and disorders such as choroidal melanoma and macular degeneration [ ] [ ] [ ] [ ] [ ] . thus, exogenous agents may contribute to chemical injury by acting as photosensitizers [ ] . these components include tetracyclines, chloroquine, nonsteroidal anti-inflammatory drugs, and psoralen, among others, reaching the ocular tissues directly or indirectly via the circulation. high ambient temperature attributable to global warming may influence thermal damage in ocular structures. bacterial keratitis caused by staphylococcus aureus and pseudomonas have been found to be more prevalent in warmer climates [ ] . fungal keratitis is more common in certain geographic areas with hotter weather. a hot environment may then potentially worsen the burden of trachoma and may trigger the formation of cataracts and the occurrence of central retinal artery occlusion (crao) [ , , ] . air pollution is a mixture of harmful substances in the air we breathe. besides of pollutant gases, airborne suspensions "particulate matter" (pm) are particularly detrimental for human health. several studies have shown evidence that pm . alters the microvascular endothelium-dependent dilation. moreover, adar et al. demonstrated significantly narrower retinal arteriolar diameters in people living in areas of elevated pollution [ ] , and cheng et al. established a possible link between pollutants and crao [ ] . a large report from the uk also found a considerable association between higher pm . exposure and the risk of ganglion cell loss and glaucoma [ ] . the usa (us) currently ranks first in healthcare spending among the developed nations of the world. it is widely recognized that waste in the healthcare system contributes to the prominent cost of medical care. several strategies have been evaluated to assist with cost reduction, estimating that the best approach was the minimization of waste, with future savings that could represent > % of the total health care expense [ ] . ophthalmology, as a surgical specialty, plays a part in generating one third of all hospital-regulated medical waste [ ] . likewise, healthcare services are responsible for nearly % of the usa's carbon footprint. a study compared the generation of greenhouse gases and expenditures of a single cataract surgery between the aravind eye care system from india and the uk. the results were eye-opening: an ecofriendly resource with comparable-to-better patient outcomes and substantial less spending. the same study reported that up to % of drugs used during elective phacoemulsification were discarded, resulting in environmental impact [ ] . the aravind eye care system and other low-income countries routinely reuse surgical materials (after proper sterilization) with minimal rate of endophthalmitis [ ] . benefit: reduction of air pollutants through medical planning the american academy of ophthalmology (aao) had provided recommendations and guidelines for ophthalmologists around the world during the covid- pandemic. initial reduction of elective procedures and less-crowded offices were inherently safer for the patient, the ophthalmologist, the staff, and the environment. disadvantage: aftermath of recession us treasury forecasted unemployment in the usa could reach % due to covid- . at the end of march , the department of labor published data showing that the unemployment rate reached . %, among all major worker groups. several comprehensive and multi-specialty ophthalmology practices closed their offices and laid-off a few of the staff (personal communications). others remained open for urgent visits and procedures or care for the patients through telemedicine services. instead, most retina practices continued seeing patients at high risk of blindness, taking all the precautions needed to prevent the spread of the disease, limiting their regularly high volume of patients. on average, retina practice volumes declined between and %. the job market seems to be uncertain in the near future too. an article published in concluded that the job market in ophthalmology is affected for - years following a recession [ ] . the healthcare industry is the second-largest greenhouse gas polluter after the food industry. although greenhouse emissions may drop after the covid- pandemic, their effect on air temperatures would take years to centuries to perceive changes considering how long the gases persist in the air. if the emissions return to the pre-pandemic levels after the resolution of the crisis, the progress made would have been undermined. anthropologically, societies are transformed if a pandemic kills a considerable proportion of the population, unleashing the economic pressures of less productivity and higher consumer prices. at the moment, the total repercussions of the covid- pandemic are yet to be determined. in the meantime, digitalization has been accelerated to optimize services and to mitigate the intrinsic difficulties of social distancing. for doctors, including ophthalmologists, the sequel might require drastic changes in how we practice medicine. nonetheless, if fundamental lessons are learned, we will start taking responsibility for climate change with promising positive outcomes for the entire humanity. in the end, resilience in the midst of a catastrophic event is the only way forward. code availability (software application or custom code) not applicable geology of mankind human domination of earth's ecosystems ipcc: global warming of . c. an ipcc special report on the impacts of global warming of . c above pre-industrial levels and related global greenhouse gas emission pathways, in the context of strengthening the global response to the threat of climate change, sustainable development, and efforts to eradicate poverty the little ice age insight from past millennia into climatic impacts on human health and survival contribution of working group i to the fifth assessment report of the intergovernmental panel on climate change impact of climate change on air and waterborne disease evolution in action: climate change, biodiversity dynamics and emerging infectious disease amid coronavirus outbreak: copernicus monitors reduction of particulate matter (pm . ) over china short-term energy outlook covid- is not a silver lining for the climate coronavirus leaves china with mountains of medical waste ozone depletion and solar ultraviolet radiation: ocular effects, a united nations environment programme perspective ultraviolet radiation oxidative stress affects eye health the environment and the eye oxidative photodegradation of ocular tissues: beneficial effects of filtering and exogenous antioxidants environmental temperature and senile cataract association between season, temperature and causative organism in microbial keratitis in the uk rise in lens temperature on exposure to sunlight or high ambient temperature ambient air pollution and the risk of central retinal artery occlusion air pollution and the microvasculature: a cross-sectional assessment of in vitro retinal images in the population based multi-ethnic study of atherosclerosis (mesa) the relationship between ambient atmospheric fine particulate matter (pm . ) and glaucoma in a large community cohort waste in the us health care system-insights for vision health people, planet and profits: the case for greening operating rooms cataract surgery and environmental sustainability: waste and lifecycle assessment of phacoemulsification at a private healthcare facility waste generated during glaucoma surgery: a comparison of two global facilities impact of the economy and recessions on the ophthalmology job market publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord- -zf jfol authors: parrish, rebecca; colbourn, tim; lauriola, paolo; leonardi, giovanni; hajat, shakoor; zeka, ariana title: a critical analysis of the drivers of human migration patterns in the presence of climate change: a new conceptual model date: - - journal: int j environ res public health doi: . /ijerph sha: doc_id: cord_uid: zf jfol both climate change and migration present key concerns for global health progress. despite this, a transparent method for identifying and understanding the relationship between climate change, migration and other contextual factors remains a knowledge gap. existing conceptual models are useful in understanding the complexities of climate migration, but provide varying degrees of applicability to quantitative studies, resulting in non-homogenous transferability of knowledge in this important area. this paper attempts to provide a critical review of climate migration literature, as well as presenting a new conceptual model for the identification of the drivers of migration in the context of climate change. it focuses on the interactions and the dynamics of drivers over time, space and society. through systematic, pan-disciplinary and homogenous application of theory to different geographical contexts, we aim to improve understanding of the impacts of climate change on migration. a brief case study of malawi is provided to demonstrate how this global conceptual model can be applied into local contextual scenarios. in doing so, we hope to provide insights that help in the more homogenous applications of conceptual frameworks for this area and more generally. the climate change-migration nexus has been the subject of research debate for decades. indeed climate change has been instigated in human migration since early humans first moved out of africa and migration has long been an adaptive strategy to climate shocks, long-term changes or cyclic climate conditions [ ] . the field of climate migration has been gaining global scientific and popular attention since roughly the s [ ] , and very much so in recent years, since the emergence of the concept of 'environmental refugees' [ ] . since the european migration 'crisis', the topic has received increasing controversy and non-evidence-based rhetoric in the media. as climate change int. j. environ. res. public health , , of continues throughout the st century, it will likely serve as a threat magnifier of other migration drivers [ ] . whilst terms such as 'climate refugee' are not recognised legally, migration and conflict are considered key mechanisms by which climate change has become a priority global health concern [ ] [ ] [ ] . indirect health impacts of climate change, such as those mediated via migration and displacement, are often under-recognised and under-researched. the ongoing covid- pandemic is a poignant example of how the special circumstances of migrant communities creates unique and extreme health vulnerabilities: whilst some migrants living in displacement camps are unable to practise good hygiene and social distancing, other migrants are finding themselves denied their right to asylum, neglected or turned away at borders due to travel restrictions and fear of new waves of infection. the lancet countdown on climate change and health created a 'climate migration' indicator [ ] whilst the newly launched lancet migration collaboration aims to explore and provide evidence for policy on the impacts of climate change on migrant health [ ] . despite these advances, conceptual frameworks for robustly exploring and understanding the impacts of climate change upon migration are lacking in some key areas, and the body of empirical studies remains thin. these gaps undermine the ability of policy makers to design effective evidence-based policy, public health interventions, and strategies to support safe and positive migration experiences. the aims of this paper are to provide a critical review of existing climate migration literature; from this, we also suggest modifications to existing conceptualisation of climate migration by providing a new conceptual model of the system of migration determinants. the model is designed to be pan-disciplinary and transferable to any geographic or social context. we advocate the systematic application of theory in climate migration studies which may help better geographic representation [ ] and improve our understanding of how climate change is impacting migration with contextual relevance to policy makers and public health interventions. finally, we apply this model to a case study of malawi to demonstrate how doing so can improve understanding of the local context and result in well-grounded and policy-relevant insights into the true impacts of climate change on migration. in order to improve conceptual modelling, several critical issues related to climate change and migration have been identified and discussed here. a key characteristic of migration is the multicausal nature of its drivers. climate change may act as a direct driver of displacement but in many cases is also inextricably linked to other, dynamic and interacting social, political, demographic and economic drivers [ ] [ ] [ ] . a popular constructed narrative is that climate change acts as a threat magnifier of existing migration drivers. this can result in many empirical studies identifying that economic factors rather than climate factors dominate the decision to migrate [ ] [ ] [ ] . however, it is possible that such studies overlook the mediated effect of climate change through other factors such as agriculture [ ] . it is now largely acknowledged that the relationship between climate change and migration is complex, dynamic and non-directional. another critical issue relates to the multifaceted nature of climate change itself. scholars typically outline several classes of climate change: a change in climate variability; changes in frequency and magnitude of fast-onset climatic events (including extreme weather events, droughts, floods, and heatwaves); and slow-onset climate change, including long term changes in average temperature, rainfall and chronic drought or flooding [ ] . this wide temporality as well as severity of climate change must be accounted for when discussing the implications of climate change on future population movements. migration itself may occur over a range of spatial scales-from movements between rural and urban areas, to international migration-as well as a range of temporal scales such as short-term migration, circular migration, to permanent moves. the decision of each individual to migrate may also carry different levels of human agency. the decision to migrate is due to an aggregation of micro-level (typically household or individual) and macro-level (societal) drivers. as such, each potential migrant has their own unique profile of factors and drivers. such individualistic situations are often described in terms of the individual's or community's vulnerability [ ] [ ] [ ] . this presents a key challenge in many existing studies, which struggle to reconcile drivers at the macro-and micro-demographic scales. a key challenge remains the paucity and compatibility of datasets regarding both migration and potential drivers thereof, and the scarcity of such data at appropriate spatial and temporal levels, particularly in low-income and in indigenous communities. the necessity for localised quantitative studies can result in fragmented analyses of specific timescales, geographies, types of migration and drivers thereof. furthermore, this can make it difficult to summarise and build a global narrative of the risks of climate change to human security [ ] . the resultant synthesis is that the impacts of climate change on migration are complex, multifaceted and dynamic. as such, increased attention on the upstream drivers of migration is called for. some authors argue that there is also some limitation in theoretical development and so in recent years there has been a push to promote a more sophisticated theoretical understanding of how climate change may interact with other drivers of migration [ , , ] . this has allowed the narrative to evolve through time: the conventional narrative suggests a more simplistic view of climate change as a blanket push driver resulting in large-scale waves of migration [ ] . however, newer frameworks appreciate the multi-driver nature of migration as well as the resilience and adaptive strategies of affected individuals and communities. nevertheless, such frameworks still struggle to capture the dynamic nature of such drivers including feedback and lag times, as well as the interactions between drivers themselves through time. the rising concept of ecological public health goes some way to attempt to address this, yet many policy-facing groups remain slow on the uptake [ ] . understanding of climate-induced migration is further skewed by the discipline of researchers: each scientific discipline-be it epidemiology, economics, political sciences or anthropology-carries with it its own intrinsic assumptions and methodologies [ , , ] which can perpetuate the fractured nature of the literature. therefore, to advance the understanding of the impacts of climate change on migration requires a truly interdisciplinary response. the collective result of these challenges is that current understanding of climate-induced migration is geographically uneven and studies are often non-transferable to other settings which presents an obstacle for policy makers and health intervention design. to aid design of interventions, scientists and decision makers (such as governments and humanitarian agencies) should engage with each other at all points of the intervention design and implementation. this can help ensure that interventions are contextually relevant and evidence based and that their impacts can be measured and evaluated. from the challenges identified in the above critical analysis, a new conceptual framework of climate migration is provided. migration, as a subjective concept, cannot carry one single definition and is highly contextualised. however, this is often neglected within most quantitative studies. in particular, the terms 'environmental migration' and 'climate migration' lack unanimous definitions across academic, ngo and political actors. migration exists as a normative behaviour in most communities globally but may also manifest as forced displacement and other involuntary or voluntary movements. furthermore, often overlooked in climate migration literature is the possible inhibiting effect of climate change on migration, resulting in reduced mobility rather than driving migration events [ , ] . many scholars advocate the need for appropriate migration typologies and several have been presented. for instance, stojanov et al. [ ] argue the need for contextualisation of climate drivers on a community in order to appropriately discern climate driven migration from normative or otherwise induced movement. renaud et al. [ ] also comment on the difficulty of identifying the environmental signal within migration drivers and present a decision-making framework and accompanying typology of environmentally induced migration. carling [ ] created the first iteration of the aspiration-capability framework, which describes voluntary or involuntary, mobility or immobility, based on both desire and ability to migrate. based on a review of a large variety of both qualitative and quantitative literature, we identify four dimensions which quantify migration: societal, temporal, spatial, and agency levels. "societal level" refers to the level of society affected, from micro scale (individual and household level) to macro scale (community, regional or population level). "temporal level" refers to the time duration of the migration, and the short term may consist of a matter of months, the long term is typically considered to be a year or more, though there is much range within empirical studies, and permanent migration represents the longest form of migration. "spatial level" refers to the physical distance covered by the migration. short distance may consider anywhere from intracommunity, intra-regional movements, to movements within the country and includes movements to between rural and urban hubs. long distance constitutes international movements across large geographical areas. whilst some cross-border movements may only require a few miles of travel and as such may be considered short distance, a large amount of international movements cover multiple countries and sometimes continents. such movements are of international political interest, though do not represent a large quantity of migrants or types of mobility [ , ] . the spatial scale, like the societal scale may also be summarised in terms of macro (generally medium or large distance) and micro (small, community level distances) and may align to climate and economic macro-or micro-level determinants. "agency level" refers to the level of choice afforded to each migrant, existing as a continuous scale between the extremes of totally involuntary (in other words, forced) to totally voluntary. it should be noted that all four dimensions are continuous variables and hence demarcations used should be contextually modulated. by applying generalised demarcations, however, we classify five key categories of environmentally induced migration. the first category is forced displacement, also referred to as distress migrants [ ] or temporary displaced migrants [ ] . the second category is adaptive migration at the decision of the migrant(s) [ ] . whilst this is a voluntary movement, a crucial caveat is applied here to note that such migration may not be truly voluntary; whilst many scholars and decision makers consider it as such, there is an emergent narrative arguing that migration due to longer-term environmental or economic degradation, or erosion of human security constitutes a type of forced migration, rather than a voluntary or adaptive movement [ ] . the third category is proactive migration at the decision of wider authority such as local or national government referred to as 'planned resettlement' [ ] . the fourth category is for trapped populations, which refers to a lack of mobility due to at-risk populations becoming trapped by environmental and socioeconomic barriers such as poverty [ ] . the final category is immobility, which represents a lack of mobility at the decision of the person(s) at environmental risk [ , ] . table summarises these classifications according to the four dimensions outlined. throughout the remainder of this paper, we shall use the term 'climate migration' for simplicity. a newly proposed conceptual framework for identifying the determinants of any given migration is presented in figure below. this framework is an updated iteration of prior models, which, over time, have converged to agree that migration is generally the result of a combination of upstream drivers, split into five categories: social, economic, political, demographic and environmental. however, consideration of interactions and evolution of these drivers through time, societal and spatial scales remains low. climate change is presented as an external driver which is expected in many contexts to act as a threat magnifier by exaggerating negative, push factors for vulnerable populations [ ] [ ] [ ] . attributes of climate change are split into three categories: physical, biological/ecological, and anthropogenic impacts [ , ] . the physical effects of climate change may be fast onset or slow onset. fast onset includes sudden events such as extreme weather or disaster events. slow onset consists of more gradual changes of mean values such as annual rainfall, rainfall variability and chronic droughting and flooding. secondary, or ecological climate aspects may include changes in land cover, flora and fauna habitats, including disease vectors and pollinators. tertiary or anthropogenic aspects include subsequent changes to anthropogenic systems such as crop yield and fish or game catch. a newly proposed conceptual framework for identifying the determinants of any given migration is presented in figure below. this framework is an updated iteration of prior models, which, over time, have converged to agree that migration is generally the result of a combination of upstream drivers, split into five categories: social, economic, political, demographic and environmental. however, consideration of interactions and evolution of these drivers through time, societal and spatial scales remains low. climate change is presented as an external driver which is expected in many contexts to act as a threat magnifier by exaggerating negative, push factors for vulnerable populations [ ] [ ] [ ] . attributes of climate change are split into three categories: physical, biological/ecological, and anthropogenic impacts [ , ] . the physical effects of climate change may be fast onset or slow onset. fast onset includes sudden events such as extreme weather or disaster events. slow onset consists of more gradual changes of mean values such as annual rainfall, rainfall variability and chronic droughting and flooding. secondary, or ecological climate aspects may include changes in land cover, flora and fauna habitats, including disease vectors and pollinators. tertiary or anthropogenic aspects include subsequent changes to anthropogenic systems such as crop yield and fish or game catch. the model aims to build upon this a priori understanding by providing deeper discussion of the complexity of driver interactions, driver dynamicity and the evolution of both drivers, and their linkages over time and spatial scales. importantly, the range of possible migration outcomes receives greater attention in this conceptual model, with recognition that different combinations of causal and contextual determinants may result in different migratory responses, including differences in the level of agency of a migrant. the model is designed with the purpose of being pan-disciplinary and, as such, relevant in any academic or non-academic context. the model presents not only a theoretical exercise, but a frame of thinking to support quantitative studies, with a view to informing future research, data collection or intervention design. drivers within each of the five classes may act as push agents-encouraging movement away from the origin, or pull agents-attracting movement to a host area. bowles et al. [ ] also identify glue and fend factors. glue factors act to cement a potential migrant in his/her home location such as cultural and family ties, whilst fend factors deter migration into an area such as hostile immigration policies. recent studies highlight that climate change may act as a glue factor in many situations, the model aims to build upon this a priori understanding by providing deeper discussion of the complexity of driver interactions, driver dynamicity and the evolution of both drivers, and their linkages over time and spatial scales. importantly, the range of possible migration outcomes receives greater attention in this conceptual model, with recognition that different combinations of causal and contextual determinants may result in different migratory responses, including differences in the level of agency of a migrant. the model is designed with the purpose of being pan-disciplinary and, as such, relevant in any academic or non-academic context. the model presents not only a theoretical exercise, but a frame of thinking to support quantitative studies, with a view to informing future research, data collection or intervention design. drivers within each of the five classes may act as push agents-encouraging movement away from the origin, or pull agents-attracting movement to a host area. bowles et al. [ ] also identify glue and fend factors. glue factors act to cement a potential migrant in his/her home location such as cultural and family ties, whilst fend factors deter migration into an area such as hostile immigration policies. recent studies highlight that climate change may act as a glue factor in many situations, rather than as a push factor as is often popularised [ ] . climate change is a sub-category of environmental drivers which may be further categorised into three classes [ , , ] . these are perhaps best described as primary physical effects, secondary biological and ecological effects, and tertiary anthropogenic effects [ , ] . climate change is segregated here from other environmental factors and framed as an externality to the determinant system. this facilitates investigation of the impacts of climate change as an upstream pressure to all five classes of drivers. each of the five categories is described in further detail below. to demonstrate the temporal nature of the system, the model is presented on a set of axes with time on the horizontal dimension with arbitrary timepoints t and t . this encourages consideration of the dynamicity of all determinants, as well as the changing nature of their interactions through time. as such, feedback implication on both host and source environments and communities of a migration decision may be decoded. externalities, such as future climate shocks or political interventions, such as climate mitigation, which may alter the system and resultant migration can also be presented and their impacts conjectured. the y axis depicting scale of impact refers simultaneously to the societal and spatial level of impact thereby encouraging the disparate nature of drivers on these scales to be considered. micro refers to small-scale, individual-or household-level factors whilst macro may be factors affecting large distances and large numbers of people. within the next section, we take a more granular view of each of the key families of drivers, and consider how each may directly or indirectly impact migration. this analysis is not exhaustive but attempts to provide a detailed summary of drivers over time and space, thereby encouraging a more nuanced and detailed exploration of the complexity of climate change. all climate factors are considered to occur at the macro spatial scale (figure ), which correlates to the macro societal impact level as described in table above. temporality of climate factors varies, and depends on the climate determinant (as shown in table ). the true speed of climate change varies geographically and so there can be no definitive definition for fast or slow onset. furthermore, some aspects may manifest across multiple timeframes. it is well established that climate change is increasing the frequency and magnitude of extreme weather events and climate shocks, which can be a direct cause of forced displacement. nevertheless, in such natural hazard events, socially constructed vulnerabilities often govern the extent and type of migration responses which occur. myers et al. [ ] , in a study of displacement due to hurricane katrina in , identified a range of social vulnerability factors which had a significant impact upon outmigration from affected places. similarly, gray and bilsborrow [ ] identified within an ecuadorian household migration survey, that household vulnerability factors such as home ownership, connectedness of household (to roads and schools), and poverty level, all confounded the environmental signal in the causes of observed migrations. less clear is the extent to which long-term or chronic climate change affects migration. chronic changes may include changes in average temperature, average rainfall, rainfall variability or extent of periodic drought and flooding. such changes often impact migration via mediating biological and anthropogenic factors such as impeded agricultural outputs [ , ] , adverse health outcomes [ , ] , or labour productivity [ ] . in such examples, the extent of climate factor as a driver of migration compared to other sociodemographic and economic factors is seen to vary greatly across studies. to better understand such relationships, we classify these indirect impacts as biological or anthropogenic (secondary or tertiary). biological or secondary impacts are as a result of physical climate change, which may lead to changes in regional geochemistry, and flora and fauna. such biological changes may alter the vulnerability of human populations. for instance, climate change may drive changes in the distribution of disease vectors [ , ] . anthropogenic or tertiary aspects of climate change comprise the resultant alterations to human systems. examples may include changes in anthropogenic land use and land availability due to sea level rise. alterations, for example, in crop yield and fish catch, may have direct implications to socioeconomic factors, for example, due to reduced agricultural output [ ] , food security [ ] , and therefore upon urbanisation rates due to rural to urban migration [ ] . such anthropogenic pathways generally act over a longer temporal scale and can lead to the climate signal being masked by more proximal factors. as such, understanding of their impact on migration remains inconclusive [ ] and less studied than direct physical impacts [ ] . furthermore, additional consideration is needed to assist the recognition of dynamic interactions between the physical, ecological and anthropogenic aspects of climate change. there are of course a range of other drivers of migration which are important to understand as well as how they may be affected by climate change. it is usually a combination of drivers that culminates in an individual's decision to migrate and in what manner. within the context of climate change, we refer to this aggregation of drivers (climatic and other) as the 'vulnerability profile' which will be unique to each individual. we now present a more detailed view of some key drivers within each of the five main classes identified. these are outlined in table below. as well as existing as intermediary drivers, each of these drivers may have direct impacts on migration decisions. for example, henry et al. [ ] using regression modelling and gray and bilsborrow [ ] using discrete time event history modelling both found that high literacy rates and economic status can act as significant push factors for migration. ezra and kiros [ ] also found marital status and poverty level acted as push factors. warner et al. [ ] clearly identified the role of government relocation policy on driving planned resettlement of communities away from flood plains in mozambique. such epidemiological methods are well utilised for analysing such direct causes. however, each analysis is limited to a specific type of migration and set of pre-assumed key drivers. it is not possible within this paper to examine in depth the nature and relationships of each driver, rather the authors focus on presenting a broad overview, elucidating the multilevel and multitemporal nature of migration drivers, as well the dynamicity of the drivers and their linkages. some key examples are used to demonstrate such complexities. table . non-climatic drivers of migration. drivers are split into five classes: social, economic, political, demographic and environmental. societal level refers to the societal scale at which drivers typically impact. some drivers may exist both as micro and macro factors. temporal scale refers to the typical timescale of change in each driver. whilst there is no set demarcations, slow change refers to a change typically over years or decades and change fast refers to changes which may occur immediately or over a short timeframe of months. static implies that factor is not usually time varying. many studies examine the importance of social factors such as migration networks [ ] [ ] [ ] [ ] . education and literacy rates have also commonly been identified as determinants of vulnerability [ , [ ] [ ] [ ] [ ] . social drivers such as education and poverty may also alter other drivers. for example, other studies have identified that in poor areas of malawi where rain-fed agricultural practices reigned, the predominant climate change adaptation approach was not seasonal migration but the introduction of irrigation techniques to increase crop yields [ , ] . however, joshua et al. [ ] concluded that increased irrigation triggered increased water insecurity and hence water conflict. this interaction between poverty and adaptation approach has significant implications for future vulnerability levels and on future social and political factors. of course, such impacts are not isolated to only impoverished communities. developed countries with lower poverty levels can also suffer compound impacts of climate change on other social determinants [ ] . however, developed countries generally have a higher capacity to mitigate or adapt to such changes resulting in different outcomes (migration and other), with different distributions across communities [ ] . closely linked to social factors are economic considerations such as employment opportunity and household wages [ ] at the micro societal level. poverty is also a key determinant of an individual's vulnerability and hence ability to migrate [ , , ] . macro-level factors such as average employment rates and average income of a community may also act as push or pull factors which have often been identified as the dominant drivers of migration [ , ] . there also exists a debate on the role of failed politicised economic models such as 'trickle down' and 'rent seeking 'as being largely responsible for the increase in wealth gaps and rising relative poverty [ ] . these economic models may contribute to future migration behaviour due to relations between poverty and mobility [ ] and the effect of inequality gradients acting as sinks for migration [ ] . for instance in the malawian example given above, findlay [ ] also comments on the additional causes of food insecurity beyond water scarcity, including soil erosion, socioeconomic factors including vulnerability to poverty, ability to financially withstand crop failures, low food utilisation and infrastructural factors such as high transport costs. political drivers are largely absent from environmental migration quantitative studies and yet present a significant category of migration drivers. possibly the most influential and most studied of this category is the role of political insecurity on migration. whilst the role of political insecurity and conflict is a well-acknowledged driver of migration, the role of climate change in driving political instability remains contested [ ] . burrows and kinney [ ] present an overview of multiple pathways through which climate change may lead to or exacerbate conflict such as through increasing rural to urban migration, resource competition or dispute between migrant and host communities. sokolowski et al. [ , ] also discuss the role of political interventions such as efforts for conflict resolution, international relief, and immigration policy such as the closing of borders, and their impact upon migration outcomes. though largely overlooked in general climate migration literature, some models do focus on political drivers of migration with relatively accurate predictions [ , ] . sokolowski and banks [ ] modelled population displacements that occurred in syria in using unhcr guidelines for factors prompting departure. indeed, the syrian conflict can be argued to contain both political and climate determinants in the mass displacement that has resulted [ ] . other political drivers include level of governance and trust in government and the level of institutionalisation and infrastructure within a community. infrastructure and governmental and non-governmental organisations are critical intervention nodes and as such their connection to environmental migration form an important area of potential study. other policies such as water, food and agricultural policy also co-interact and may result in a range of normative and adaptive migration approaches. for example, loevinsohn [ ] studied the malawian food crisis and identified primary causal factors to be both environmental drought and the underinvestment by the national government in agricultural stock. loevinsohn further identified that % of households interviewed during had migrant family members working seeking alternative income [ ] . crackdown on immigration policies in western countries such as britain, the usa and across the eu will also have significant impact upon future migration trends. with climate change expected to impact the numbers of both internal and international migrants in the future, existing dichotomies between the evidence on migration drivers and the political response to it will undoubtedly renew pressure on migration issues [ ] . demographic factors at the micro level (such as age, gender, ethnicity) as well as at the macro level (such as average living conditions, affluency, diaspora presence) can act as push or pull factors as well as interact with other factors. the combined effect of climatic drivers and demographic drivers have resulted in many developing countries being the most vulnerable nations to climate change and has helped to drive research and narratives around climate justice [ ] and climate refugees [ ] . rapid urbanisation is often a trend in such locations, leading to slum development, poor infrastructure and high vulnerability to future climate change, not to mention other shocks such as the covid- pandemic. in developed countries, different demographic challenges such as population ageing may also impact upon population mobility and health. for example, an older population may result in a reduced willingness to move and increased mental health burden of doing so [ ] . conversely, countries with aging populations can benefit from the 'healthy-migrant' effect [ ] . as such, appreciating the demographic factors, their dynamics and interactions is essential to understanding climate risk on future sustainable development and population changes. when modelling future environmental migration, it is therefore essential to take into account the demographic situation of the study area. climate change is a key driver of environmental change. environmental degradation, such as desertification, permafrost melt and coastal erosion, undermines livelihoods and therefore acts as a push driver for migration away from these regions. in the short term, there may be positive environmental changes such as increased precipitation and improved agricultural production in many parts of the globe which may act as a migration pull factor [ ] . environmental determinants such as rainfall and vegetation cover are commonly used in quantitative studies of climate change though other ecosystem attributes and ecosystem degradation appears somewhat overlooked in migration studies, such as food availability from natural sources and pollution of water. many environmental factors occur independently of climate change and may be influenced by other socio-political factors, often overlooked in environmental migration studies. for example, changes in land use, urbanisation, overexploitation of natural resources, environmental pollution and geophysical natural hazards may each be key determinants of migration. such environmental changes often have strong feedback loops-for example, rural to urban migration has significant repercussions on environmental degradation, air and water pollution, energy consumption and greenhouse gas emissions [ ] . environmental drivers have been found to be critical in many development studies. the environmental kuznets curve ("ekc") hypothesis purports that the early stages of economic development are coupled to environmental degradation and has been found to be true in many contexts [ ] . in the context of urbanisation led by adaptive migration, this hypothesis suggests that urbanisation will result in further environmental degradation, with significant implications for future development [ , ] , health [ ] , political security [ ] , and internal migration [ ] . we now provide a brief example of applying the conceptual model to a case study. we select rural malawi as a pertinent example of a climate-vulnerable society. malawi is a land-locked country in southern africa whose main economy is small-scale, rainfed agriculture, employing approximately % of malawians [ ] . as such, many people's livelihoods as well as key source of food is highly climate sensitive. already malawi has witnessed an annual mean temperature increase of . • c since the s, and whilst local rainfall patterns are difficult to accurately model, there has been an observed increase in frequency and magnitude of drought and flood events [ ] . by conducting an in-depth literature review of malawi's political, demographic, environmental, social and economic makeup and then applying the conceptual approach described above by considering the impacts of climate change (primary, secondary and tertiary) to each key factor, we arrive at the case-specific model shown in figure below. we now provide a brief example of applying the conceptual model to a case study. we select rural malawi as a pertinent example of a climate-vulnerable society. malawi is a land-locked country in southern africa whose main economy is small-scale, rainfed agriculture, employing approximately % of malawians [ ] . as such, many people's livelihoods as well as key source of food is highly climate sensitive. already malawi has witnessed an annual mean temperature increase of . °c since the s, and whilst local rainfall patterns are difficult to accurately model, there has been an observed increase in frequency and magnitude of drought and flood events [ ] . by conducting an in-depth literature review of malawi's political, demographic, environmental, social and economic makeup and then applying the conceptual approach described above by considering the impacts of climate change (primary, secondary and tertiary) to each key factor, we arrive at the case-specific model shown in figure below. a key advancement of this malawi-specific model is that each variable is quantifiable using observational datasets. as such, it demonstrates how the application of the generalised conceptual model in figure , to a local context, allows the creation of an astute, practical and measurable model, from which well grounded, policy-relevant research questions may be formulated and tested. by applying this methodological process, the malawi-specific model that is generated is based on wellgrounded assumptions and it holistically captures key variables that may be of relevance for future testing. additional information about each variable can be found in supplementary information table s . based on this conceptual model, the next step in the method would be to identify appropriate study and modelling techniques such as epidemiologic, mathematical, or integrated models to quantify the extent of each relationship depicted by the arrows in figure . the insights from such models may therefore make possible evidence provision which can be particularly relevant for national adaptation, economic development and public health plans. a key advancement of this malawi-specific model is that each variable is quantifiable using observational datasets. as such, it demonstrates how the application of the generalised conceptual model in figure , to a local context, allows the creation of an astute, practical and measurable model, from which well grounded, policy-relevant research questions may be formulated and tested. by applying this methodological process, the malawi-specific model that is generated is based on well-grounded assumptions and it holistically captures key variables that may be of relevance for future testing. additional information about each variable can be found in supplementary information table s . based on this conceptual model, the next step in the method would be to identify appropriate study and modelling techniques such as epidemiologic, mathematical, or integrated models to quantify the extent of each relationship depicted by the arrows in figure . the insights from such models may therefore make possible evidence provision which can be particularly relevant for national adaptation, economic development and public health plans. complexities naturally arise when taking an upstream, systems-thinking approach to migration determinants. there are two key complexities identified. firstly, the acknowledgement of multilevel and interactions and feedbacks between drivers. secondly the dynamicity of drivers and their connections over time and space. despite these complexities, models must be transparent and provide results from which simplicity may be derived in order to be useful for decision makers and intervention planning. to aid reflection upon such interactions, figure depicts a simple representation of the interactions between individual and classes of drivers. each class of driver is represented by a funnel, from which a combination of both macro and micro drivers is filtered from an interconnected reservoir where drivers from different classes interact on a range of temporal, spatial and social scales. the combination of drivers at the individual migrant level results in a unique vulnerability profile and context which determines the migration decision made by each potential migrant. climate change is again presented as an externality, cross-cutting all other driver classes and acting across the temporal and societal levels. as in figure , each driver may vary over both time and spatial dimensions. however, modelling such dynamicity requires simultaneous understanding of drivers, their interactions, and their evolution through time and space. the insight that such dynamic modelling would allow may enable the effective identification of suitable intervention nodes for public health, land use and immigration policy to name but a few. connections over time and space. despite these complexities, models must be transparent and provide results from which simplicity may be derived in order to be useful for decision makers and intervention planning. to aid reflection upon such interactions, figure depicts a simple representation of the interactions between individual and classes of drivers. each class of driver is represented by a funnel, from which a combination of both macro and micro drivers is filtered from an interconnected reservoir where drivers from different classes interact on a range of temporal, spatial and social scales. the combination of drivers at the individual migrant level results in a unique vulnerability profile and context which determines the migration decision made by each potential migrant. climate change is again presented as an externality, cross-cutting all other driver classes and acting across the temporal and societal levels. as in figure , each driver may vary over both time and spatial dimensions. however, modelling such dynamicity requires simultaneous understanding of drivers, their interactions, and their evolution through time and space. the insight that such dynamic modelling would allow may enable the effective identification of suitable intervention nodes for public health, land use and immigration policy to name but a few. the concept of a vulnerability profile allows for the acknowledgement that each migrant has a unique set of drivers due to the multilevel and multitemporal combination of factors he or she is subjected to. in this way vulnerability may be conceived as a meta-driver of migration. the concept of vulnerability describes the ability of an individual or community to withstand and recover from a risk such as a disaster event [ ] . other meta-drivers include resilience and adaptive capacity [ ] [ ] [ ] . whilst vulnerability is a commonly used meta-driver in much climate migration literature, resilience is often the currency of choice in the fields of disaster management and climate change adaptation [ ] . however, these terms are broad and often overlap and are even used interchangeably, rendering their distinction and usefulness within scientific analysis questionable. despite this, such meta-drivers are the dialogue of choice for policy makers and must be utilised for research to have political relevance. however, care should be taken when referring to such meta- the concept of a vulnerability profile allows for the acknowledgement that each migrant has a unique set of drivers due to the multilevel and multitemporal combination of factors he or she is subjected to. in this way vulnerability may be conceived as a meta-driver of migration. the concept of vulnerability describes the ability of an individual or community to withstand and recover from a risk such as a disaster event [ ] . other meta-drivers include resilience and adaptive capacity [ ] [ ] [ ] . whilst vulnerability is a commonly used meta-driver in much climate migration literature, resilience is often the currency of choice in the fields of disaster management and climate change adaptation [ ] . however, these terms are broad and often overlap and are even used interchangeably, rendering their distinction and usefulness within scientific analysis questionable. despite this, such meta-drivers are the dialogue of choice for policy makers and must be utilised for research to have political relevance. however, care should be taken when referring to such meta-drivers and the contributing drivers as explored above must be contextually relevant and carefully selected. previous conceptual models explore the linkages between climate change and migration with different assumptions and perspectives. the foresight report identifies five key families of drivers and concludes that migration may be an adaptive strategy in the face of climate change and represents possibly the best to-date, globally accepted conceptual model for climate migration [ ] . the report disputes the long-time argument that migration represents a failure to adapt in situ. this conclusion, however, fails to consider several key aspects of migration: firstly, the agency and social well-being of migrants involved at each stage of the migration process (prior to movement, in transit, and at host destination). secondly, the level of agency afforded to would-be migrants during the migration decision-even as a supposedly proactive adaptation measure. finally, the delicate line between forced and voluntary movement, based upon a composition of drivers and the bias of the person(s) awarding the classification. the ongoing lancet commission on climate change and health also presents an interesting framework where migration as a result of climate change is appropriately framed as a health challenge, and a public health opportunity [ ] . this framework, however, does not give a large amount of consideration to intermediate drivers and various pathways by which climate change may drive migration or produce trapped communities. helping to close this gap, and drawing on a range of political and economic, as well as health literature, the model presented by sellers, ebi and hess considers a puzzle of immediate and longer-term drivers of social instability, with both climate shocks and migration as contributing factors and possible outcomes [ ] . mcmichael et al. [ ] also present a foundational model whereby the basic links between climate change and migration are presented though driver interactions and dynamics are not discussed in depth. whilst this and other conceptual models encourage an upstream approach to environmentally induced migration, putting such thinking into practise presents further challenges. the paucity of empirical studies limits our understanding of how global climate change may threaten development and public health, particularly regarding the indirect impacts of climate change. lack of suitable data and quantitative metrics needed to conduct such studies remains a perennial challenge. it is essential that these challenges be overcome through future data collection and empirical modelling. migration datasets are largely based on cross-sectional survey and census data whilst information about health and well-being, disaggregated by migration status, is largely lacking. furthermore, collecting and disseminating such data present significant ethical and privacy concerns. for many drivers, proxies may be used. for instance, the normalised difference vegetation index (ndvi) may act as a proxy for natural resource availability [ ] . henderson et al. use a simple count of manufacturing industries as a proxy for urban industrial capacity when analysing the relationship between climate change and urbanisation in an african context [ ] . lu et al. [ ] suggest the possibility of using out-migration rates as a proxy for changes in habitability. neumann and hilderink [ ] present a range of possible datasets such as glasod for soil degradation and lada for biomass production of earth observation land degradation data. however, each of these datasets has its own challenges concerning spatial and temporal resolution, uncertainty and effectiveness as a proxy. furthermore, misalignment of datasets at the spatial, temporal and social levels creates further challenges in appropriately modelling migration determinants. other, more squidgy drivers such as perceived political stability and social networks remain elusive to measurement and under-represented in quantitative studies. the availability and quality of data in turn create methodological challenges for empiricists. some studies utilise a range of statistical and epidemiological methods. however, traditional epidemiological methods each have their short-comings. cross-sectional analyses do not allow for the temporal nature of drivers. timeseries analyses are often impeded due to lack of sufficient data and the ability to control interactions between drivers across a range of temporal and spatial resolutions. gravity models can capture linear push and pull factors at the macro level, though may struggle with ecological fallacy and in modelling of the more nuanced relationship between driver and migration outcome. recent developments in mathematical models offer useful insight. such models include improved agent-based modelling (abms) and multiagent systems approaches [ , , , ] . study approaches must be chosen appropriately based on the assumed relevant determinants and their interactions, as choice of methods may have significant impact on the study results. the advantage of such systems approaches is that driver dynamics and interactions may be inbuilt and allowed to alter in timesteps. the individual nature of human decisions may also be captured through abms. however, abms require high-resolution data and are generally only applicable for small geographical scales. economic approaches such as economic bargaining theory can also be used to explain some micro-level migration decisions such as the 'healthy-migrant' effect, whereby young and fit-for-work individuals may be more likely to move in search of work and remittance opportunities [ ] . however, since climate change exists only as a macro factor, such micro-level considerations within current models of climate migration are often lacking. other approaches have been proposed to deal with complexity and dynamicity. barbieri et al. [ ] use a combined economic-demographic-climate model to understand the interactions between different classes of drivers over time (using appropriate proxies) in the northeast region of brazil. another emerging method is the use of shared socioeconomic pathways (ssps) to provide a combined set of scenarios for future population, urbanisation and wealth factors [ ] . the ssps are designed to be used in conjunction with climate change representative concentration pathways (rcps) for future radiative forcing emission scenarios. application of this approach can be seen within the groundswell report who combine the rcps and ssps into three scenarios and use gravity modelling to provide a view of internal migration for three global regions [ ] . finally, we make a crucial note regarding the overall approach by scientists towards climate migration. care must be taken when treading the literature of various typologies and terminologies which are necessarily subjective and vary by author and by discipline. furthermore, climate migration may be studied through a variety of academic lenses. as such, the impact of different epistemologies on conclusions is complex and often overlooked [ ] . politically impactful research should attempt to transcend traditional research boundaries and avoid tribalism in science [ , ] . indeed, in the pursuit of improved global health, research of climate migration should be contextually relevant, and politically pertinent and timely. one way to help achieve this is to adopt a pan-disciplinary approach such as the one demonstrated within this paper. table elucidates this point by demonstrating a selection of fields which contribute to the study of climate migration as an aspect of global health. table . an overview of the range of scientific disciplines which contribute to the study of climate migration, its drivers and impacts. human geography offers a range of frameworks and tools for studying human migration and its drivers. through the study of human behaviour, anthropological methods offer a deeper insight into the decision-making process behind migration, as well as the impacts of migration upon individual and societal well-being. ethnography offers a unique and rich insight into people's opinions and decision making. political sciences political sciences may be used to explore the effects of policy on immigration, as well as the geographic, economic and social drivers of migration policy and sentiment. both macro and micro economics can be used to quantify migration as well as study the economic drivers and impacts of migration. for example, in the case study of malawi, econometric modelling could be applied in the study of the impact of failed crops on household wealth and as such on migration. mathematics a range of mathematical models are used in the study of migration such as system dynamic models, agent-based models, gravity models, and diffusion models. environmental epidemiology can be used in the study of migration and its drivers. for example, the field of ecological public health supports the exploration of the relationships between the biological and material realms [ ] . disaster risk reduction sciences disaster risk reduction relates mainly to sudden-onset events and short-term, forced displacement and as such provides cross-over to the field of migration science. computer sciences computer science is used in migration studies to model and simulate migration and its quantifiable drivers and impact. sociology can be used to study migration and its impacts at the societal level, with special interest in demographic makeup and the social structure of migrant (and non-migrant) communities. the study of population dynamics and structure places migration as a core component. ultimately, climate change may have critical impacts upon future migration across the globe and has significant implications for public health, human security and sustainable development. climate change is already and will continue over the coming decades to contribute to large numbers of displaced persons [ ] , refugees [ ] , internal migrants [ ] , international migrants [ ] and immobile and trapped persons [ ] . as such, better understanding of the relationship between climate change and migration is essential for effective future policy planning in all sectors. this can be achieved through the systematic and homogenous application of robust conceptual frameworks to local contexts. a lack of data, particularly for low-income and indigenous settings, is a key set back which obstructs furthering our understanding. it also hinders the ongoing desire of academia, national and international policy makers to identify who are the climate migrants of today and of the future and count how many there are. this paper has attempted to demonstrate the need for a flexible and pan-disciplinary approach to environmentally induced migration. research which cross-cuts traditional discipline boundaries, in accordance with the planetary health viewpoint, is encouraged when using such a conceptual framework in the study of climate migration [ ] . in this way, traditional pitfalls may be avoided, better reconciliation of macro and micro determinants may be achieved and more visibility of the dynamics of drivers and hence a more accurate understanding of their role in driving migration may be elucidated. however, the review within this paper is non-exhaustive and draws lightly on a wide range of literature and academic standpoints. as such, it is designed to demonstrate a nuanced approach to climate migration theory and application, rather than present a comprehensive "how-to" guide. finally, it was beyond the scope of this paper to fully apply our conceptual model to mathematical and epidemiological quantitative models, providing instead a simple overview, though this is the natural progression of the research. table s : a descriptive table providing an overview of each variable included 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study of water resources management along the likangala river in zomba district preparing for internal climate migration future climate impacts on maize farming and food security in malawi climate risk assessment and agricultural value chain prioritisation for malawi and zambia working paper no. . . wageningen, the netherlands linkages between vulnerability, resilience, and adaptive capacity migration as an adaptation to climate change geographies of resilience: challenges and opportunities of a descriptive concept climate change, human health, and social stability: addressing interlinkages has climate change driven urbanization in africa? engø-monsen, k.; et al unveiling hidden migration and mobility patterns in climate stressed regions: a longitudinal study of six million anonymous mobile phone users in bangladesh the influence of climate variability on internal migration flows in south africa agent-based model simulations of future changes in migration flows for burkina faso might climate change the "healthy migrant" effect? glob. environ climate change and population migration in brazil's northeast: scenarios for - the shared socioeconomic pathways and their energy, land use, and greenhouse gas emissions implications: an overview places, people and perpetuity: community capacities in ecologies of catastrophe climate change's role in disaster risk reduction's future: beyond vulnerability and resilience internal displacement monitoring centre unhcr. the environment and climate change of the rockefeller foundation-lancet commission on planetary health this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- -mrrctuxt authors: freeman, hugh james title: review: adult celiac disease and the severe “flat” small bowel biopsy lesion date: journal: dig dis sci doi: . /b:ddas. . .d sha: doc_id: cord_uid: mrrctuxt classification of architectural changes in the small intestinal biopsy may be clinically useful to define the cause of diarrhea or suspected malabsorption, especially in adults. pathologic changes may include severe (flat) or variably severe (mild or moderate) abnormalities. for some disorders, small bowel biopsy findings may be very distinctive and lead to a specific diagnosis. for others, like adult celiac disease, biopsy changes are less specific. indeed, it is becoming increasingly appreciated that several conditions can produce similar histopathologic changes. serological assays, including endomysial antibodies and tissue transglutaminase antibodies, may be very useful tools for screening and case finding in clinical practice. however, demonstration of characteristic changes in the small intestinal biopsy is critical, along with a gluten-free diet response. to reveal a specific cause. while detailed lists of tests and algorithms appear in medical texts, these are costly and time-consuming. moreover, some function tests are patient-dependent and based on timed collections that are difficult to do with desired laboratory precision. often, some can be circumvented by small bowel biopsy or done later if additional documentation is necessary (e.g., fecal protein loss studies, -hr fecal fat measurements, barium radiographs). some patients with malabsorption may have no significant diarrhea. instead, weight loss or anemia associated with iron deficiency may lead to a biopsy to exclude a small intestinal cause. or, there may be other unexplained abnormal chemistries (e.g., low serum carotene, folate, or proteins, particularly albumin). finally, a positive screening celiac antibody blood test (e.g., antibodies to endomysium or tissue transglutaminase) should lead to biopsy to confirm the serologically based suspicion of celiac disease and define pathological changes in the small bowel before treatment ( ) . even a normal small bowel biopsy may be useful, in most instances, to exclude structural small bowel causes of diarrhea, particularly those with diffuse and severe changes in the proximal small intestine, such as classic celiac disease. normal or abnormal biopsy appearances, freeman however, should be interpreted cautiously depending on the biopsy site. for example, endoscopic specimens from the proximal duodenum may normally appear to have shorter villi with "pseudoflattening" over regions of brunner's glands (i.e., brunner's gland artifact) but crypt hyperplasia is not present. in addition, biopsies showing histologic evidence of apparent "duodenitis" or nonspecific inflammation in the duodenal bulb may indicate peptic-related disease. indeed, a broader spectrum of disease might be considered (e.g., crohn's disease) if similar changes are found in more distal small intestine. for this reason, screening biopsies (even if the duodenal mucosa is visually normal) to exclude a proximal small bowel mucosal cause for diarrhea should be done distal to the duodenal bulb. biopsy interpretive artifacts may result from biopsy handling. technical trauma may result from the biopsy instrument (i.e., biopsy crush artifact) or during transfer of the biopsy from forceps to filter paper or mesh. poorly oriented specimens may lead to tangential sections outside of the core of the paraffin-embedded tissue block. endoscopic pinch biopsies are now most commonly submitted to hospital pathology laboratories, but these are smaller than suction biopsies and more difficult to accurately orient for serial sectioning. however, clinical experience has demonstrated that acceptable biopsy material may be obtained for routine diagnostic purposes ( , ) . usually, two or three biopsies with regular-sized forceps from the second or third portion of the duodenum are sufficient. in special circumstances (e.g., recurrent symptoms in established celiac disease), more biopsies from multiple sites along the length of the small intestine (using a longer instrument) may be required. for example, in celiac disease and suspected lymphoma, only of small bowel biopsies proved to be positive for malignant cells ( ) . for clinical purposes, abnormal small bowel biopsies may be classified based on the degree of disturbed architecture together with specific diagnostic features ( ). multiple biopsies from separate sites are most useful since some disorders reveal only focal changes (e.g., crohn's disease), while others may show diffuse, but more variable severe abnormalities (e.g., giardiasis). sampling of normal-and abnormal-appearing small bowel mucosa should be done, especially if erosive or ulcerative macroscopic changes are endoscopically evident, since some pathologic features may be easier for the pathologist to appreciate in biopsies from less inflamed or less reactive epithelium. for clinicians, there may be limited time to examine biopsies, so the single most important imperative is clear communication of the clinical problem to the patholo-gist. good-quality biopsies should be submitted and their sites precisely defined to optimise histologic evaluation. a clinically relevant pathological interpretation based on the degree of severity of architectural disturbance may be very helpful and can be especially valuable for clinical management ( , ) . during endoscopy, macroscopic changes may be appreciated. in celiac disease, for example, absence of normal structures or a smooth tubular mucosal surface has been described ( ) . also, a so-called "scalloped" or "ridged" endoscopic appearance has been noted ( ) . these macroscopic changes are not specific to celiac disease, however, and should never be relied on for definitive diagnosis. indeed, in most patients with celiac disease, no significant endoscopic alteration is recorded despite the presence of severely abnormal histologic abnormalities. often, changes of celiac disease may be reported later by the pathologist even if the disease was not clinically or endoscopically suspected. even very experienced endoscopists estimated that celiac disease was diagnosed "unexpectedly" in over % simply because screening biopsies were done from normal duodenal mucosa ( ) . a visually normal endoscopic appearance alone (without biopsies) is simply not sufficient to exclude small bowel mucosal disease, including celiac disease. diarrhea, weight loss, and malabsorption of a broad range of nutrients occur in most patients with classic celiac disease. in addition, significant histopathologic changes are usually present in the proximal small bowel, the so-called severe "flat" mucosal lesion (figures and ) . indeed, the most common cause of this biopsy abnormality is untreated celiac disease ( ). these changes appear to be similar to those described as the so-called flat destructive or marsh type lesion ( , ) . several features should be present. villi are absent or rudimentary. increased lamina propria lymphoid cell elements and increased intraepithelial lymphocytes are seen and the surface epithelium appears more cuboidal (rather than columnar). crypt cell hyperplasia (not hypoplasia) with an increase in the crypt epithelial cell mitotic index (i.e., the number of mitoses per crypt epithelial cell) is present. in celiac disease, treated with a strict gluten-free diet, these changes revert toward normal ( figure ). longterm studies have shown that biopsies from comparable proximal small bowel sampling sites will eventually show improvement. villi reappear and crypt mitotic indices normalize. surface epithelial cells become more columnar. the cellularity of the lamina propria diminishes and the absolute numbers of intraepithelial lymphocytes fall. changes also occur in another dimension-along the length of the small intestine. these may not be so well appreciated in the modern era since endoscopic biopsies are usually obtained under direct vision from the proximal small intestine alone (rather than hydraulic suction biopsies from multiple sites in more distal jejunum and ileum). the extent and severity of these abnormalities through the length of the small intestine seem to correlate better with the patient's clinical status than changes seen with repeated sampling from similar proximal small intestinal mucosal sites. in celiac disease with clinically significant malabsorption, small bowel changes may be severe, with architectural changes extending beyond the proximal jejunum. farther along the small intestine into the ileum, less severe, often patchy, rather than diffuse, architectural changes may be present. possibly, this is because with clinically more severe disease, longer lengths of small intestine are exposed to higher concentrations of dietary gluten. or, alternatively, some pathological changes, particularly in more distal small intestinal sites, may be more "indirect" and driven by recirculating memory t cells ( ) . even in the most distal ileum, however, earlier human studies demonstrated that the mucosa was very sensitive to gluten-containing peptides infused experimentally through a long small bowel tube ( ) . this proximal-todistal gradient in severity of mucosal disease makes it more difficult to detect changes of celiac disease with ileal biopsies (obtained at colonoscopy), and, more importantly, this gradient is altered with a gluten-free diet. with more extensive small bowel disease, diarrhea and malabsorption of many nutrients result. after treatment with a gluten-free diet, clinical improvement occurs, with resolution of diarrhea and weight gain, reflecting improved absorption along the length of the intestinal tract. concomitant with these clinical improvements, it appears that resolution of the abnormal histologic changes occurs in a distal-to-proximal direction. conceptually, this is potentially very important in the clinical and histologic assessment of the response to a gluten-free diet. prolonged periods of gluten restriction may be required, even up to several months or years after diarrhea resolves, to show normalization of biopsies from the most proximal small intestine ( ) . repeated biopsies from the same proximal small intestinal sites after only a few weeks on a strict gluten-free diet may not be sufficient to show a convincing histologic response, even if the patient is clinically improved (i.e., resolution of diarrhea and weight gain). a clinical diagnosis of classic celiac disease, therefore, requires that two criteria be fulfilled: first, typical biopsy changes of untreated celiac disease in the proximal small bowel should be present; and second, improvement should occur with dietary gluten restriction. most often in adults, the clinical improvement may be sufficient to be convinced that celiac disease is responsible, especially if diarrhea resolves and weight gain results. in some patients with few clinical symptoms, however, repeated biopsies may be needed to show histologic normalization of the small bowel mucosa. serologic screening tests (e.g., endomysial antibodies or tissue transglutaminase) are inadequate to diagnose celiac disease or even define compliance to a gluten-free diet ( ) ( ) ( ) . false-negative serological tests occur. some are due to serum iga deficiency, occasionally associated with celiac disease ( ). any adult with chronic diarrhea or malabsorption suspected to be caused by celiac disease should have a small bowel biopsy to exclude the disorder. on the other hand, if one or more serological screening tests are positive, a biopsy should be done to determine if the test was correct since false-positive serological results also occur ( ). recently, even strongly positive tissue transglutaminase antibody assays were recorded using a commercially available test kit in patients with no other disease detected and entirely normal small bowel biopsies ( ) as well as in a patient with a severe flat lesion not histologically responsive to gluten restriction ( ) . thus, positive serological results, even if accompanied by biopsy changes consistent with untreated celiac disease, are not sufficient to diagnose celiac disease. a convincing response to a gluten-free diet must still be documented. in some patients with diarrhea or malabsorption, only mild or moderate degrees of altered villous architecture are present ( ). in the mild lesion, villi remain unaltered or only minimally altered in size. the epithelium, however, is very abnormal, with loss of polarity and a marked increase in intraepithelial lymphocytes. sometimes, there may also be an accompanying increase in lamina propria cellularity. these changes appear to be similar to those classified elsewhere as an infiltrative or marsh lesion ( ) . in the moderate lesion, there is also a definite change in villous architecture. these findings may be classified as an infiltrative hyperplastic or marsh lesion ( ) . often, these variably severe (i.e., mild or moderate) changes are not specific and may be detected in a number of disorders, including celiac disease, and with the previously documented glutensensitive small intestinal changes of dermatitis herpetiformis ( ) ( ) ( ) or intestinal lymphoma ( , ) . similar histologic changes have also been reported in some asymptomatic first-degree relatives of celiac disease patients ( , ) and, therefore, may, in some instances, represent part of the pathological spectrum of celiac disease ( ) . histologic changes less severe than those typical of celiac disease, however, should prompt investigation for a nonceliac cause, especially to exclude infectious agents (e.g., giardiasis, cryptosporidiosis). in a recent report ( ) , however, intraepithelial lymphocytosis with normal small bowel mucosal architecture was recorded in of patients. in these, a favorable clinical response to a gluten-free diet was described in (about %). although the changes in these patients may have been related to gluten sensitivity, repeated histological studies were not recorded. others have also suggested that celiac disease might be diagnosed without typical villous structural change, using immunohistochemical markers to label intraepithelial lymphocytes ( ) ( ) ( ) . additional studies will be required to confirm these intriguing observations. detection of celiac disease may be delayed, even into later adult years ( ) . in some with clinically silent celiac disease, only very limited morphologic changes may be detected, with only mild or moderately severe abnormalities in villous structure, whereas others may have severe histologic changes but limited to the most proximal small intestine. as a result of this limited involvement of the most proximal small intestine, only isolated deficiencies of specific nutrients, such as iron, may be present with little or no clinically detectable diarrhea or weight loss. a number of disorders have now been recognized as a possible clue to this form of clinically unsuspected or occult celiac disease including isolated iron, folic acid, or calcium deficiencies ( ) , dermatitis herpetiformis ( ) ( ) ( ) , autoimmune types of thyroid disease (i.e., hashimoto's thyroiditis) ( ), pediatric insulin-dependent diabetes mellitus ( ) , small intestinal adenocarcinoma ( ) , and some lymphomas ( , ) , particularly, although not exclusively, t-cell forms of lymphoma in intestinal ( ) and nonintestinal sites, including the liver ( ), spleen ( ) , and thyroid gland ( ) . rarely, the clinical presentation may be dramatic, with free intestinal perforation due to a complicating malignant lymphoma ( , ) . in addition, celiac disease has been detected in patients with an initial diagnosis of a microscopic form of colitis, such as lymphocytic or collagenous colitis ( , ) , lymphocytic gastritis ( ) , and lymphocytic sclerosing cholangitis ( ) . interestingly, determination of endomysial antibodies or tissue transglutaminase antibodies did not increase detection of celiac disease in small numbers of serologically screened patients with either lymphocytic or collagenous colitis ( ). in addition, feeding high-gluten-containing diets to two patients with lymphocytic colitis did not elicit small intestinal changes of celiac disease ( ) . in contrast, a recent prospective biopsy evaluation of consecutive collagenous colitis patients revealed that over % also had underlying and unrecognized celiac disease ( ) . another category of clinically silent celiac disease is latent celiac disease, reported in patients with dermatitis herpetiformis or small intestinal lymphoma ( , ) . in this condition, histologic assessment of the small bowel architecture is initially normal. following a high-glutencontaining diet, however, histologic changes of variable severity may be induced, indicating that the small intestinal mucosa in these possibly genetically predisposed patients is gluten-sensitive, as reflected in this latent small intestinal mucosal lesion. these changes in the small intestinal mucosa do not occur in otherwise normal volunteers fed high-gluten-containing diets. demonstration of histologic improvement in these gluten-induced changes in the small intestinal mucosa with a gluten-free diet in patients with latent celiac disease was also documented ( , ) . not infrequently, biopsies with minimal changes are labeled compatible with celiac disease. in this setting, a number of issues immediately result. a patient having florid symptoms or laboratory test abnormalities suggestive of malabsorption with celiac disease should have more than just mild nonspecific changes in small bowel biopsies. if only minimal morphologic changes are present, there are at least two possibilities: first, another disorder may be causing the symptoms, and, second, normal mucosa is being erroneously diagnosed as mild chronic inflammation. the consequences of a false-positive diagnosis of celiac disease are not minimal. major disruptions in culinary lifestyle may result and there are implications related to a number of possible associated conditions (including lymphoma). rarely, additional studies may be required. some patients with only mild changes in villous architecture could still have celiac disease ( , ) . a trial of gluten-free diet followed by a high-gluten diet challenge might be done with biopsies to determine if the mucosa is gluten-sensitive. a final diagnosis of celiac disease should only be made with certainty if the mucosal abnormalities can be shown to be gluten-sensitive. there also seem to be some patients with symptoms attributed to gluten ingestion (or, more often, ingestion of grains such as wheat) without abnormal small bowel biopsy changes. often, despite reintroduction of dietary gluten, even for prolonged periods, repeated small bowel biopsies are normal. likely, some form of functional disorder is present. without evidence of gluten-sensitive mucosal changes, a diagnosis of celiac disease cannot be established. in some patients with well-defined celiac disease and well-documented histologic improvement on a gluten-free diet, clinical symptoms of diarrhea or malabsorption may recur. these may be associated with recurrent and severe histologic changes usually seen in untreated celiac disease. in most, poor compliance with a strict gluten-free diet or inadvertent ingestion of dietary gluten can be documented, sometimes from a ubiquitous source, such as pill capsules or communion wafers. sometimes, evaluation by an astute dietitian or even hospitalization may be required to identify the source of gluten. in others, another cause for diarrhea or weight loss may develop (e.g., infectious diarrhea). for these celiac disease patients, treatment of the infection often will resolve the recurrent symptoms. alternatively, some may develop histologic changes in the small intestine directly related to malabsorption of a specific nutrient and superimposed on the changes of celiac disease. this might result in refractory clinical symptoms (e.g., zinc deficiency associated with ongoing malabsorption) or independent morphologic changes, in-cluding macrocytic epithelial cells and crypt epithelial hypoplasia (e.g., folic acid deficiency). in others, a related cause, such as pancreatic exocrine insufficiency with pancreatic calcification, may develop in long-standing celiac disease with long-standing malnutrition ( ) . sometimes, reevaluation of the original diagnosis is necessary to ensure that the correct diagnosis was initially established. finally, some may develop diarrhea caused by a recognized association of celiac disease, such as collagenous or lymphocytic colitis, or a serious complication, such as lymphoma ( ) . rarely, an unusual disorder, collagenous sprue may occur. this was originally described in a patient with celiac disease ( ) . in most patients, severe panmalabsorption with diarrhea, weight loss, and marked nutritional and electrolyte disturbances may develop. many of these patients may eventually require ongoing nutritional support to survive. interestingly, iga endomysial antibodies have been detected in collagenous sprue, providing additional evidence of a direct link with preexisting celiac disease ( ) . finally, as in celiac disease, rare collagenous sprue patients have also been recently reported to develop lymphomas ( , ) . in a small number of patients with well-documented celiac disease, no specific cause for refractory symptoms appears evident. some, but not all, of these patients may have an unusual and poorly understood syndrome characterized by recurrent or persistent small bowel histologic changes of variable severity, splenic hypofunction, and a peculiar form of pathologic cavitation of mesenteric lymph nodes ( ) . some of these refractory patients eventually develop or are found to have a concomitant intestinal lymphoma ( ) . if rebiopsy is done in patients with refractory or recurrent symptoms, the pathologist should examine biopsies for evidence of collagenous sprue or a lymphoma. pretreatment biopsies should also be reviewed to determine if originally there was truly convincing improvement on a gluten-free diet. without evidence for biopsy improvement with gluten restriction, other causes for diarrhea and/or malabsorption should be considered, since celiac disease may not even be present. detection of a persistently severe flat lesion in duodenum and jejunum, however, may not mean that refractory celiac disease is necessarily present since histologically severe changes may persist for prolonged periods even after more distal intestinal mucosa has improved. similarly, in patients with celiac disease, the appearance of iron deficiency may not necessarily reflect refractory celiac disease due to impaired iron absorption in the proximal small intestine. other causes, including superimposed occult blood loss, may still require exclusion. the terms unclassified sprue or sprue-like intestinal disease have been used to describe patients who may have a severe (flat) or variably severe mucosal lesion but have not been shown to respond to a gluten-free diet. this possibly represents a heterogeneous group of small intestinal disorders, a "wastebasket group" with no specific cause. some possibly represent the atrophic hypoplastic or marsh lesion described elsewhere ( ) . most remain severely symptomatic with malabsorption and profound wasting in spite of a gluten-free diet. some could have a "clinically resistant form" of celiac disease, whereas others eventually prove to have a difficult-to-diagnose intestinal lymphoma. in patients recently reported with the label of "refractory sprue," an abnormal subset of intraepithelial lymphocytes was described with morphologically normal, but phenotypically abnormal lymphocytes ( ) . most died with uncontrolled malabsorption despite steroid therapy and parenteral nutrition. in a subsequent report by the same group ( ) , partial trisomy of the q region was recorded. additional studies are needed to determine if this intriguing observation will prove to be a specific prognostic marker of possible lymphoma development. another usual disorder, originally described in children ( ), with associated enterocyte or goblet cell antibodies, has been recently described in adults ( ) ( ) ( ) ( ) . pathologically, this so-called autoimmune enteropathy is a form of unclassified enteropathy and fails to respond to a glutenfree diet. some studies have suggested that this intriguing intestinal disorder has a very distinct pathogenesis ( ) . other causes of a severe (flat) or variably severe small intestinal biopsy lesion may be associated with diarrhea or malabsorption. table lists some of these causes with their treatment, if available. although some believe that oats may be consumed safely by celiac patients ( ), even for prolonged periods ( ), a recent report has documented that oats alone may have induced villous atrophy ( ) . certain infections have very distinctive findings that may lead to their recognition (e.g., giardiasis). in some, treatment with a specific antimicrobial agent may cause rapid symptom resolution and complete normalization of biopsy changes. often, pathologic changes are present but no specific infectious agent can be detected. in children, for example, severe changes may occur, possibly related to a viral agent or an escherichia coli infection, that may spontaneously resolve without treatment ( ) ( ) ( ) . more often, however, especially in adults, severity of architectural changes in the small intestine is variable and limited. in giardiasis, for example, only % of small bowel biopsies were severely abnormal. most biopsies either were normal or showed only mild to moderate changes. a number of other protozoan agents may cause small intestinal inflammation. detection of mature adult organisms, their trophozoites, or some intracellular component of the life cycle within the surface epithelium or on the epithelial surface may lead to a specific diagnosis. isospora belli ( , ) , cryptosporidium parvum ( , ) , cyclospora cayetanensis ( ) , and the microsporidiosis agents (i.e., enterocytozoon bieneusi and encepalitozoon intestinalis), may be seen in small bowel biopsies ( ) , and in some patients, specific treatment may resolve the diarrhea or malabsorption. in others, if there is no immunologic compromise, spontaneous resolution of the infection may occur. most often, however, these infectious agents are detected in patients with acquired immune deficiency syndromes, following transplantation or after hiv infection ( ) . often, in this setting, the presence of multiple agents makes definition of the precise cause of pathologic changes in the small intestine impossible. parasites may also cause altered architecture and small bowel lesions with a morphologically distinctive agent (e.g., strongyloides stercoralis, hookworm, schistosoma or capillaria species). in some patients, there may be morphologic similarities to eosinophilic gastroenteritis, a diagnosis that can only be established after parasitic disease has been excluded. viral agents, like cytomegalovirus, have been detected in small intestine, particularly from immunocompromised patients ( ) . often, it is not clear if the viral agent is the cause of the diarrhea or the intestinal pathologic changes. similarly, hiv-infected patients may develop a wide range of mucosal changes from severe (flat) to variably severe changes; in these patients, it is not known if the viral agent per se directly causes the pathologic abnormalities or if changes are indirect, possibly related to some immunologic alteration, malnutrition, or another infection ( ) . still, other viral agents, including the sars coronavirus ( ), appear to cause no histological alteration in the small intestine, even though scattered viral particles may be detected on the surface of microvilli of the enterocyte. fungal organisms have also been found in the small intestine, including candida species and histoplasmosis. patients are usually immunocompromised ( ) . in addition, stasis in 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-ijrghpco authors: bein, thomas; karagiannidis, christian; quintel, michael title: climate change, global warming, and intensive care date: - - journal: intensive care med doi: . /s - - - sha: doc_id: cord_uid: ijrghpco nan in the last five decades, human activities have resulted in the release of increasing quantities of carbon dioxide and other greenhouse gases, thus contributing to global climate change by additional heating of the atmosphere. the world has warmed up by approximately . °c. in particular, the last decade has been the warmest since [ ] , and the frequency and intensity of natural disasters (such as earthquakes, devastating storms, forest fires, prolonged heat waves, droughts, and floods) have increased manifold. between and , climaterelated and geophysical disasters killed . million people and left a further . billion injured, homeless, displaced, or in need of emergency assistance [ ] . climate change scenarios include a change in the spread of infectious diseases with warming and changes in outbreaks associated with extreme weather events after floods or as a result of water heating. furthermore, warmer climates provide more favourable conditions for the survival and completion of the life cycle of the vector that transmits pathogens [ ] . natural disasters and extreme events lead to traumatic deaths and injuries, mental illnesses, and infections, while global warming per se promotes heat-associated illnesses (cardiovascular strain, pulmonary diseases, exsiccosis, mental disorder [ ] ). the world health organisation (who) expects approximately , additional deaths per year between and from extreme heat, natural disasters, and changing patterns of infections, mostly in people at risk (people living in coastal regions or mega cities, children, the elderly, people with multiple and/or severe comorbidities, and-last but not least-people living in regions with weak healthcare infrastructures) [ ] . the impact of global warming on diseases requiring intensive care has been extrapolated from some existing data regarding a change in the spread of infectious diseases [ ] , an (further) alteration of the function of the respiratory system-especially in patients suffering from chronic lung diseases-an expected increase in kidney diseases, an expansion of cognitive disorders due to heat waves, particularly in the elderly, and some adverse effects on the cardiovascular system. an overview of health-related changes requiring intensive care is given in fig. . according to statistical models to estimate the global burden of infectious diseases due to climate change, in , there could be a % increase in diarrhoeal diseases, primarily in young children, and the population at risk for malaria could increase by - % [ ] . of late, in germany, the first incidence of meningitis caused by mosquito-borne west nile virus infection was reported in a man who never had travelled abroad [ ] . in consequence, in future, the european intensivist-who already should be aware of uncommon vector-borne diseases due to global tourism-must be further sensitized to presently uncommon or tropical mosquito-borne diseases such as malaria and dengue, or other vector-borne viral, fungal, or bacterial transmissions like coccidioidomycosis or avian influenza. what should we expect regarding the impact of climate change on respiratory health? lungs in a warming world [ ] become more vulnerable, especially those that are yet damaged. in patients with serious lung diseases like asthma or chronic obstructive lung disease (copd), an aggravation of severity or increased mortality has been described as a consequence of global warming. increasing concentration of greenhouse gases, air pollution, forest fires, prolonged heat waves, droughts, and floods contributes to increased respiratory morbidity and mortality. during the heat wave in portugal, for each °c increase copd morbidity increased by . %, especially in women and in persons aged over years [ ] . furthermore, a rapid spread of viral respiratory infections (e.g. hantavirus, respiratory syncytial virus) as well as fungal respiratory infections (coccidioidomycosis, aspergillosis) can be expected [ ] . dehydration and volume loss could be the other consequences of climate-related extreme heat exposure, leading to chronic kidney diseases or acute renal failure [ ] apart from that caused by diabetes, hypertension, or glomerulonephropathy. a recent study [ ] has recognized an epidemic of chronic kidney disease of unknown aetiology in central america ('mesoamerican nephropathy'), which has been attributed to recurrent dehydration due to heat stress. several studies have addressed potential cardiopulmonary health effects associated with climate change parameters [overview in ] , and these effects are expected to further increase in the coming decades. it seems that a rise in mortality, emergency room visits, and (intensive care) hospitalization due to cardiopulmonary diseases attendant on heat stress, extreme weather conditions, or air pollution are more certain than worst-case horror scenarios. but what is about climate change and intensive care medicine? do we have enough awareness (and answers) in the face of all these challenges for the healthcare system? in a recent survey on climate change and health on the members of the american thoracic society [ ] , the majority of the respondents stated that climate change is a fact that it is relevant for patient care. they confirm that physicians and physician organizations should play an active role in educating patients, the public, and policy-makers about the effects of climate change on human health. do we play such an active role yet? are we prepared to confront the increased frequency of critical illnesses about which, in some cases, we have to learn first? [ ] to our knowledge, there is presently no specific activity in intensive care medicine to face current and future challenges in the context of global warming and climate change, although it is expected that intensive care medicine will need more specialized capacities, better knowledge on the part of the intensivists, and better preparation for worst-case scenarios (heat stroke waves or infectious outbreaks) to manage the consequences of climate change adequately. it has been shown that acute and intensive care services vary substantially across economic regions, in highincome as well as in low-income countries [ ] , and it is debatable whether the current structure of intensive care is yet armed for needs related to climate change. icu bed capacities represent by far not the only challenge; changes in knowledge and organization are required to be prepared ( table ). the intensive care staff will in the near future need specific knowledge about 'uncommon' diseases and heat stroke management (especially in the elderly), and the icu must be organized so as to be able to effectively take care of larger numbers of rapidly incoming critically ill patients in the context of natural disasters. especially in megacities and coastal regions, the number of icu beds should be checked so that they are sufficient in times of natural disasters. a functionally adequate capacity for isolating patients with critical infections is indispensable for successful infection control management. expert support from psychiatrists or psychologists must be available in situations of devastating floods, storms, or heat waves. preparing intensive care medicine for the consequences of climate change does not mean neglecting the basic principle of medicine-the best cure is prevention. therefore, supporting efforts that reduce or ideally stop global warming are the need of the hour. increasing number of critically ill patients due to heat waves, natural disasters, air pollution or forest fire increase capacity of icu beds, especially in coastal regions or megacities mass casualties of critically ill patients due to rapid weather changes, floods, heat attacks implement a 'reserve' of staff and icu beds, which can be easily activated increasing incidence of 'uncommon' infectious or non-infectious diseases provide sufficient capacities of patient isolation instruct the icu staff in the diagnosis and management of 'uncommon' diseases increasing number of nephropathy during heat waves provide sufficient machines for renal replacement mitigation of climate change. contribution of working group iii to the fifth assessment report of the intergovernmental panel on climate change united nations office for disaster risk reduction, centre for research on the epidemiology of disasters ( ) economic losses, poverty, and disasters global warming and its health impact medical aspects of global warming global climate change and infectious diseases climate change and infectious diseases: what can we expect? lungs in a warming world: climate change and respiratory health excess mortality and morbidity during the july heat wave in porto climate change and respiratory infections climate change and the emergent epidemic of ckd from heat stress in rural communities: the case for heat stress nephropathy ckd of unknown origin in central america: the case for a mesoamerican nephropathy environment, global climate change, and cardiopulmonary health survey of international members of the american thoracic society on climate change and health prioritizing health in a changing climate international forum of acute care trialists. access to urban acute care services in high-vs middle-income countries: an analysis of seven cities key: cord- - eu zlba authors: shirley, dennis title: vectors of educational change: an introduction to the twentieth anniversary issue of the journal of educational change date: - - journal: j educ change doi: . /s - - - sha: doc_id: cord_uid: eu zlba this article introduces a special, th anniversary issue of the journal of educational change. the special issue edictoras have organized significant international contributions to theory-building into three areas. these concern diverse modalities of educators’ professionalism, debates around “getting to scale” with successful innovations, and conflicting views of social justice in schools and societies. each of these areas comprises an independent vector of disagreement and debate, with differing meaning and interpretations based upon the cultures and histories of the given systems under review. the article asks what kinds of new research, and what kinds of affiliated theories in these topic areas, can best help to move the field of educational change forward in the coming years. anyone had imagined when state-wide testing was introduced in the us in the s. standards were promulgated with increasing force. exit options like charter schools in the us and academies in the uk spread like wild-fire. the traditional grammar of schooling (tyack and tobin ) , was back with a vengeance. such phenomena now have been documented in tens of thousands of schools in the us and beyond. accountability pressures provided new rationales for educators to return to forms of frontal instruction and curriculum delivery that innovators had sought to surpass with more active, student-initiated forms of learning. as evidence accumulated, many began to suspect that it was no longer a matter of tinkering here and there to upgrade the new reforms, some of which we had tacitly supported, but rather of critically interrogating them. it became important to determine what the purpose of education was in the first place, and then to assess whether the new reforms were attaining espoused goals or distracting from them. this became an increasingly moral imperative as surveys and teacher retention data showed that educators themselves were increasingly disenchanted with what was transpiring under the name of "reform," while the ratcheting up of pressure and sanctions produced at best modest results. at times, it began to feel that the entire enterprise of public education was under siege. still, those of us working away in the proverbial "trenches" knew that the problems in schools had predated recent reforms. many issues couldn't be pushed off on policy makers. some of the most tenacious problems-outdated teaching strategies, poorly designed curricula, tracking, and unfair assessments that discriminated against the poor, the learning disabled, and students of color-were squarely within the locus of control of the profession. these would have to be confronted and resolved by educators themselves. in this regard the journal of educational change, in its different iterations over two decades, has endeavored to stake out an independent position on contemporary educational issues while at the same time being open to all kinds of dissenting perspectives. in a retrospective review of the first years of the journal published in , juan cristobal garcía-huidobro and his colleagues identified periods in the journal's history that reflected the major transformations in the field. in the first of these, "assumed universality in educational change," from to , journal authors wrote with enthusiasm about learning across borders and its potential for improving the human condition. this was followed by a second phase from to , emphasizing "equity and context" in diverse jurisdictions. from to the journal published numerous articles on international large-scale assessments. in the next phase, from to , articles conveying predominantly "anglo-american skepticism" prevailed. finally, a fifth phase from to ensued of "emergent internationalization and empiricism." (garcía-huidobro et al. , pp. ) . there is real value in this type of literature review. the historical periodization rings true, and helps readers to understand where the field has been in order to ascertain where we might go next. still, there is a danger that subtle but important differences of opinion could be overlooked in the formulation of generalizations. mindful of tendencies towards groupthink that can inhibit even the best of journals, incoming editor corrie stone-johnson and i have wanted to do something different with this twentieth anniversary issue of the journal. we have sought out authors we believed could make significant contributions to theory-building and have clustered their topics into three areas. these are: in what does it reside, and how is it evolving over time? . getting to scale once a successful change model is identified, how we can ensure that it is accessible to each and every learner who could benefit from its approach. . justice given the striking inequalities evident in almost all school systems in almost all countries, what can educators do to contribute to teach and empower those students who are most needful of a quality education? consider each one of these topics-educators' professionalism, getting to scale, and justice-to constitute an independent vector of disagreement and debate. what kinds of new research, and what kinds of affiliated theories in these topic areas, can best help us to move the field of educational change forward? the first vector pertains to debates about the meaning and import of educators' professionalism. our opening article in this special issue is written by andy hargreaves, the editor-in-chief of the journal for its first years. in it, hargreaves draws upon new data from ontario, canada, to argue that it isn't simply the case that high-stakes testing is damaging to pupil learning. this has been done many times. what is new here is that hargreaves argues that even mid-stakes testing has deleterious effects. ontario tests its students at grades , , and , and it is precisely at those grade levels where educators report the least innovation and the most traditional forms of instruction prevail. while students at other grade levels can flourish, and their teachers enjoy far-reaching professional discretion, students in those grade levels are subjected to the most anachronistic forms of rote learning and memorization. this is done even though teachers themselves know that they could do better, which makes for an insidious form of compliance in one's own de-professionalization. if ontario wishes to maintain its position as a global leader of educational change, hargreaves argues, it is time to abolish its mid-stakes testing system and to restore the professional integrity of its educators across the system. what might come after testing? in her essay entitled "creating capacity for learning-are we there yet?," louise stoll invites us to reposition learning as the central purpose of schools. this might seem so obvious as to go without saying. still, the challenges of "low trust and punitive accountability" in schools often provokes defensive reactions among educators that counteract the deep and mindful teaching and learning of which we are capable (shirley and macdonald ) . "spirals of inquiry" (halbert and kaser ) provide one way of thinking collectively about learning, rather than public relations management, as the core moral purpose of schools. while open to engaging with the kinds of concerns espoused by hargreaves and stoll, amanda datnow, my predecessor as editor-in-chief of the journal of educational change, suggests that an intermediate strategy that draws upon data selectively but is not enslaved to it, could be explored by teachers in more schools. drawing upon her research with vicki park ( ), datnow argues that much potentially useful data often is not studied by teachers in any meaningful way, because they do know how to leverage it to improve their teaching. she finds that when teachers have adequate professional development, administrative support, and collaborative opportunities with colleagues, that data can take their rightful place in a broad portfolio of materials that teachers can use to improve their instruction and to lift their students' learning results. while scholars should be rightfully critical of an overemphasis upon testing and accountability systems, then, there is no inherent reason why teachers should fail to draw upon relevant findings, especially when these can help those students who need assistance most to get on with their learning. the heart of the matter is "professional collaboration with purpose" driven by a genuine "mindset of learning" rather than a soulless "bureaucratic task or an overly linear model of instructional improvement." like datnow, jenny weiner acknowledges problems with testing yet still wishes to preserve what she sees as some benefits of testing and accountability. her research supports the findings of incoming journal editor corrie stone-johnson that younger teachers as a generational cohort don't especially mind testing, and even find their older colleagues' preoccupation with its negative features rather quaint and eccentric. weiner calls these educators' approach a new form of "nuanced professionalism" that is superseding an older variant of "occupational professionalism." this new form of professionalism recognizes that teachers' autonomy has sometimes been abused in the past by those unwilling or unable to put in the long hard hours planning lessons, managing unruly students, and grading assignments. observing these problems, younger educators appear to be willing to trade off some kinds of discretion in favor of a more collective way of conducting work, even if this entails some top-down direction from time to time. as weiner points out, her perspective is similar to stone-johnson's description of a "parallel professionalism" ( , p. ) in which educators find standards and curriculum frameworks to be useful points of reference that relieve them of the burden of planning everything from scratch day-in and day-out. the differences between the findings of hargreaves and stoll on the one hand, and datnow and weiner on the other, indicate that a range of opinions on the essence of educators' professionalism can be found in the journal of educational change. while there is a shared concern for ongoing transformations in teachers' work, the findings point in different directions when it comes to how policy should be understood and negotiated in situ in the schools. the differences should not be overstated, but they should be noted and taken as points of departure for further research. it is perhaps not accidental that in addition to different perspectives, the lines of division here reflect different" generational interpretive frameworks" (stone-johnson , p. ). a second vector of disagreement can be found regarding the topic of spreading educational change. in santiago rincón-gallardo and brahm fleisch brought together a truly global ensemble of articles for a special issue of the journal on the topic of "bringing effective instructional practice to scale." the underlying concept of the special issue was to "bring together original articles and scholars who have developed and/or studied relatively successful approaches to large scale instructional change in a variety of contexts." (rincón-gallardo and fleisch , p. ). these included colbert and arboleda ( ) , writing on the escuela nueva movement in colombia; banerji and chavan ( ) writing about literacy reforms in india; and rincón-gallardo and fleisch themselves, reporting on their findings on education as a social movement in mexico and the teaching of reading in south africa respectively. it was a fascinating special issue, not the least because the author credited with the notion of "getting to scale," richard elmore, wrote a blistering critique of his own concept as marked by "unthinkable presumptuousness and naivete," in an afterward to the volume (elmore , p. ) . hang minh le would be likely to agree with elmore on the dangers of thinking in terms of "getting to scale," especially when it comes to the dissemination of reform models internationally. in her article on "where be the 'magic bullet' for educational change? vietnam and the quest of policy borrowing from abroad" in this issue, le criticizes the tendency of policy makers in the global south to seek solutions from other countries rather than on developing indigenous capacity to address educational challenges. she asserts that a "coloniality of knowledge production" influences policy makers, who are controlled in many ways by transnational funders such as the world bank. le finds that this influence of coloniality is so powerful that even highly successful grass-roots mobilizing efforts in the global south, such as the escuela nueva movement in colombia, that have been recognized for their achievements by the yidan prize in hong kong and the wise prize in qatar, are complicit in the oppressive legacy of colonialism. for vietnamese educational reformers, the attraction to escuela nueva appears to reside in its adaptation of western educational ideals such as student-centered classrooms, active learning, and parent and community engagement. for le, these are not universal features of good teaching and learning. they are manifestations of western values that should not be imposed on vietnam, however attractive they might appear to policy makers eager to make their mark as effective educational leaders. since vietnam already scores very highly on the oecd's pisa tests, she notes, it hardly needs to engage in the "fast policy making still rooted in coloniality" that is common in much of the global south. le's analysis of escuela nueva would appear to offer a critique of the enthusiasm expressed by rincón-gallardo for this model of educational change recently in his book ( ) entitled liberating learning. in his article in this special issue, rincón-gallardo draws upon his leadership and research with the "learning communities" approach developed by gabriel cámara and his colleagues in remote rural middle schools in mexico, which shares many affinities with escuela nueva. he asks that greater scholarly attention be devoted to reform efforts such as these two latin american examples as well as "activity based learning" in india and community schools in egypt. these diverse efforts, he argues, are "more similar in their genesis and development to social movements than to conventional education programs or policies." taken together, they present a "feasible alternative" to the default culture of schooling that has been exported by the global north and imposed upon the global south. brahm fleisch, for his part, expresses no particular concerns about strong stateled initiatives to improve education in the global south. he argues that western commentators generally have failed to acknowledge the contributions of vigorous, top-down leadership to "systems as diverse as hong kong, singapore, korea, japan, shanghai, and vietnam." fleisch might agree with le that the sensitivity of educators from the "anglosphere" of the global north for child-centered pedagogies, project-based learning, and teacher autonomy has little relevance for countries endeavoring to extricate themselves from the powerful legacies of colonialism. instead, fleisch sees value in "strong central control" by governments motivated by "strong egalitarian impulses." from this perspective, elmore's retraction of the concept of "getting to scale" is overstated. whether through the kinds of confidence-building, state-led leadership strategies favored by fleisch, or the bottom-up social movements described by rincón-gallardo, educators have moral obligations and professional responsibilities to engage with all kinds of diverse populations to promote learning to enhance the human condition. even le's criticism of policy borrowing from one nation of the global south to another could be seen as an admonition to practice "getting to scale" with indigenous philosophies and practices within vietnam. self-criticism is good. self-paralysis is not. finally, a third vector of disagreement may be found in conceptualizations of justice in educational change. helen janc malone argues for community schools as expressions of social justice that help schools to "engage in diverse intentional partnerships, authentic grassroots family and community engagement, and collaborative governance and leadership structures." while many have argued that we each now live in a "deterritorialized context" (appadurai , p. ) with a concomitant "decline of the local" (foster , p. ) , malone sees local communities as indispensable educational resources, especially for equity purposes, as "funds of knowledge" (moll et al. ) ordinarily bracketed out of schools are appropriated for learning. however much we have come to rely on our cell phones and the internet, in the end we are all embodied and need contact with other human beings unmediated by technological tools. given the unprecedented disruptive power of the coronavirus, many would agree that local communities and their schools have proven themselves to be more crucial than ever. alison skerrett, on the other hand, asks educators to consider how they are serving growing numbers of transnational students, who, "through a mixture of necessity and choice, live their lives across two or more countries." she calls for a "transnationally-inclusive approach to literacy education" as an expression of social justice, that could help students to understand their different languages and distinctive cultural blends as assets rather than deficits. since educational systems are bound up with concepts of nation-building, skerrett sees formidable obstacles for the future of transnational education. she cites research by hamann and zuñiga ( ) showing that it nonetheless is possible to blend transnational identity formation for youth with nations' legitimate concerns to educate students capable of thriving within their given geographical boundaries. can we find a way to combine the social justice concerns for community schools advanced by malone and the similar values upheld by skerrett for transnational students? one promising case is provided by franco-ontarian educators in canada who have found new ways to blend their traditional and endangered language minority culture with a new transnational population of french-speaking students from countries as diverse as haiti, senegal, cameroon, and france itself (shirley and hargreaves ) . by building strong interdisciplinary teams of educators, including staff with expertise in cultural diversity, linguistics, and curriculum development, these educators have shown that an inclusive and creative approach to all of our diverse students can indeed be forged which creates a new kind of community appropriate to our current circumstances. we need not make a forced choice between local community and transnational students, then. as educators, we have the collective intelligence and can marshal the willpower to forge new kinds of learning communities that are as diverse and culturally rich as the students we find before us. in its first years, the journal of educational change has served as a fulcrum for fascinating debates on many more topics than the themes of professionalism, getting to scale, and social justice than are featured in this special issue. as of this writing (june ) the world is in the grip of a global pandemic that has killed more than , people and infected more than million others. nine out of children worldwide have been kept home from school. millions of these had no internet access at the time, depriving them of access to educational resources even as others, more fortunate, bounded ahead. unemployment leapt off the charts to its highest levels since the great depression in many countries, and food insecurity soared. at the same time as dark clouds loomed, silver linings could be detected. some students genuinely liked learning at home, free from the distractions of disruptive classmates and ill-timed interruptions to instruction. while many apps designed to promote learning were poorly designed, others were exciting and they connected students with new digital friends from around the world. nature rebounded as flights and commutes to and from work ground to a halt. random acts of kindness popped up in unlikely places. many agreed that they now were taking each other less for granted. now and then it was possible to glimpse a better future. modernity at large: cultural dimensions of globalization improving literacy and math instruction at scale in india's primary schools: the case of pratham's read india program bringing a student-centered participatory pedagogy to scale in colombia data-driven leadership getting to scale …" it seemed like a good idea at the time the decline of the local: a challenge to educational leadership spirals of inquiry for equity and quality schooling and the everyday ruptures transnational children encounter in the united sttes and mexico funds of knowledge for teaching: using a qualitative approach to connect homes and classrooms. theory into practice liberating learning: educational change as social movement bringing effective instructional practice to scale: an introduction community organizing for urban school reform valley interfaith and school reform: organizing for power in south texas student engagement: beyond relevance, technology, and fun parallel professionalism in an era of standardization generational identity, educational change, and school leadership the "grammar" of schooling: why is it so hard to change? the journal of educational change has a role to play in shaping that better future. in its years, it has forged bold new spaces for debates on professionalism, "getting to scale," change, and social justice. the coronavirus pandemic is just one signal that the scope of these debates has ample new room for development in the years to come. in this, my final introductory essay for the journal, i ask that all of us affiliated with the journal-its board members, authors, and reviewers-provide our wholehearted support to incoming editor-in-chief corrie stone-johnson. key: cord- -m ig hii authors: sharp, mindy mcgarrah title: if you’re ready, i am ready (but the wait is harming us both) individual risks in institutional conversions date: - - journal: pastoral psychol doi: . /s - - - sha: doc_id: cord_uid: m ig hii rambo, adele, and a religion professor walk into a coffee shop. what in the world do they have in common? this essay integrates lewis rambo’s scholarship on conversion with pop singer adele’s popular song “send my love to your lover” to understand and redress inequity in individual risk within processes of institutional conversion. the author focuses on practices of inclusion in institutions of higher education using the specific example of theological schools with aspirational school mission statements that embrace equity and diversities. she argues that bringing rambo and adele into conversation with anecdotal and published research on institutional (in)justice illuminates four practices that institutions could adopt to better align practice with promise and thereby promote wellness for all who are impacted by institutional health. album for my birthday. we listened to it together in the car while driving to and from school and various activities and commitments. the song entitled "send my love to your new lover" quickly became a favorite in family car sing-alongs, especially its final overlapping chorus. the chorus has two different phrases sung in different melodies at the same time, and we loved each having a part in singing the harmonies. we talked about what the song could mean in contexts beyond a coupled love relationship but generally focused more on joining our voices with the powerful voice of adele. my family is not alone in being drawn to the power of adele's voice and this song in particular. as of the drafting of this paper, the youtube version of the music video for just this song registered over million downloads. for a stretch of time when it was our family's # car song, i found myself humming it between meetings, classes, reading, and writing. not surprisingly, during this time adele's "send my love to your new lover" song would often be an earworm throughout my day. one ordinary workday while waiting on a notoriously slow copy machine, i realized that "my" line "if you're ready, i am ready" was playing in my head. i was struck by hearing the words as descriptive of what's at stake in relationships where partners fail each other, where people get wounded, and where possibilities for transformation could lead to change in future relationships. i thought about this in the context of collective conversion in institutional life in theological education, what can go wrong, and which bodies get more or less wounded in the process. i was pondering the age-old theological and philosophical debate of the difference between is and ought. why is what is happening so different from what ought to be happening? why is there so often such a chasm between is and ought? what kinds of changes or conversions are needed to close the gap? as a theological educator, i have been fascinated by the aspirational mission statements of seminaries and theological schools that stress prophetic witness, truth, and justice. yet, my studies in postcolonialism have exposed histories of educational and religious institutions, methodologies, and curricula as deeply flawed, at times serving as instruments of discrimination and oppression (hooks ; smith ) . i wrestled with these thoughts in front of the copy machine while adele's chorus "if you're ready, i am ready" played in my mind. what does "being ready" mean in the context of theological education? ready for what? i heard adele calling for readiness in her lyrics, including readiness to live into who we say we are in our educational mission statements. adele sings of readiness to learn from relational failures and change one's future practices for the better. what does it mean to do better and learn from relational failures on an institutional level? these kinds of questions became a secondary earworm influencing how i bear witness to, interpret, and participate in some of the more difficult aspects of institutional life. in this paper, i weave three unlikely conversation partners-adele's song "send my love to your new lover," lewis rambo's theory of conversion, and a practical theological engagement with inclusive and equitable institutional practices. while they may seem unrelated, weaving adele, rambo, and institutional practices together yields insights into practices of institutional conversion that could address gaps between the inequities and injustices that exist and the aspirational educational missions that many schools claim. in order to redress ways in which institutions of education, including religious and theological education, are shaped by oppressive and exclusive histories and habits, institutions must embrace and expect change, what rambo calls conversion. one sign of change would be robust diversities represented in curricula, faculties, administration, students, boards, and other stakeholding groups and aspects of teaching and learning. but, change is hard, and institutions, like individuals, can resist or be slow to embrace equity and inclusion in practice (sharp ) . at the copy machine, i thought of friends, once relieved at the promise of an academic job after years of unpaid doctoral studies, who were now leaving tenure-track faculty positions to start over at other schools or leaving the academy altogether. nearly all who came to mind inhabited minoritized identities. the online publication the feminist wire's scathing article "black academic women's health forum" had already been haunting me and helping me recognize institutional dynamics across higher education where friends were and were not struggling in academic vocations (cox et al. ) . the forum editors amplified one anonymous reader's comment on the forum: with every piece i read, i felt like a layer of my skin was being pulled back, like more and more was finally revealed. i've spent so many years on the margins of this institution that i got used to making myself invisible, on purpose. i am seeing myself again. i will demand to be seen. thank you. (cox et al. ) most folks i knew and read about who were suffering serious health consequences related to institutional stress were underrepresented in various ways due to race, gender, sexuality, nationality, religious identity, and more, and had been hired with great joy by institutions who celebrated their initial inclusion. yet, the relationship with their institutional contexts had not lived up to the promises of inclusive institutional practices. stress was increasingly manifest in more minoritized bodies while other dominant-culture colleagues appeared to be just fine. for some community members, the way things had always been seemed to be working-or folks long ago had adapted to a normalized system and become more change averse. other community members seemed to be holding on by a thread. in my particular field of study, there was growing recognition among pastoral theologians of the spiritual and material costs of institutional demands in theological education-the "maceration" of faculty members-often if not always borne disproportionally (miller-mclemore ) . the feminist wire forum editors aimee meredith cox, aishah shahidah simmons, and tamura a. lomax summarized the strong resonance of readers with the online forum mentioned above with the declaration: "we exhaled into each 'peace' and they breathed back into us. we let the world know loud and clear: we want to be well! we will be well!!" (cox et al. ) , echoing novelist and activist toni cade bambara's haunting question in her novel the salt eaters: "are you sure, sweetheart, that you want to be well?. .. just so's you're sure, sweetheart, and ready to be healed, cause wholeness is no trifling matter. a lot of weight when you're well" (bambara (bambara / . just what does an institution of theological education that supports wellness equitably look like? and who decides? colleagues who came to my mind in front of the copy machine had experienced much heartbreak and many conflicting narratives swirling in and beyond various communities, but this was not the last word. when the institution is slow to change, individuals can make declarations of collective wellness in public profession. in interviews, adele also declared wellness on her own terms, reporting that , her third album, was the making-up album after the heartbreak album and her breakout record , each named for her age at the time of recording the album (epworth and adkins ; adele n.d.) . the song "send my love to your new lover" is, like many adele songs, set in a strong and confident voice, pointing to a coherent sense of self that is able to recover and reflect on relational dynamics gone wrong-or certainly hopes or pretends to do so while still carrying wounds of letting go of what once was. adele's song "send my love to your new lover" is described by the singer herself as communicating that she "can finally reach out a hand to my ex. let him know i'm over it" (lamont ) . while resolved, "send my love to your new lover" is not static but rather emotionally complex, evoking layers of movement (bacle ) . truth-telling regarding both past heartbreak and future resolve, the lyrics charge a former lover to do better with their next relationship, to break a cycle of harm, to change. the song's narrator describes the release that comes with letting go and freeing oneself from a toxic relationship that doesn't support well-being. i hear adele's lyrics not only in contexts of interpersonal relationships but also as descriptive of the general experience of individuals caught up in systems not structured to support their wellness who have to choose between either staying or protecting their wellbeing, especially when institutions fail to deliver on promises of change. adele's "send my love to your new lover" can be read as an anthem for unmasking individual risk in institutional conversion. the song's lyrics can help clarify dynamics involved in institutional deep change. moving from systemic harm toward institutional wellness is a kind of conversion process, leading me to interrogate adele's powerful sung call for change with scholarship on conversion. lewis rambo's classic book-length treatment of conversion, understanding religious conversion ( ), provides language for making sense of discernable aspects within the sometimes ambiguous and usually unending life-long process of conversion. bringing adele and rambo together, i argue, offers particular insights for understanding and living in the midst of institutional change. in the following sections, i will describe four elements of institutional conversion that flow from the conversation between lewis rambo's decades of research on religious conversion and what i was hearing as a pointed call for change in adele's "send my love to your new lover." i locate these insights in the realm of theological education, where i work, but imagine they could help illustrate similar dynamics in other areas of higher education and institutional life. life is full of change and needs for change. the question is, do we want to marshal this change in the service of being well? theological education is in a precarious moment, wrestling with institutional, ecclesial, and cultural changes while being called to conversion to just, equitable, inclusive practices that honor and celebrate multiple diversities. in short, institutions of learning are presently in a decolonizing conversion process that is yet to be fully realized. educational infrastructure built centuries ago to prepare young white men for mainline religious leadership in the united states and missionary service abroad now hosts an ecumenically and internationally diverse faculty, staff, and administration, as well as a diverse student body preparing to serve in a large variety of faith-based and other creative forms of leadership in a world increasingly recognized as globalized and interconnected. tensions between tradition and innovation abound while corporate identity shapeshifts at the level of school, denomination, and beyond. member schools of the association of theological schools, whose mission statements often indicate aspirations of hospitality and inclusion, are being called to conversions through transformations to inclusive, just, and prophetic policies and practices. there is not necessarily agreement on what this means, but it is increasingly clear that much is at stake. practical theology and pastoral psychology are well positioned to help integrate theory and aspirational commitment with practice. connecting popular religiosity with scholarly theology is at the heart of these disciplines that weave theory and practice, learning what works and doesn't work to support individual and collective well-being. connecting adele's "send my love to your new lover" and rambo's study of conversion is illuminating. conversion scholar rambo's theory of discernible elements within conversion processes can help assess where theological schools may be stuck or slowed in their decolonizing conversion process to more just and inclusive institutional practices. for the purposes of this paper, i use marc baer's definition of conversion as "an intensification of belief and practice of one's own religion.. .. [w]here one did not give other than cursory thought or attention to the theology of one's faith or engage in keeping wholeheartedly to its requirements, one devotes one's mind and body fully to understanding and embracing the religion" (as quoted in rambo and farhadian , p. ) . using this overarching definition of conversion as "intensification," i consider rambo's seven stages of conversion experiences in institutional settings, bearing in mind that "spiritual transformation affects work place culture" (paloutzian , p. ; see also sandage et al. ) . within theological education at the institutional level, rambo's first two stages of conversion, contemporary contexts and crises, are clearly present. his next three stages of conversion, what he names quest, encounter, and interaction, present sticking points in a decolonizing conversion to more just practice within theological education. published reports from accrediting bodies, ethnographic studies, and theoretical engagements espouse wide agreement that present structures of theological education are not as just, inclusive, and equitable as they/ we profess and aspire to be (association of theological schools ; muhs et al. ; cox et al. ). further, tenure and retention of diverse faculties hasn't changed much even while faculty hires have diversified (association of theological schools ), it is clear that seminaries that want to practice more equity, inclusion, and diversity are not necessarily sustaining these aspirations over time at the level of faculty, whose responsibility includes embodying institutional mission through curricula. like many stage theories, commitment and consequences, what rambo names as the final two stages of conversion, come after addressing stuck places in earlier phases. a quick review of the mission statements of the seventeen institutions represented at the new directions in pastoral theology meeting where i originally presented this paper suggest aspirational missions of transformative education; well beyond this sample, association of theological schools member schools report "diversity" as one of the top six priorities for theological schools (gin , p. ) . what does it look like in practice to strategically prioritize diversities? what kinds of institutional change or conversion are required? while reviewing rambo's seven stages of conversion, it becomes clear that adele's "send my love to your new lover" is one resource that can help put language to what is at stake in remaining stuck in the middle of a conversion process at an institutional level. who bears more and who bears less individual risk of woundedness and wellness? to be sure, more minoritized scholars are often scapegoated along the way when theological schools that profess readiness to inhabit more just practices that are hospitable to greater diversities find themselves/ourselves stuck or prematurely professing achievement regarding the fullness of conversion. in conversation with rambo's work on conversion and with reference to three verses and the chorus of adele's song as representing voices of more minoritized scholars, i identify four practices to confront frustrated change. institutions who expand their welcome to include more previously underrepresented members-in an effort to become more just, diverse, equitable, and inclusive communities-can fall short of these goals and thereby cut the conversion process short by failing to sustain the hard work of deep change. bringing adele and rambo into conversation, i distinguish four practices that could loosen institutional stuckness in order to motivate institutions who still want to keep moving through a decolonizing conversion process: ( ) untangling initial inclusion from completed conversion, ( ) truth-telling about desires not to convert, or resistances, ( ) detangling toe-dipping from being in over our heads, and ( ) (re)engaging a commissioning call and response in order to (re)commit to conversions in practice. without engaging these practices in an institutional decolonizing conversion process, more minoritized community members are often left to shoulder a disproportionate measure of individual risk. adele's song "send my love to your new lover" can serve as a fresh prophetic anthem for the limits and opportunities for theological schools engaged in a conversion process of transformations in just practice. a conversation between conversion scholar rambo and music superstar adele can lead to recommendations for strategic practices to inspire institutions and we who inhabit them to move more deeply into practicing the best of what is professed. rambo has been writing about conversion for decades. across his abundant scholarship, conversion most simply means change and transformation. while conversion can and often does denote change from one religious tradition and set of practices to a different religious tradition or set of practices, conversion can also mean change and transformation within the same profession of faith. this latter aspect of conversion is my focus, "intensification [that] takes place within a tradition" and its "already available" practices (rambo , pp. , - ) . already complex as institutions, mission and identity statements of theological schools often have a prophetic character and calling, beckoning regular conversation and discernment at individual and institutional levels (van dyk ). how do schools that are committed to mission-driven theological education engage conversion from actual practice more deeply into aspirational mission? such conversion involves interpersonal and intercultural interactions that unfold in both the foreground and background of lived human experiences. what exactly is institutional conversion? what does it involve? and, why is it so difficult? writing during a s rise in religious practices alongside increasing recognition of pluralism, rambo notes how institutional fraying co-exists with freedom to move across diverse religiosities (rambo , p. ) . with resonances with womanist and feminist calls for scholarly accountability also rising in the early s (russell and clarkston ) , studying conversion involves reflexivity, professions of faith, and academic commitments (p. ). by the s, womanist and feminist theologians around the world were calling for conversion as an ongoing "seeing and acting in new ways in a fundamental process of transformation" (oduyoye , p. ) . by the s in pastoral theology, scholars were also paying closer attention to systems, communities, and cultures, now identified as emerging communal-contextual and intercultural paradigms (ramsay ) . as a white woman who is partnered, a mother, a lay united methodist, a u.s. citizen, and an employed academic pastoral theologian with international work experience and teaching and learning commitments across borders of identity and geography, my academic commitments are to pastoral theology's intercultural paradigm, requiring ongoing assessment of practices at intersecting individual, communal, and global dimensions (sharp ) . this is the location from which i read rambo's work. using an interdisciplinary methodology, rambo weaves academic discourses of psychology, anthropology, sociology, and religion with extensive interviews and ethnographic engagement around the world ( , pp. xi-xii, ) . what is conversion? conversion is many things. rather than one hegemonic definition, rambo delineates factors and energies, movements and moments involved in conversion as a process. multidimensional, conversion involves empathy, personal experiences, human predicaments, careful observation within and across cultural contexts, and a deep desire for self-and communal understanding alongside conceptual explanation (pp. xiv, - ) . emotionally complex, conversion can elicit joy, astonishment, release, anguish, turmoil, despair, conflict, guilt, and more, often all at once (p. ). human yearnings, emotions, and moral commitments shape individual and collective discernment through what rambo calls an emerald web of seven stages or periods of change (p. ). in the following paragraphs, i will briefly summarize rambo's seven conversion stages-context, crisis, quest, encounter, interaction, commitment, and consequencesthrough the lens of the interplay between institutional conversion (intensification of already established religious commitment in practice) and embodied individual risks and vulnerabilities. context involves an internal and external "force field of people, events, experiences, and institutions [that] operate on conversion. .. the total environment in which conversion transpires" (pp. , - ) . complex and dynamic, context can dissuade and/or encourage conversion depending on how conversion may meet needs and yearnings within particular contexts (pp. , , ) . context involves large multi-systemic forces and local daily life practices (p. ). context is a great resource for conversion, given that cultures are constantly in motion (p. ) even while tradition and familiarity infuse common resistances to change. rambo acknowledges that "most people say no to conversion" (p. ). contextual factors that make conversion difficult for groups of people include ways in which increasing religious choice is interpreted as a gift and/or threat of pluralism, as a gain and/or loss of identity, as fracturing and/or expanding capacities of persons and communities (pp. , , ) . what would it mean for institutions to enter a process of converting to pluralism, where religious mandates lead to practicing multiple diversities as a creative good (bidwell ; lartey ) ? further, when communal conversion does occur, however incrementally or slowly, how do groups of people reckon with ways that previous practices, even after being renounced and transformed, linger? rambo reminds us that "even symbols. .. directly and explicitly rejected can remain a powerful part of [the] psyche" (p. ). conversion prompts many such conflicts and crises of faith within contexts. crisis, explains rambo, forces "individuals and groups to confront their limitations and can stimulate a quest to resolve conflict, fill a void, adjust to new circumstances, or find avenues of transformation" (p. ). he notes that it can be hard to tell whether crisis precedes and leads to conversion or follows a glimpse of a different way of life than one is accustomed to practicing-or perhaps both (p. ). crisis is co-extant with new possibilities (p. ) as "all conversions implicitly require a leaving-behind or a reinterpretation of some past way of life and set of beliefs" (p. ). quest awakens the resourceful human yearning to make meaning and find life purpose (pp. , ). conversion can be very difficult because it involves grief, loss, sadness, severed relationships, altered rituals, disconnections, dislocations, and more (pp. - , ). "the past," rambo reminds us, "is powerful because that is the world in which we dwelt for years, and it lives on in our minds and hearts. there is no easy escape from the past, no easy transition to the future" (p. ). no wonder conversion also includes a quest for meaning, a yearning to be persuaded that new possibilities will also bring release of tension and increase of joy. motivated by a variety of complex and interacting factors, potential converts turn to communities of support for affirmation of new possibilities that are at once attractive and repulsive (p. ). when it comes to institutional conversions, how long can groups reside in this threshold between recognition of loss and promise of release, and at what cost? can tensions be held collectively rather than foisted on a few? encounter involves negotiations between advocates of transformative conversion and would-be converts who often co-reside in shared and/or overlapping contexts. in encounters, advocates for change meet advocates for remaining (p. ). within encounters, significant wisdom resides with "marginal persons [who] are often the earliest converts to a new movement" that promises to support more justice, meaningfulness, emotional gratification, practical wisdom, leadership, and power (pp. - ). indigenous people, notes rambo, often inhabit models of movement and are culturally pliable as a consequence of both internal commitment and outside force (p. ). within encounters, consulting others only goes so far as each person in such interpersonal dramas must find what is at stake for themselves "to enter personally into this new story, to own it" (p. ). encounters raise issues of trust, diverse affirmations of human dignity, and mutual curiosity, especially in complex institutions where forces of tradition meet forces of innovation. interactions that support conversion processes are mutually educational and therefore particularly important for schools. following encounter, deeper interaction foregrounds learning and hospitality. "potential converts," writes rambo, "learn more about the teachings, lifestyle, and expectations of the group, and are provided with opportunities, both formal and informal, to become more fully incorporated into it" (p. ). rambo points to multiple relationships, rituals, rhetoric, and roles evident in interactions. because conversions are both deconstructive and constructive (p. - ), folks can struggle to speak and represent what is going on in the middle of change and transformation. struggle itself is a persistent theme of conversion and, like many conversion elements, can be cast positively and/or negatively, including struggle between self-awareness and selfdeception. for example, while i have been shaped by reading literature by black women academics such as bambara mentioned above-learning what i've never been required to learn about my need to convert from complicities in oppression-as a white woman, i must both read and learn, never stopping at just reading and quoting. "most white academics," writes antiracist scholar audrey thompson, "do not read widely across races and, even if we do, we tend to use the writings of scholars of color to bolster rather than to interrogate our work" ( , p. ) . beyond mere encounter, conversion interactions challenge and/or affirm role expectations and role enactments (p. ). interactions go deeper. rambo powerfully reminds us that "people who convert and remain the same are not really on a spiritual path of transformation" (p. ). rather, conversion is deep change. highlighting this theme, womanist theologian kelly brown douglas calls institutions to convert in terms of who counts as a full person and to radically expand the notice of place ( ; see sharp where i engage dr. brown douglas' profound work more deeply). a willingness not only to learn but indeed to be transformed by a learning process is required. rambo puts it this way: "the relationship [that supports conversion] is based on one person knowing more and being willing to share it with an interested person who is seeking assistance, who is willing to play the role of novice" (p. ). persons who know more about what needs to be learned do not always have access to teaching roles denoted as more informed. conversion interactions birth new relational networks, new rituals, new ways of speaking and listening, and new roles (p. ). "conversion of hearts is not enough," declares ethicist m.t. davila, commending the kind of learning interaction that will support lasting change. she specifies: conversion of hearts for effective social and structural transformation requires listening to the people and the movements that most clearly call out the forms that structural sin take in today's world. convicted hearts receive extensive training in direct social action and advocacy, and they also engage in ongoing education about the ways the sin of racism continues to dominate many of our political and economic structures today (davila ) . to underscore this point, other scholars had also correlated conversion and role change at the time of rambo's text. also in , ethicist emilie townes published a troubling in my soul: womanist perspectives on evil and suffering, an edited volume that included womanist theologians (townes ) . across the chapters, contributors noted ways in which institutions as currently structured perpetuate structural evil and therefore must be engaged with bold resistance and suspicious ambivalence if and when theological schools and faithdriven institutions seek to be more inclusive. roles in institutions don't shift easily, but they must, argued novelist activist toni cade bambara nearly years ago. why? because we are "abusing each other, aborting each other's nature-in the teeth of experiences both personal and historical that should alert us to the horror of a situation in which we profess to be about liberation but behave in a constricting manner" (bambara , p. ) . she called for conversion from sinful western models that have distorted roles and are in need of transformation and recovery, "irresistible revolution" not outright rejection (bambara , pp. , ; bambara bambara / . conversion interactions negotiate, ritualize, speak about, and embody truth-telling and power. what kind of commitment can support such mutual investment, and how do we know that learning creates lasting change? commitment "is the fulcrum of the change process" (rambo , p. ) . expressed in vows, contracts, covenants, and mission statements, commitment involves both word and deed. practicing commitments, argues rambo, involves decision-making, ritual promises, surrender, testimony, and moral wrestling with motivation (p. ). rambo's training in psychology and religion is helpful as he aptly describes the deep soul wrestling involved in each of these elements of practicing commitments (p. ). decision-making involves reflection on one's own life experiences, listening to and believing the life experiences of other people-both stranger and trusted friend-confronting desires and fears, and negotiating between questions of meaning and practical problem-solving (pp. - ). these practices need ongoing mutual accountability and support because individuals resist change while institutions can fall prey to substituting talk of change for transformed practice (p. ). power and control flow through resistances and accountabilities, with promise and threat continually at play (pp. - ). public profession and communal assessment support a commitment to conversion. often absent from revised mission statements and vows, commitment involves articulating what will be released to make room for changed ways of being. rambo writes that such "surrender requires [a] person to confront directly what [they] will be giving up for the benefits of the new option. this process is never easy. .. anguish is keenly felt" (p. ). while it can take a "leap of faith" to believe, surrender "is the very point at which energy becomes available for a new life" (p. ). on an institutional level, backlash or what sara ahmed ( ) calls brick walls appear just when lasting change seems most promising. why is institutional change so difficult? rambo writes, [o]n a more profound level, surrender . . . continues over a lifetime. it is an inner resolve to shift loyalties, and the process is never complete. old urges return, sometimes with greater power than before. few people experience surrender as a final achievement, without reservations or backsliding. surrender is a process in which the person disconnects [themselves] from old ways and patterns and gradually is able to consolidate the new life into a firmer, growing commitment ( , p. ). space for and ritualization of public testimony remain important beyond any initial commitment or aspirational change. testimony "becomes more than a story of individual change; it also reflects the ongoing process of institutional change" (p. ). in the commitment stage, testimony is a cooperative truth-telling process about how conversion manifests not only the desire to change but also the intention to live into lasting consequences. consequences, according to rambo, are the practiced effects of transformation and change, the living out of conversion experiences over time since "more profound changes may come in the months, and even years, after the initial conversion has taken place" (p. ). for lives shaped by institutions, multiple conversions and multiple consequences unfold simultaneously. rambo suggests practices of accountability around a simple question: what is "the quality of life produced by these institutions" (p. )? one assessment tool used by several institutions is the crossroads anti-racist spectrum for institutions invested in multicultural affirming practices, a whole stage theory within the consequences stage. crossroads' development of practice moves from more exclusive to more inclusive practices described as exclusive ➔ "club" ➔ compliant ➔ affirming ➔ transforming ➔ fully inclusive anti-racist organization in a transformed society (crossroads n.d.). rather than assess total conversion (not that that is likely or advisable), rambo suggests using developmental moments as "lens[es] through which to observe conversion [where] one can make evaluations as to whether the convert has progressed, regressed, or remained the same developmentally as a result of conversion" (p. ). again, his advice to prioritize voices of more institutionally minoritized peoples in all stages of conversion is also relevant here. institutions should find ways to assess quality of work life. where do folks experience deeper relationships, relief from guilt/sin, celebration of communal belonging, better understandings of what is going on, authentic participation in a "profound revolution," and increased courage, security, and peace (pp. - , )? deep and radical transformations take a long time and require multiple conversions; therefore, partnerships of accountability, regular opportunities for public witness and testimony, and periodic assessments of the quality of life together are warranted. rambo's seven interconnected stages slow down discrete moments in a long process of change, thereby showing why conversion can be so very difficult, even undesirable. these stages help organize change among diverse groups of folks and institutions that resist change. the transformative change of conversion, writes rambo, "is precarious; it must be defended, nurtured, supported, affirmed [by and in] community" (p. ). institutions both support and dissuade conversions with processes, policies, and practices that "support systems and suppression, access and repression, encouragement and discouragement" (p. ). yes, conversion is possible and happens all the time "in all directions" (rambo and farhadian , p. ) . and yes, conversion is challenging in the best of circumstances, requiring an ongoing investment in struggle for a purpose. rambo both presents an in-depth study of conversion and invites conversation partners and new resources to contribute to the meaning of conversion and the phenomenon of change. this is similar to what i am doing in this article by highlighting a perhaps unlikely conversation partner, the pop singer adele. while rambo supports understanding conversion as a long-term process filled with as many if not more difficulties and reservations as possibilities and transformations, i have found the lyrics of adele's "send my love to your new lover" help put language to what is at stake for theological institutions invested in long processes of conversion, especially institutions who need to slow down and assess where they are in change processes and who is more and who is less impacted in daily life struggles for transformation. conversion scholars are still calling for expanding conversion studies to account for global forces, to include religious diversities, and to traverse geographies (rambo and farhadian , pp. - ) . theological schools are also trying to be more diverse and inclusive. schools should be able to support mutual learning processes of conversion, yet rambo shows just how much folks in conversion processes often drag their feet in changing bits of tradition, from a meeting time to a convocation program, not to mention practicing radical transformation. turning to the arts may help loosen our collective imaginations in what is at stake given that conversion is such a long and winding process, as rambo helps us understand. rambo invites such studies that highlight that "the importance of culture in shaping persons, communities, and religions must be given more weight in conversion studies" (rambo and farhadian , p. ) . conversion scholar diane apostolos underscores that "as human beings we come to know through our natural and primary aesthetic sensibilities-sight, sound, movement-through icon, image, and illustration, through myth and story, through dance and ritual" (apostolos , p. ). further, she argues that "art impinges upon, narrates, and supports conversion" (apostolos , p. ). regarding the transformative power of art, rambo notes that "the greatest testimony to the power of a work of art is its ability to elicit sustained reflection and engagement on many levels. .. [and] engage us intellectually, emotionally, and spiritually" (rambo , p. ) . as i described in the prelude, it struck me one day in front of a notoriously finnicky copy machine that the lyrics to adele's song "send my love to your new lover" may have such transformative potential. singing since age seven, adele laurie blue adkins grew up humbly in tottenham, london, in a "very big family of a lot of women who did everything on their own" (robinson ) . she transformed from a beloved local hipster singer to an international superstar known for stunningly made-up eyes and an even bolder voice, having already received some of the music industry's highest accolades by age . before long, "[h]er music was the most-requested [sic] in karaoke bars, the most played at funerals, 'the best for nervous flyers,' 'the most popular to fall asleep to'" (lamont ) . after her emotionally raw and resonant albums and (recorded when she was ages and ), adele's album "track[s] personal regrets, broken relationships and dashed expectations with. .. crushing emotional power" (npr ) . in her own words, adele reflects that "my last album was a break-up record and if i had to label this one i would call it a make-up record. i'm making up with myself. making up for lost time. making up for everything i ever did and never did" (adele n.d.) . adele describes seeking a co-writer to help her write what would become "send my love to your new lover" as a quest to have someone bring out of her the song she knew she could sing (npr ) . although written during the aftermath of a love relationship gone bad, this song can also be read to examine other contexts. when read through the lens of stalled institutional conversions to just practices and the inequitable costs involved at the level of individual risk, four practices emerge that i believe could help loosen impediments to practicing the transformative change that theological schools so often profess. in what follows, i will quote a few lines of "send my love to your new lover," followed by a practice these lines suggest and questions they could raise for theological schools invested in transformation. this was all you, none of it me you put your hands on, on my body and told me mmm, told me you were ready for the big one, for the big jump i'd be your last love, everlasting, you and me mmm, that was what you told me untangling initial inclusion from completed conversion process this practice involves the temptation to be lured into believing that conversion can be quick and easy. "conversion," writes rambo, "is very rarely an overnight, all-in-an-instant wholesome transformation that is now and forever" (rambo , p. ) . it is particularly dangerous to think about the start of change as the end of a conversion process. the same institutions that celebrate increasingly diverse students, faculties, staff, and community partnerships can end up being short-term gigs for the most minoritized community members. a celebrated new scholar can arrive, only to leave before long for what is often described in-house as a better opportunity, a better fit, a hundred reasons other than the institution's lack of hospitality to diverse thriving born of structural oppression. there is a difference between admission and retention, outlined poignantly in a forum for theological explorations report that asked theological institutions to consider: "are we measuring what matters? are we asking the right questions? how might we begin to ask questions of our field and institutions that are not only concerned with whether people of color increase numerically, but that also call our attention to whether these growing numbers of students and scholars are thriving vocationally?" (forum for theological explorations ). when schools seek to diversify faculties, expanding who is invited in the door in the first place is a necessary step. however, practices of learning and hospitality also need to pay serious attention to supporting a conversion process that leads to lasting institutional transformation. inclusion is not enough to sustain a more diverse community. understanding an institution's growing pains can be helped by interviewing the people who have left over the past years, listening well and taking seriously what is heard, and being willing to respond in good faith even and especially when the leavings went poorly. i have heard this advice from colleagues in and outside of academia. as adele's lyrics above note, initial inclusion communicates readiness for change but doesn't mean that institutions are ready for ongoing transformation. the above lyrics highlight at least three aspects of conversion as it relates to the burden of individual risk often placed on more minoritized community members in institutions of higher education (muhs et al. ) : ( ) aspirational organizational mission, ( ) responsibility and accountability, and ( ) embodied risks. first, i suggest that "the big one, the big jump" that is "everlasting" is descriptive of an institution's aspirational organizational mission. in hiring processes, search committees often attempt to discern whether a particular candidate is committed to the school's expressed aspirational mission statement. for current faculty, my school ritualizes collective missional recommitment in an annual convocation service in which faculty vow to support the school's aspirational mission, promising to do so with collegiality and integrity. "that was what you told me" can serve as an assessment tool to measure a community's practices in relation to its aspirational mission. the desire for education to be transformative drives mission statements that call on schools to also be constantly transforming, learning, growing, persuaded to the disciplines and accountabilities of lifelong learning. whose fault is it when things go wrong? "this was all you, none of it me" is a difficult charge, painting a stark calculus of responsibility and placing the blame of harm solely on one party. power and privilege can affect possibilities of mutual interactions when assessing desired and demonstrated change and transformation. at the same time, a growing chorus of voices is calling theological schools to dismantle structures of white supremacy that clearly oppress all persons yet can threaten and actually wound more minoritized scholars in acutely embodied ways (e.g., hill fletcher ; james jennings ; sharp ) . who is responsible for the institutional conversion that can and should occur in theological schools committed to lifelong transformational learning? what are the reliable accountability practices when an institution is serious about midwifing change and experiencing conversions in justice and mutual concern? in addition to aspirational organizational mission and an assessment of responsibilities and accountabilities, conversion is also an embodied experience with embodied risks. embodied risks can feel heightened for all in the enticing and terrifying professions of readiness for deep change and transformation, and yet it is simply the case that some bodies bear more risk than others in an institution that carries tenure, rank, salary, and load differences among the employed community and dynamics of grading, vocational discernment, and curricular decisions that determine the boundaries of learning and opportunities for the future between faculty and students. "you put your hands on my body." deep change and transformation of conversion to a more just, equitable, and inclusive community will require paying attention to the embodied risks of a community's made more and kept more minoritized members, putting into place accountable practices that assess and uphold structures of thriving for all persons, and being willing to engage in the difficult yet transformative process of assessing the community's current practices in relation to its aspirational organizational mission statements. truth telling about desires not to convert, or resistances this is a second practice suggested in adele's song lyrics. it is not surprising that an institution with an aspirational, even prophetic, organizational mission falls short in its daily practice. misunderstandings, mistakes, apologies, and wrong turns are all predictable in long processes of conversion that are lasting and transformative. how do people who inhabit institutions remain steadfast in collective commitments, willing to hear and believe what is not working and committed to creating the conditions where all community members can, are expected to, and do thrive? i contend that deep engagement with resistance to change is needed. when i researched empowerment, i quickly realized that recognizing personal and collective investment in disempowerment is a necessary first step to empowerment (sharp ) . change that matters always elicits resistance because it is long and difficult work (pressfield ) . grappling with resistance is important in contexts that can include grueling decision processes where community members who are not thriving due to structural racism and other forms of structural oppression decide to leave in order to survive. rambo notes that conversion requires learning and hospitality. adele goes further. conversion also requires freedom, freedom from oppression and freedom to thrive. institutions more resistant to change constrain avenues of belonging and frustrate the collective labor of teaching and learning that conversion requires. the illusion of change can quickly become more compelling than the long labor of birthing change, including caring for tender newborn practices. across the board, theological institutions across hundreds of association of theological schools member schools desire to be more diverse. hiring pools and invitations of initial inclusion have diversified. yet, retention of historically excluded, more minoritized scholars has not changed much. entire structures must shift along with every practice of the schools. to use a theological metaphor, it's not just pulling up more chairs to an old feast. when the institution and its internal relationships are toxic and wedded to old colonial models based on hierarchies of power, access, and relative worth, the healthiest choice can be to leave or divest and choose to be well, letting go and even forgiving oneself for falling for an institution for falling short ofdesiring to be changed through sustained conversion. i was too strong, you were trembling you couldn't handle the hot heat rising (rising) mmm, baby, i'm still rising i was running, you were walking you couldn't keep up, you were falling down (down) mmm, there's only one way down detangling toe-dipping from being in over our heads this is a third practice suggested by adele's song "send my love to your new lover." once, when i was crossing between two very different contexts and sets of practices, a mentor advised me to remember the feeling of "toe-dipping" because it quickly fades into the illusion of swimming in the deep end when we've not even waded beyond the water's edge. toe-dipping is an embodied experience. as the analogy goes, the water is cold and, for a moment, one's body inhabits the in-between, bordering two realities. it is difficult to sustain toe-dipping and move unless movement is oriented along the water's edge. as heidi park writes, the water's edge is a place of great danger and postcolonial creativity in a world shaped and mis-shaped by borders from shore to wall (park ) . sustaining toe-dipping requires practices of self-awareness, translation, advocacy, and movement across shifting, sometimes militarized ground. sustained toe-dipping, as my mentor advised me, can be an exercise in humility, a posture oriented toward learning and questioning, a place where insight is not blocked by any illusions of having already arrived. yet, staying in toe-dipping unaccompanied by personal, communal, intercultural, and global awareness and learning can lead to a total lack of movement on the one hand or a false narrative of arrival on the other. adele's lines quoted above are illustrative for institutions who desire to be changed while still in early phases of change. the phrase "in over our heads" refers to robert kegan's book of the same title, written around the same time as rambo's text. kegan suggests that the support needed for negotiating the demands of "modern life" have lagged behind the challenges presented by it. "people grow best," kegan writes, "where they continuously experience an ingenious blend of support and challenge" (kegan , p. ) . i can certainly relate to the feeling of overwork, overwhelm, and information overload, and i confront these dynamics in my colleagues, students, and communities. is it possible for institutions of learning to sustain toe-dipping as an irresistible beginning of work instead of a premature conclusion to it? are we really in over our heads in decolonizing and/or resisting change in the institutional life? to what extent? conversion, rambo reminds us, is a long process-the deep end of the work remains on the horizon for many. rambo emphasized "how far someone has to go socially and culturally in order to be considered a convert" (rambo , p. , emphasis in original) . at the beginning of this paper, i made the claim that theological education may be stuck in a long process of conversion (or set of conversions) to decolonized practices of living and learning in communities that support wellness and thriving for the most minoritized community members and by extension for all people. while i join my voices with other theologians calling for such deep change (sharp ) , for the purposes of this paper, artists' definitions of decolonizing conversion processes are illuminating. poet nayyirah waheed puts it this way in her poem "the release": "decolonization / requires /acknowledging / that your / needs and desires / should / never / come at the expense of another's / life energy. / it is being honest / that / you have been spoiled / by a machine / that / is not feeding you freedom / but / feeding / you / the milk of pain ( , p. ). beautifully knitting feeding practices and processes of decolonizing transformation, chef/storyteller/gardener/ land lovers luz calvo and catriona rueda esquibel draw inspiration from indigenous practices to support the theory that decolonization is a multi-layered process of recovery-or set of recoveries. conversion as recovery looks like moving from white supremacies and americanization programs to respect for indigenous knowledge and cultural revitalization, from disavowal to blessing, from thoughtlessness to gratitude, from refined foods to whole foods, from wasteful to resourceful, from advertising and marketing fads to ancestral knowledge and oral tradition, from pesticides and monoculture to permaculture, intercropping, and organically grown, from gmo seeds to heritage seeds, from agribusiness to locally controlled fair trade, from food for profit to food to sustain life, and from assimilation to resistance and resilience ( , p. ) . what could it look like to practice these kinds of multiple conversions within institutional theological education? send my love to your new lover treat her better we've gotta let go of all of our ghosts we both know we ain't kids no more send my love to your new lover treat her better we've gotta let go of all of our ghosts we both know we ain't kids no more . . . (re)engaging a commissioning call and response; (re)committing to converted practices this constitutes a fourth practice suggested by adele's song to support institutional conversion without foisting individual risk inequitably within theological schools. threaded throughout the three verses of this song is the chorus headlined by the song's title. "send my love to your new lover"-the love you had hoped or planned or promised to invest in me but that was not delivered-and deliver it the next time. learn, turn, and give abundantly to the next person; treat them better. adele models this refrain by breaking off a harmful relationship. in her music video for this song, adele recorded versions of singing the song that appear layered on top of each other all at once. the superimposition of multiple images expresses a range of emotions, signaling a complex layering of letting go of the ghosts that haunt relationships and embodying her artistry in a deeper way (bacle ) . extending the above lyrics to the context of institutional change, conversion that lasts recognizes and redresses harmful practices. as a call to conversion, imploring that folks do better next time invites institutions to engage in the work of conversion with accountability to consequences in practice so as not to harm future partners in the work relationship. such commitment could make institutional change irresistible rather than make leaving and stress inevitable. ghosts linger in this process. what relationships with ghosts could best accompany such a shift? what could or would it look like to let go of the stranglehold of ghosts while acknowledging the hauntings that linger as motivations to change? conversion scholars have studied this part of the conversion process. decolonizing conversion into aspirational missions of learning to lead in love for transformation, both possible and precedented, involves "renunciation of cruelty and violence in favor of nonviolence and neighbor love" (rambo and farhadian , p. ). adele sings a simpler yet still difficult song: learn from the past and do better next time. how will we who live and work in institutions know that we are ready to treat each other better? we will know when there are tangible and regular structures of support. when there is a tangible want or need alongside reliable doubt that the current system can support the challenge, transformation is made more possible through accessible and known contextual resources that can support conversion, with barriers to change identified and set apart or satisfied and with change that is implemented (paloutzian , pp. - ) . bringing adele and rambo into generative conversation helps to clarify four practices of conversion for theological institutions who seek and need to change to align institutional practices with aspirational missions. the timing is urgent as schools profess a unified focus on increasing diversity as an institutional priority at the same time that many minoritized scholars still bear greater individual and professional risk in the change process. first, recognize the misunderstanding that the beginning of conversion is not its conclusion. schools can do this by detangling rightly celebrated initial inclusion from the deep practices of learning and hospitality needed for retention and sustained change over time. second, choose to tell the truth about desires not to change, institutional and human resistances that are part of every change process. schools can do this by sharing the responsibility of confronting resistances instead of scapegoating new community members when difficulties arise. third, detangle toe-dipping from being in over one's head. this practice reveals a catch that is part of deep change: forays into real change are embodied experiences and can disorient all community members. however, this initial border-crossing disorientation is not to be confused with swimming capably in the deep end of the work. schools can work on this with dedicated practices of awareness and assessment, often needing sustained outside facilitation. fourth, engage or re-engage a call and response process that recommits to conversion. schools can ritualize ongoing conversions in word and in deed with both public profession and accountability in practice. what could it look like to incorporate ongoing conversion into already existing annual rituals of convocation, commencement, and institutional cycles of self-assessment, performance review, and accreditation? commissioning a reinvestment in conversion is a way for schools to believe in the possibility of conversion even here, even now. these four practices of conversion could help to loosen stuck places in change and share the risk and beauty of change more equitably to support institutional and personal workplace wellness. an enduring question within the study of conversion as change and transformation is the basic question of religious ethics: "what is a human being? is it possible for a human being to change?" (paloutzian , p. ) . i extend this question to ask whether it is possible for a group of human beings to change, why it can be such a difficult and long process, and what is at stake in the process of discerning and engaging this question in practice within institutions of theological education. conversion is a long process with many elements that intertwine individual risk and institutional desire for transformation. as rambo warns, [a]t the same time that a potential convert may be attracted to. .. the new. .., [they] may still be enmeshed in old ways of life. vacillation between two worlds can be very painful. the decision to cross the line into a new life, on the other hand, can be an occasion for tremendous joy, for generating a new feeling of freedom that can itself be a powerful experience confirming the theology being embraced. (rambo , p. ) . as participants in academic pastoral theology and psychology, we are involved in thresholds of life and death, processes of multiple conversions and divestments (streib , p. ). yet institutional conversions are warranted wherever individual risks differ based on intersectional embodied differences such as race, class, ability, gender, citizenship, and more, which are characteristic of so many contemporary institutions that dis/order our lives. harmful institutional structures can themselves become earworms, involuntarily shaping the tunes we hum and believe about how our life together must be organized. adele and rambo can help imagine another way. the stakes are high in terms of living well together. do we want to be well? living a feminist life seeing religious conversion through the arts women in leadership conference presentations adele's send my love to your new lover video: director patrick daughters goes behind the scenes. entertainment weekly on the issue of roles the salt eaters. vintage (contemporaries ed.). new york: vintage books foreword to the first edition when one religion isn't enough: the lives of spiritually fluid people stand your ground: black bodies and the justice of god decolonize your diet take care: notes on the black (academic) women's health forum. the feminist wire crossroads ministry the conversion of hearts and the sin of racism create conditions for scholars of color to thrive the priorities you named the sin of white supremacy: christianity, racism, and religious diversity in america. maryknoll: orbis. hooks, b. ( ). teaching critical thinking the christian imagination: theology and the origins of race in over our heads: the mental demands of modern life adele: 'i can finally reach out a hand to my ex postcolonializing god: an african practical theology contemplation in the midst of chaos: contesting the maceration of the theological teacher presumed incompetent: the intersections of race and class for women in academia conversion psychology of religious conversion and spiritual transformation postcolonializing practical theological methodology as cartography.of boundary dynamics the war of art: break through the blocks and win your inner creative battles the oxford handbook of religious conversion archaeology of memory: personal reflections on de staebler's sculptures understanding religious conversion pastoral care and counseling: redefining the paradigms cover story: adele, queen of hearts dictionary of feminist theologies the psychology of religion, spirituality, and diversity creating resistances: pastoral care in a postcolonial world the living me: unmasking poisonous pedagogies by playing and postcolonializing in pastoral theology decolonizing methodologies: research and indigenous peoples the oxford handbook of religious conversion tiffany, friend of people of color: white investments in antiracism a troubling in my soul: womanist perspectives on evil and suffering the scope of our art: the vocation of the theological teacher educational design: when tweaking the system just won't do. reflective teaching (blog) how do earworms start? classifying the everyday circumstances of involuntary musical imagery (earworms) publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations acknowledgments i am grateful for and acknowledge the support i received for this paper through conversation with new directions in pastoral theology conference participants, conference respondent kelly bulkeley, lewis rambo, faculty colleagues at columbia theological seminary, and writing group partners. key: cord- -a wl f authors: lawler, j. j.; spencer, b.; olden, j. d.; kim, s.-h.; lowe, c.; bolton, s.; beamon, b. m.; thompson, l.; voss, j. g. title: . mitigation and adaptation strategies to reduce climate vulnerabilities and maintain ecosystem services date: - - journal: climate vulnerability doi: . /b - - - - . - sha: doc_id: cord_uid: a wl f abstract increasing temperatures and altered precipitation regimes associated with human-caused changes in the earth s climate are having substantial impacts on ecological systems and human well-being. maintaining functioning ecosystems, the provision of ecosystem services, and healthy human populations into the future will require integrating adaptation and mitigation strategies. adaptation strategies are actions that help human and natural systems accommodate changes. mitigation strategies are actions that reduce anthropogenic influences on climate. here, we provide an overview of what will likely be some of the most effective and most important mitigation and adaptation strategies for addressing climate change. in addition to describing the ways in which these strategies can address impacts to natural and human systems, we discuss the social considerations that we believe must be incorporated into the development and application of mitigation or adaptation strategies to address political situations, cultural differences, and economic limitations. adaptation adjustment in natural or human systems in response to actual or expected climatic stimuli or their effects, which moderates harm or exploits beneficial opportunities. various types of adaptation can be distinguished, including anticipatory and reactive adaptation, private and public adaptation, and autonomous and planned adaptation (ipcc ) . climate change a significant and lasting change in the statistical distribution of weather patterns over periods ranging from decades to millions of years. we focus largely on changes in temperature and precipitation, as they are changing in many regions and have direct impacts on ecosystems and human well-being. mitigation actions that reduce the effects of humans on the climate. resilience amount of change a system can undergo without changing state (ipcc ) . sensitivity it is the degree to which a system is affected, either adversely or beneficially, by climate-related stimuli. the effect may be direct (e.g., a change in crop yield in response to a change in the mean, range, or variability of temperature) or indirect (e.g., damages caused by an increase in the frequency of coastal flooding due to sealevel rise) (ipcc ) . vulnerability the degree to which a system is susceptible to, or unable to cope with, adverse effects of climate change, including climate variability and extremes. vulnerability is a function of the character, magnitude, and rate of climate variation to which a system is exposed, its sensitivity, and its adaptive capacity (ipcc ) . virtually all regions of the earth are experiencing rapid changes in temperature, precipitation, and/or other aspects of climate. human-caused changes in the earth's climate are greatly altering ecological systems, with consequences for human well-being. for example, recent studies have documented shifts in the distribution of plants and animals, advances in the timing of key ecological processes, and extinctions of wildlife populations and species that are likely linked to recent increases in temperature (parmesan ) . such changes are creating regional combinations of environmental conditions that, within the next - years, may have no current-day analog. the resulting changes in ecosystem function will undoubtedly affect the provision of ecosystem services for human communities. maintaining or increasing ecosystem services into the future will require integrating adaptation strategies (actions that help human and natural systems accommodate changes) and mitigation strategies (actions that reduce anthropogenic influences on climate) ( figure ). mitigation strategies are those that reduce the magnitude of climate change that will occur. the vast majority of these strategies involve either reducing greenhouse gas (ghg) emissions or sequestering carbon, although they can address any human climate forcing. by contrast, adaptation can be defined as alterations to human or natural systems that are designed to reduce the negative impacts ofor exploit the opportunities created byclimate change (ipcc a) . adaptation strategies are much more varied than mitigation strategies, as they are more often specific to particular systems or locations. mitigation and adaptation strategies designed to address changes in climate may have additional positive or negative effects on ecosystem functioning, ecosystem services, and human well-being. for example, replacing fossil fuels with renewable energy sources reduces ghg emissions while simultaneously reducing air pollution, acid rain, and smog, as well as asthma and respiratory ailments of humans. conversely, coastal armoring, the building of seawalls, breakwaters, and jetties, may reduce damage to built structures from the combined effects of sea-level rise and storm surges but such actions can negatively affect coastal ecosystems. in this chapter, we provide an overview of what will likely be some of the most effective and most important mitigation and adaptation strategies for addressing changes to the climate system stemming from increased ghg emissions. we attempt to cover the entire spectrum of life, from plants and animals to humans, as well as the geophysical properties of our planet that support these organisms. in relation to human populations, we focus primarily on human health as an index of well-being. we also introduce concepts of climate justice. many of our examples are taken from the united states, although we also include other regions of the world in our analysis as appropriate. we discuss various ways in which mitigation and adaptation strategies can help reduce the magnitude and the impacts of the changes we are likely to experience, as well as improve human health and directly or indirectly affect ecosystem functions and services. finally, although our analysis sets priorities and orders of importance for an ideal world unimpeded by political considerations, cultural differences, or economic limitations, we include a discussion of the social considerations that we believe must be incorporated into the development and application of mitigation or adaptation strategies. in the following sections, we briefly discuss what will likely be some of the most effective mitigation strategies for addressing climate change, largely focusing on strategies to reduce co and other ghg emissions ( table ) . we have grouped these strategies into five categories: transportation, shelter, food, energy, and carbon storage and bioengineering. although we focus on mitigation strategies that will reduce the magnitude of future climatic changes, we also discuss how these strategies can improve ecosystem functioning and, in some cases, human health. transportation, the movement of people and goods, significantly impacts climate through the emission of ghgs. in , transportation accounted for % of the world's energy-related ghg emissions and road transportation represented % of the total carbon dioxide (co ) emissions from transportation (ipcc b) . in addition to being a major source of emissions, transportation also significantly affects ecosystem functions and services. pollution from vehicles affects ecosystem services such as clean air and water directly, and indirectly through impacts on the plants and animals that regulate ecosystem function. as species are lost from or move into ecosystems the functioning of those systems can change, resulting in altered ecosystem services. human health is likewise affected by pollution from vehicles (e.g., through chronic lung disease). transportation networks and moving vehicles also fragment landscapes, reducing the flows of energy and biota resulting in isolated populations and potentially making these populations vulnerable to extirpation and impairing ecosystem functions. mitigation strategies that address transportation can be placed into three basic categories: those that increase the efficiency of particular transportation approaches, those that result in a shift in the mode of transportation, and those that reduce the amount of transportation that is needed. here, we briefly table examples of mitigation strategies aimed at reducing ghg emissions or, in the case of the last category, sequestering carbon to reduce co concentrations in the atmosphere mitigation strategies barriers to implementation a large reduction in ghg emissions can be accomplished by increasing the fuel efficiency of vehicles. for example, increasing the efficiency of light-duty vehicles (cars and small trucks) has the potential to reduce their carbon emissions by up to % by (ipcc b) . such efficiencies can be attained by a combination of reducing vehicle weights, increasing aerodynamics, increasing engine efficiency (particularly diesel engine efficiency), and increasing the use of biofuels and electric and hybrid vehicles. in , the international energy association projected that the proportion of transportation energy supplied by biofuels could be increased to % (iea a) . although there is good evidence of successful biofuel markets and programs (e.g., ethanol from sugarcane in brazil, goldemberg ) , there are also negative consequences of increased biofuel production for biodiversity and carbon storage (fargione et al. ; fletcher et al. ) . one opportunity for transportation mitigation strategy is the promotion of behavioral changes of drivers. efficiency of road vehicles could be improved by - % by changes in driving styles, improved maintenance, more efficient tires, reduced idling, and traffic design and maintenance (ipcc b). a second approach to reducing emissions and many of the other effects of transportation involves changing the ways in which people and goods are moved. one such approach is to increase the use of public and human-powered transportation. increasing the amount of walking, biking, and use of public transit will depend largely on the available infrastructure and the capacity to provide it. supporting the development of 'walkable' cities and neighborhoods, bike-friendly roadways and bike paths, carpooling and car-sharing programs, and efficient and safe mass transit are all approaches for shifting the mode of urban transport. present transportation alternatives vary by country, with europe, japan, and many developing countries having the highest levels of public transportation use, and the united states having the greatest use of personal automobiles. in some developing countries, such as vietnam or china, personal automobile use is a recent phenomenon, and personal motorized transport is a high aspiration because it conveys increased social status. this disparity in the historical development of the transportation sector will mean that mitigation strategies, barriers, and solutions will have to be location specific and may be dynamic over time. in addition to the reduction in emissions and the other environmental benefits of shifts to more human-powered transportation, such changes also have clear human health benefits resulting from increased exercise, weight management, and decreases in air pollution (nemet et al. ) . simultaneously improving the quality of public transport and leapfrogging transport-intensive stages of economic growth is needed in many countries to increase higher levels of mass transit use and human-powered transportation (wright and fulton ) . further reductions in emissions can be made by changing modes of transport for other transportation such as moving more freight by train and ship and less by truck. the third way to reduce transportation-related ghg emissions is to reduce the amount of transportation that is needed. decreasing the demand for transportation largely involves changes in human behavior, urban and landscape planning, and changes in technology. sprawling, low-density urban neighborhoods, such as those found in the united states, perpetuate automobile dependency and transform ecologically functional landscapes into subdivisions. compact, highdensity communities with local urban centers allow residents to work and live in close proximity, and help build social capital among residents and protect natural areas. in conjunction with human-centered transportation infrastructure such as sidewalks, bike lanes, and public transportation, compact neighborhoods reduce car dependency while simultaneously improving the physical fitness of residents. public green space and urban forests sequester carbon while improving air quality and mental well-being. increased vegetation, shade trees, and less asphalt combat urban heat-island effects and reduce energy demand for cooling buildings. advances in communication technologies also have the potential to reduce the need for transportation. the internet, online meeting, document sharing, and conference tools have led to an increase in telecommuting and web-based meetings. similarly, online meetings have begun to replace some inperson meetings and have the potential to dramatically reduce air travel, which can account for a significant portion of an individual's carbon footprint (fox et al. ). the most significant barriers to implementing mitigation strategies in the transportation sector are long-established personal transportation behaviors and the lack of government interest or capacity to invest in new technologies. changing the way and the amount that people move around will be very difficult. in the united states and elsewhere, people's relationships with the automobile involve issues of convenience, independence, safety, and social status. changing these relationships will involve making forms of public transportation more convenient, more effective, and more appealing. rising fuel costs and commute times may provide some opportunity for implementing transportation-based mitigation strategies. failing automobile industries and economic crises may provide another. similar to the projects undertaken by the civilian conservation corps in the united states in the depression of the s, modern-day work relief programs could be used to transform transportation systems. short of such crises, major changes in transportation will take a combination of regulatory action, education, and incentive programs. housing is one of the most idiosyncratic and regionally variable aspects of human culture. the design of structures is dependent on local climate, land access, available materials, and regional architectural histories. much of the recent human-caused change in climate stems, in large part, from the way we design, construct, and inhabit the built environment. the following section focuses on a representative selection of design strategies that mitigate the built environment's contribution to carbon emissions and, in many cases, provide cobenefits to human health and local/regional ecological resilience. building material extraction can have profound impacts on ecosystems and ecosystem services. mining degrades natural landscapes, destroys habitat, pollutes freshwater environments, and can expose human populations to toxins. poorly managed lumber extraction leads to deforestation, eliminates suitable habitats, erodes soils, and diminishes the earth's carbon sequestration capacity. mines that observe best management practices such as land reclamation have fewer negative impacts on climate, local ecosystems, and human health. sustainably harvested lumber can act as a long-term carbon sink and contribute to the productive and ecological resilience of forests. rapidly renewable lumber alternatives such as bamboo can offset the demand for slow-growing trees species, rebuild soils, and set the stage for reforestation. however, as with any renewable resource, expanding the production of bamboo in monocultures has the potential to eliminate and/or degrade existing ecosystems. eight percent of annual carbon emissions worldwide can be attributed to building material extraction, production, transportation, and installation (mazria and kerschner ) . choosing building materials with low embodied energy (the cumulative energy of extraction, production, transport, installation, and disposal) and/or that use alternative energy sources reduces the building sector's carbon footprint. locally sourced materials produce fewer carbon emissions than those transported long distances and both durable materials and those with recycled content help reduce demand for virgin materials. fully recyclable materials can be reprocessed and repurposed with low energy inputs indefinitely. removing toxic compounds from material production processes and building products facilitates the recycling or biodegradation of building components. it also helps prevent air and waterborne industrial pollution and associated human illness. in some cases, it may be possible to choose building materials that actually sequester more carbon than is emitted in their production. the production of portland cement, the most commonly used cement in construction, is a relatively large source of co emissions. magnesium oxide-based cement is as strong as normal portland cement, but can be manufactured at low temperatures using renewable energy sources. magnesium oxide is an abundant mineral and is easily recyclable (smith ) . as it cures, it absorbs more co than is released during its production, resulting in a net negative carbon budget. the average lifespan of buildings in the united states is less than years (o'connor ) . design for adaptationdesign that easily accommodates changes in configuration and useextends the life of buildings and stems demand for new construction and virgin materials. when buildings are ultimately decommissioned, design for disassembly facilitates the breakdown and reuse of their constituent components. both design for adaptation and design for disassembly act upstream from recycling as part of a multicyclic system that repositions well-weathered buildings and building materials as resources rather than wastes, reducing carbon emissions in the process. building operations such as heating, cooling, and lighting account for % of electricity consumption in the united states (mazria and kerschner ) . passive design strategies can drastically reduce a building's dependence on carbon-intensive energy production. in cold climates, airtight, well-insulated building envelopes can work in tandem with building orientation, thermal mass, and passive solar radiation to provide thermal comfort and reduce heating bills. in hot climates, external shading, light colored/reflective roofs and walls, and passive ventilation strategies can reduce cooling loads. daylighting strategies such as light shelves and shallow floor plans bring natural light into interior spaces, reducing the demand for electrical lighting while, at the same time, contributing to the well-being and productivity of building occupants. similar to the use of building materials to sequester more carbon than they produce, buildings can generate more energy than they consume. buildings equipped with photovoltaics, wind turbines, and fuel cells are some of the energy producing technologies that make this possible. recent advances in computer-aided design software help designers address building performance issues early in the design process and optimize building design for energy use and production. improvements in digital monitoring and analysis allow occupants to scrutinize building performance and optimize energy use once buildings are operational. in the future, smart buildings and systems will likely respond dynamically to human occupation, weather, daily cycles, and seasonal change, further reducing their energy footprint. compact communities (discussed in the transportation section, above), land-use planning, and high-density zoning have the potential to reduce co emissions, reduce sensible and latent heat fluxes, and improve human health and ecosystem function. centralized infrastructure and zoning laws that segregate industrial, commercial, agricultural, and residential land use can be beneficial to public health within localities. however, these practices also create a cognitive disconnect between human action and its environmental consequences. they facilitate the transfer of local environmental burdens to regional and global scales, contribute to climate change, and undermine the health of vulnerable populations. integrated land use and responsibly managed distributed ecological infrastructure have the potential to safeguard public health without the transference of environmental burdens. synthesizing commercial, industrial, agricultural, and residential precincts, reimagining landscape as infrastructure, and bringing utilities into the public realm raises awareness about the causal relationships that link human activities, environmental degradation, and human health. urban agriculture, for example, reduces dependence on carbonintensive industrial food production, minimizes the carbon footprint of food transport, teaches urbanites how to grow their own food, and provides nutrition to communities. carbon-conscious materials, regenerative buildings, compact communities, and integrated landscapes have the potential to mitigate the worst effects of climate change. their success will depend on the ability to implement them not only in developed countries but also in the burgeoning cites of the global south. we must adapt these strategies to diverse climates, contexts, and cultures and pursue them in tandem with issues of economic development and social equity. in modern history, land distribution and regulation have always been the prerogative of governments. for this reason, national, regional, and local governments have a large role to play in housing mitigation strategies. governments control land-use planning, zoning, and building-code regulations. the ability to act on the mitigation strategies identified above, therefore, is largely dependent upon the regulatory and enforcement environment in a region. in a democratic society, these will in turn be dependent upon the inclination and ability of the state to assume progressive and proactive attitudes toward mitigation. agriculture releases considerable amounts of ghgs into the atmosphere. in , it accounted for - % of the total global anthropogenic ghgs including approximately % of nitrous oxide (n o) and % of methane (ch ) of the anthropogenic totals (rosenzweig and tubiello ; smith et al. ; burney et al. ) . land-use change, nitrogenous fertilizer applications, livestock production, rice farming, and biomass burning are among the major direct sources of ghg emissions. indirect sources of ghgs from agriculture include the production and/or applications of fertilizers and pesticides, the operation of farm machinery, and the transportation of agricultural products (burney et al. ). there are multiple opportunities for mitigating climate change impacts through modifications in agricultural practices and food consumption. with respect to agricultural production, mitigation strategies include soil carbon sequestration and the reduction of ghg emissions through altered production methods. with respect to food consumption, society can mitigate the impacts of climate change through changes in diet and by reducing the amount of transportation needed to deliver food (e.g., increasing local food production). below, we discuss several of these mitigation strategies. the topic of increasing local food production is covered in previous sections on transportation and shelter. unlike housing, food is a sector that is potentially much more amenable to individual decision-making and personal choice. this will depend on the relative proportion of food in a household budget. while americans spend only % of their incomes on average on food, the figure for indonesia is %. this indicates that people from wealthy countries would have greater flexibility to support alternative and nonindustrial agriculture through consumer preferences than those from countries where food is a larger part of household budgets. agricultural and degraded soils can act as large carbon sinks with the potential to sequester - gt of carbon globally (lal ) . soil organic carbon (soc) is the major component ( %) of this large soil carbon pool (lal ) . soc accumulation is facilitated by adding biomass to the soil and minimizing soil disturbances. the increased soc improves the physical, chemical, and biological properties of the soil and in turn enhances crop productivity, water holding capacity, and land sustainability under a low-input cropping system. several agricultural practices facilitate accumulation and retention of soc in cropland soils, e.g., conservation tillage, mulches, cover crops, manuring, crop rotations, and agroforestry (lal ) . the benefits of these agricultural practices go beyond carbon sequestration. they have the potential to reduce erosion, limit positive feedbacks in drought cycles, and benefit human health. over the past years, % of the world arable land has been taken out of production as a result of erosion (pimentel ) . conventional agricultural practices involving frequent tillage result in soil erosion, which in turn increases concentrations of airborne particulates. these particles can carry pathogens causing infectious diseases and can provide positive feedbacks to drought conditions. recent climate modeling has demonstrated a positive feedback in the 'dust bowl' drought of the s in the united states (schubert et al. ; cook et al. ). cook et al. ( ) discovered that a reduction in vegetation cover and the addition of eroded soil dusts to the air resulted in a high temperature anomaly over the northern united states and intensified the drought. as a result of the dust bowl, more than % of the surveyed farms comprising . million acres were seriously eroded, and the economic loss was valued close to us$ million (aaas ) . a combination of warming, drought, and conventional agricultural practices together have the potential to re-create the dust bowl of the s in the great plains of the united states (rosenzweig and hillel ) . the livestock industry is a main contributor to three major climate-related problems associated with global food systems: ghg emissions, reactive nitrogen mobilization, and plant biomass appropriation (pelletier and tyedmers ) . increases in livestock production and meat consumption are also blamed for the evolution of new and virulent emergent diseases. any improvement in the environmental sustainability of livestock production will concurrently have a benefit for human health. pelletier and tyedmers ( ) estimated that globally the livestock sector contributed % of anthropogenic ghg emissions, % of reactive nitrogen mobilization, and consumed % of human-appropriated biomass as of . reduction in ghg emissions in agriculture can be achieved by developing strategies involving improved production efficiency, land-use changes, cropland and rangeland management, and livestock and manure management (rosenzweig and tubiello ; smith et al. ; burney et al. ; pelletier and tyedmers ) . for example, enhancing production efficiency through agricultural intensification has been credited with the avoidance of gt of carbon emission globally since (burney et al. ). this figure highlights that the investment in agricultural research to improve the environmental sustainability of global food production systems (i.e., livestock, crop, and aquatic systems) should be a prioritized mitigation strategy (lobell et al. ; burney et al. ; steinfeld and gerber ) . another mitigation strategy to reduce emissions involves shifting livestock production regimes from ruminants to more efficient and lower impact monogastric species (e.g., poultry), well-managed fisheries and aquaculture, and promoting plant-based protein sources (pelletier and tyedmers ; steinfeld and gerber ) . changing livestock production from ruminants to monogastric animals and reducing livestock production overall both require significant changes in diet in many parts of the world. this is, of course, where tradition and culture play an important role, yet it is not impossible to induce dietary changes. initiatives that reduce the consumption of meat could have dramatic effects on ghg emissions particularly in developed countries (mcmichael et al. ) . such policies would also reduce impacts on ecosystems and promote other ecosystem services such as a reduction in the clearing of amazonian rain forests, reduced nitrogen deposition, and a reduction in the application of high-phosphate fertilizers used to grow feed. reduced meat consumption could also improve human health in developed countries particularly when coupled with incentive structures and educational measures to replace high-fat, sugar-rich foods with more complex diets based on plant proteins. reduced red meat consumption will likely lower the risk of obesity, diabetes, and several types of cancer (and specifically colorectal cancer), and may reduce the risk of ischemic heart disease (mcmichael et al. ) . a shift from meat-based proteins to plant-based proteins has the potential to substantially increase the total food calories available for consumption worldwide. worldwide educational efforts, altered trade policies, shifts in farm subsidies, public health campaigns and new environmental stewardship programs will be needed to achieve a fundamental change in the consumption of meat to achieve large-scale cobenefits for the environment and human health. energy consumption and production affect carbon emissions, which in turn affect human health and ecosystem functioning and services. these effects can be divided into the influence of energy consumption and production. while human well-being is in many ways positively affected by the increased availability of energy, there are also negative health effects resulting from the energy consumption and production. below, we describe potential mitigation strategies for the energy sector. the us energy information administration reports on energy consumption in the united states in four sectors: residential, commercial, transportation, and industrial. in , consumption across the four sectors accounted for , , , and % of overall energy consumption, respectively (us energy information administration ). overall energy consumption, as well as co emissions from energy consumption, continues to rise. total us co emissions from energy consumption were million metric tons of carbon in and million metric tons in (research and innovation technology administration ). reducing ghg emissions through declining energy consumption can be accomplished through a combination of technological advances and changes in human behavior (including the adoption of new or alternative technologies). several mitigation strategies for addressing energy consumption have been provided in the previous sections on transportation and shelter. in addition to those strategies, increasing the efficiency of appliances and lighting is likely to provide significant reductions in ghg emissions. the most efficient appliances in use today use half to one-fifth of their most inefficient counterparts currently in use (ipcc b) . in developed countries, substituting smaller and more efficient refrigerators, washing machines, and other appliances for less efficient ones can substantially reduce energy use. in addition, in developed countries, improvements in the efficiency of electronic devices with inefficient standby modes and power supplies (or more simply unplugging these devices when not in use) can significantly reduce energy consumption. in developing countries where biomass (e.g., wood, dung, and charcoal) is used for cooking fuel, substantial reductions in ghg emissions and improvements in human health may be attained by increasing the efficiency of biomass stoves, fostering a shift to cleaner burning liquid and gaseous fuels, and improving access to electricity (ipcc b). significant reductions in emissions can also be made through the use of more efficient lighting. the emissions from electric lighting worldwide are equivalent to roughly % of those generated by light passenger vehicles (iea b). substituting more efficient lighting technology (e.g., compact fluorescent bulbs and occupancy detectors) can reduce residential energy use by a factor of four or five (ipcc b). programs designed to increase the energy efficiency of appliances, vehicles, and lighting (e.g., the energy star and vehicle fuel economy (cafÉ)) also have the potential to reduce ghg emissions. although such programs have not decreased energy use in the united states, they may be partially responsible for halting the growth of per capita energy consumption (us energy information administration ). methods for reducing the impacts of energy production on atmospheric co concentrations, human health, and the environment focus primarily on improvements in an increased generation of renewable energy. renewable energy sources include those based on waves and tides, biofuels, solar, wind, and hydropower. a shift to renewable energy sources has great potential to reduce ghg emissions, reduce impacts on ecosystems and ecosystem services, and improve human health. nonetheless, the production of renewable energy will have adverse effects on some ecosystems. below, we discuss some of the benefits and potential side effects of some sources of renewable energy. in regions of large tides and tidal currents, such as the united kingdom, tidal energy could supply as much as % of the country's energy supply (blunden and bahaj ) . two types of systems are in use or are proposed, the tidal barrage and tidal turbines. tidal barrages allow water to flow into a bay or estuary during flood tide, then they release the water back during ebb tide. as water is released it flows through turbines generating energy. the impact of barrages on fish may be substantial (aprahamian et al. ) . power can also be extracted in the marine environment by turbines in regions of high wave and current energy. however, the ecological impact of the extraction of energy in highly energetic environments is not clear (shields et al. ) . the effects of such installations include alteration of benthic (ocean bottom) environments through changes in sediment transport and detrimental impacts on intertidal species through altered currents and nutrient flows. biofuels (discussed briefly in section . . . transportation) include corn-and sugarcane-based ethanol, woodbased methanol, biodiesel from soy, rapeseed oil, and switchgrass. biomass is also used in cofiring (combining with coal or other fossil fuels in the burning process) or burned alone as a fuel. crops can be grown explicitly for biofuels, or residues from farming or forestry can be used as biofuels. biofuels can also be converted into gasses and then used to fuel gas engines. nonetheless, many biofuels require significant amounts of land and water to grow them, which brings fuel production into conflict with food production as well as potentially result in a climate effect from the alteration in the surface fluxes of heat and moisture into the atmosphere. in addition, fuel plants may not be as effective as others at absorbing ghgs, and more natural lands converted to agriculture for biofuels will result in increased erosion, decreased water quality, and a loss of wildlife habitat and biodiversity. solar energy can be used to generate electricity, heat water, and heat buildings and can be a highly effective form of renewable energy in some regions. although photovoltaic power generation only accounted for . % of world power production as of , expansion is occurring at roughly % per year, largely in developing countries (ipcc b) . hot water production is the second single largest use of energy in residences in both the united states and china. solar water heating is a cost-effective alternative to other fuel sources in many locations. similarly, passive solar heating of buildings (mentioned in the section . . . shelter) is an effective heating measure in many regions. finally, solar energy can be used to generate power with solar thermal electric plants that focus solar rays to heat a liquid, which is then used to generate electricity. passive solar space heating and hot water production likely have the lowest environmental costs. decentralized photovoltaic use on residences and buildings incurs environmental costs in terms of the production process, but likely have few other negative effects. conversely, photovoltaic fields and solar thermal electric plants can have large footprints, adversely affecting plant and animal habitat. like solar power, wind-generated power is more effective in some areas than others. wind produced . % of global electricity in but has been growing at a rate of approximately % per year since (ipcc b ). there are potential adverse effects on birds, through collision, displacement due to disturbance, barrier effects, and habitat loss, with more research needed in all areas (drewitt and langston ; fox et al. ). in addition, the impact of wind turbines on views is perceived to be a substantial barrier to implementation in some communities (wolsink ) . as of , hydropower accounted for % of global electricity (ipcc b) . together, large hydropower plants and small and micro hydropower systems can supply power to large municipalities and isolated rural communities with relatively few emissions. hydropower is not carbon neutral, however, and some studies have estimated significant emissions from hydropower reservoirs (ipcc b) . in addition, dams and reservoirs fragment river networks, alter natural flows of water and sediments, and change stream temperatures. these effects are discussed in section . . . human-dominated systems. there are several other major renewable energy sources including nuclear, hydrogen, and geothermal. as with the renewable sources described above, there are both benefits and costs to using these other renewable energies. most of the mitigation strategies discussed above are focused on reducing ghg emissions. additional strategies can be used to increase carbon sequestration, removing co from the atmosphere. below, we discuss a few strategies that have been proposed for increasing carbon sequestration and storage as well as much riskier geoengineering proposals. deforestation is one of the largest sources of emission accounting for over gt co per year in (stern ) . although there are many other uses of natural resources that result in carbon emissions, here we focus on mitigation strategies that focus on deforestation. given the slow rate at which trees absorb carbon dioxide, simply planting new trees in place of those removed does not immediately offset the removal of a tree. a more rapid approach to mitigation is to reduce deforestation. unlike the majority of emissions that emanate from industrialized countries, most emissions from deforestation are generated by developing countries. deforestation also itself alters the climate system (pielke et al. ). reduced emissions from deforestation and forest degradation (reddþ) is a program of the united nations that seeks to mitigate climate change by providing incentives to conserve forest cover. given recognized challenges in redd (dickson et al. )including country by country differences in who owns the forest, who should receive the monetary benefits of preserving the forest, and the reliance of various indigenous groups on forests for livelihood and cultural identitythe program was modified to reddþ in to minimize negative consequences and respect the rights of indigenous peoples and members of local communities (kanninen et al. ) . assumed cobenefits include the suite of ecosystem services such as watershed protection; reduction of soil erosion; flood control; provision of fuel, timber, and foods; and cultural and spiritual amenities for local communities. biodiversity is presumably also enhanced by protecting natural systems. it is possible that forest-dependent communities will be able to maintain access to forests through this initiative. potential coharms are increased demand for conversion of other ecosystems if forests are protected, replacement of mixed stands with plantations, and benefits accruing to those not responsible for care and maintenance of the forests. prevention of deforestation mitigates atmospheric co concentrations by preventing the release of stored carbon that occurs when forests are harvested and protecting forest stands that absorb carbon, as well as limits changes in surface energy and water fluxes. as of , south america and africa continue to have the largest net loss of forests and large decreases in carbon storage (fao ) . although tropical forests have been the focus of early redd efforts, recent evidence indicates that a large amount of carbon can be stored in mangrove forests (donato et al. ) , peatlands (dise ) , and boreal forests (carlson et al. ) , making these potential targets for conservation efforts. urban forests have also been largely neglected as a potential carbon sink. escobedo et al. ( ) review the services and disservices of urban forests combined with human health and pollution mitigation as well as carbon sequestration. in some locations, urban forests are on par with other techniques for carbon sequestration and reduction. in addition to capturing carbon in plants, it may be possible to increase carbon storage in the water and in the earth's crust with various methods. these proposed approaches are still on the scientific fringe and considered to be highly unproven and/or potentially quite risky. one such approach is to capture carbon in the oceans through iron fertilization. in regions of the ocean where there are ample nutrients to drive primary production (photosynthesis) but observations show that chlorophyll is relatively low, the lack of iron is thought to limit the productivity. over the last two decades, several experiments have attempted to determine whether the addition of elemental iron into the water could cause an increase in algal production and ultimately, as photosynthesis fixes carbon into organic molecules that then sink, the sequestration of carbon in deep ocean sediments. however, the results of these experiments are mixed (de baar et al. ) , with the export of carbon being difficult to assess and likely quite modest. at the same time, the impacts on local ecosystems and thus ultimately fisheries in potentially highly productive regions of the subpolar oceans are not yet known. other carbon sequestration approaches have also been proposed including pumping air down into the oceans, injecting carbon into the ground, and incorporating biochar into soils. given the magnitude of projected changes in climate and the difficulty in implementing meaningful mitigation measures, some scientists have argued for large-scale efforts to reduce the amount of incoming solar radiation. these approaches are collectively referred to as geoengineering. at present, these approaches are unproven, risky, and, at best, will only address some of the impacts of the human influence on climate. one such geoengineering approach would inject sulfate aerosols into the stratosphere. these aerosols would reflect incoming solar rays and reduce warming. although such an approach might reduce average global temperatures, it would also likely decrease average global precipitation (bala et al. ) as well as alter atmospheric and ocean circulation patterns. for a given change in radiative forcing, solar forcing is more effective at changing the hydrological cycle than forcing from carbon dioxide owing to differences in the surface latent heat (and thus evaporation) response. while the impact of carbon dioxide fertilization could increase net primary production in terrestrial ecosystems (govindasamy et al. ) , ecosystems could continue to be vulnerable due to changes in water resources. in addition, solar radiation management would not address the potentially significant problem of ocean acidification to marine ecosystems. the majority of both natural and human systems will have to adapt to climate change in some form or the other. a broad array of adaptation strategies has been proposed for reducing the impacts of climate change in both more natural systems and human communities (ipcc a; heller and zavaleta ) ( table ) . many of these strategies are either general concepts or strategies that apply to most ecosystems and regions. we begin by describing general concepts and strategies. we then describe some of the strategies that have been proposed for four broad types of ecosystems including freshwater, terrestrial, coastal, and human-dominated systems. for the freshwater, terrestrial, and coastal systems, we focus on strategies that would lessen negative impacts of changing climate on ecosystem functioning and ecosystem services. for the human-dominated systems, we discuss the ways that some of the most often discussed strategies for climate adaptation in these systems will impact (either negatively or positively) ecosystem functioning and the provision of ecosystem services in these and in surrounding areas. developing adaptation strategies to address future shifts in climate will require expanding the temporal and spatial scales of plans, policies, and management (kareiva et al. ; lawler three-year, -year, and even -year plans will fail to address many climate impacts. future planning will need to address both short-term and long-term changes in systems. to address long-term changes, plans and policies will need to look at least years into the future and will be more useful if they look ahead or more years for many ecosystems and ecosystem services. it will often also be necessary to expand the spatial scale at which systems are managed and policies and plans are developed. as species move and ecosystems change, it will be necessary to think well beyond neighborhood, park, refuge, state, or even national boundaries. planning will need to occur at multiple, integrated scales and will need to involve local, regional, and sometimes national participants (kareiva et al. ) . while expanding the temporal and spatial scale of climate adaptation strategies, it will be necessary to keep in view local histories, politics, and economies. scale and locality also play a formative role for issues of 'environmental' or 'climate' justice. environmental justice describes the tendency for poor communities to be more vulnerable to environmental degradation. poor communities tend to bear not only the unequal burden of polluting, destructive, and destabilizing industries but also a disproportionate share of the blame for environmental destruction and responsibility for ameliorating environmental change. for example, polluting industries are often located in poor communities due to cheap land values and the inability of poor communities to politically resist their presence, or a desire to develop jobs at any cost. for instance, swidden agriculturalists in southeast asia have been blamed for forest fires despite satellite data pinning large fires to governmentsponsored oil palm plantations (harwell ) and southeast asian fishers living near toxic riparian runoff from mining have been blamed for polluting local waters by cyanide fishing (lowe ) . the broad spatial scales that are required to address the effects of a changing climate necessitate cooperation and coordination among managers, planners, and policy makers from different jurisdictions, agencies, and management units (kareiva et al. ) . they require agencies with diverse mandates and cultures to work together in ways that they have not done in the past. as an example, the us climate change science program is a federal organization that reaches across federal agencies. the broad scale of climate impacts also requires cooperation across sectors of society and different types of organizations including nongovernmental organizations, local and regional governments, citizen groups, and industry. the us fish and wildlife service's landscape conservation cooperatives are an example of a system designed to bring together diverse groups of scientists, stakeholders, policy makers, and managers to, in part, address climate change. in addition, as emissions from one locale affect the climate in other locales, addressing the global nature of climate change will involve cooperation at an international level (hannah ) . although, as the above examples demonstrate, some regional, national, and international efforts have been put in place to foster collaboration, more such efforts will need to be made to adequately address climate change. in addition to cooperation among scientific and political leaders, local and regional stakeholders need to work together as a unified force for social and environmental change. representatives from the global north need to appreciate perspectives emanating from the global south and vice versa. climate adaptation can be brought into many other environmental and social justice agendas among community advocacy groups and should not remain the purview of the community of knowledge leaders with scientific expertise. there are three fundamental approaches to managing systems in a changing climatepromoting resistance, resilience, and transition. resistance-based adaptation strategies focus on maintaining the current state of a system in the face of climate change or climate-driven changes in the environment. building a seawall to prevent erosion as sea levels rise and storm surges intensify is an example of a resistance-based adaptation strategy. resilience can be defined as the ability of a species or system to return to its current state following a perturbation (e.g., holling ) . resilient systems will be able to maintain ecosystem functions and processes and avoid a transition to a new state as climates change. most of the discussion of climate adaptation to date has focused on managing ecosystems for resistance or resilience. most of the general adaptation strategies discussed below and the more specific strategies discussed for each of the four types of ecosystems are intended to increase resilience. the magnitude and rate of projected climatic changes over the coming century will likely make both resistance-and resilience-based strategies insufficient or ineffective in the long run. managing many systems will require promoting change to a new state. strategies for promoting change in human-dominated systems may include planting new crops, redesigning road networks, and relocating people. strategies for fostering change in more natural systems include assisted colonization of threatened plants and animals (see below) and shifting management efforts from one species to another. the outcomes of these more aggressive and forward-looking strategies will be more uncertain, and their implementation will be more controversial. change is especially hard in social contexts where it is uncomfortable, disadvantageous, expensive, or where the old system is remunerative or beneficial to elites, decision makers, or other small influential groups. adaptive management is a flexible approach to managing a system in which the management actions are treated as largescale experiments (holling ; walters and hilborn ) . it is an iterative process in which the system is monitored following the initial management action and new actions (again treated as experiments) are designed and implemented in response to the results of the initial actions. adaptive management was conceived for managing highly uncertain systems, and thus it has great potential for managing systems in a changing climate (arvai et al. ) . adaptive management approaches designed to address climate change will likely have four basic steps (kareiva et al. ) : ( ) assessing the potential impacts of climate change on the system in question, ( ) designing management actionsin the form of experiments with testable hypotheses or predictionsto address the potential impacts, ( ) monitoring the system for climatic changes and system responses, and ( ) using the results of the monitoring to evaluate the effectiveness of the management actions and redesign them as necessary before repeating the four steps. it is generally accepted that more diverse systems are more resilient to environmental change than less diverse systems. thus, restoring or protecting the biological diversity of a system will likely increase resilience of that system to climate change (glick et al. ). this diversity can be in the form of species diversity, environmental diversity, or genetic diversity (schindler et al. ) . a second, general approach to increasing resilience of species and systems involves reducing the impacts of other factors that reduce the overall resilience of the species or system. for individual species, this might mean reducing harvest, restoring or protecting habitat, eliminating diseases, or reducing human interactions. for ecosystems, this may mean reducing pollutants, restoring vegetation, reducing human use, and controlling invasive species. as climates change, many species need to move to track suitable climatic conditions and changing habitats. in some cases, it may be possible to facilitate movement by increasing connectivity of freshwater and terrestrial landscapes (see sections . . . freshwater systems and . . . terrestrial systems). however, many species with limited dispersal abilities or that face significant barriers to dispersal will be unable to respond to changing climates. assisted colonization is one strategy that has been proposed for facilitating climate-driven range shifts (hunter ; mclachlan et al. ); also referred to as managed relocation and assisted migrationassisted colonization is the translocation of species outside their native range to facilitate movement in response to climate change. the concept is controversial for both ethical and ecological reasons. some researchers have highlighted the potential for negative, ecological, evolutionary, and economic impacts, as well as ethical concerns (ricciardi and simberloff ; sax et al. ). advocates argue that assisted colonization will be essential for cases in which species will have to move to find suitable environments but will be unable to do so, that many of the potential risks are overblown, and that guidance can be developed for the strategic and informed use of assisted colonization. several decision-making frameworks and prioritization strategies for applying assisted colonization have been proposed (hoegh-guldberg et al. ; richardson et al. ). although assisted colonization will likely be a useful strategy in many systems, there will be specific factors and risks that will need to be considered for particular systems (e.g., in freshwater systems, olden et al. ). virtually all freshwater systems are already impacted by human activities (e.g., vörösmarty et al. ) . under current climate projections, virtually all freshwater ecosystems will face significant impacts by the middle of this century. most of these impacts will be detrimental from the perspective of existing freshwater ecosystems and the human livelihoods and communities that depend upon them (poff et al. ) . discussions have broadly focused on rising water and air temperatures, increasing and decreasing precipitation, increasing evapotranspiration, and changing regimes of groundwater and surface runoff. there are four basic types of adaptation strategies that have been proposed for addressing these impending impacts. we define these as ecological water management, connectivity, protected areas, and restoration strategies. below, we briefly describe some of the specific strategies that have been proposed in each of these areas. increased water scarcity associated with decreased precipitation and increased warming in some regions is likely to prompt the modification of dams and reservoirs to improve storage capacity (see section . . . . managed aquatic systems). traditional water management has generally sought to dampen the natural variability of river flows to attain steady and dependable water supplies for domestic and industrial uses, irrigation, navigation, and hydropower, and to moderate extreme water conditions such as floods and droughts (postel and richter ) . however, the extraordinary species richness and productivity characteristic of freshwater ecosystems is strongly dependent upon, and attributable to, the natural variability of their hydrologic conditions. thus, large changes to natural flow regimes can have large impacts on species, ecosystems, and society (postel and carpenter ; iucn ; wcd ) . the water needs of humans and natural ecosystems are commonly viewed as competing. however, water managers and political leaders increasingly recognize that there are limits to the amount of water that can be withdrawn from freshwater systems before their natural functioning and productivity, native species, and the array of goods and services they provide are severely degraded ). this has led to a growing realization that society derives substantial benefits both from out-of-stream extractions of water as well as by maintaining adequate flows of water within freshwater ecosystems. in recent years, the water needs of river ecosystems are receiving increasing attention in water-supply planning, offering hope that many rivers can be protected before their health and ability to provide important goods and services is seriously compromised by water development. restoring or protecting some semblance of the natural river flow conditions necessary to support ecosystem function (called environmental flows) into the future is one of the most important climate change adaptation strategies for flowing waters. recent regulatory mandates and policy decisions calling for restoring and preserving environmental flows have led to a reexamination of dam operations for better protection of river health (watts et al. ) . for example, the south african national water act ( ) calls for the creation of a reserve of water in each river basin to meet both basic human needs and protect river ecosystem health. similarly, other national directives and international agreements such as the water framework directive of the european union are providing mechanisms for river protection, including the provision of adequate environmental flows (postel and richter ) . maintenance of environmental flows (flows that sustain river ecosystems) is likely to be the highest priority adaptation response for freshwater ecosystems in most contexts. this will require policies and implementation mechanisms to protect (and, if necessary, restore) flows now, and to continue to provide environmental flow regimes under changing patterns of runoff associated with climate change. as air and water temperatures warm, many freshwater species will be forced to rapidly adapt to their new environments, to migrate to more suitable habitats, or face extinction (heino et al. ). responses of aquatic organisms to climate change will be constrained because they are limited to dispersal along pathways of connected water. consequently, the linear nature of river systems makes them highly prone to fragmentation from waterfalls, dams, and water withdrawals. such fragmentation can not only limit dispersal and the ability of species to track climate change but also disrupt the completion of life cycles for many freshwater organisms. for many streams, especially in arid and semiarid ecosystems, there is concern that more frequent and severe droughts will lead to the building of more dams and water diversions, further fragmenting stream networks (seager et al. ) . increasing the connectivity of freshwater systems is one key way to help aquatic systems adapt to climate change. species responses to climate change will be limited by human-engineered structures, including hundreds of thousands of dams, diversions, and impassable road culverts that exist globally (nilsson and schopfhauser ) . in the united states alone, there are over large dams and an estimated . million or more small impoundments that disrupt the dispersal of aquatic organisms. in recent decades, however, new water laws and programs have helped to increase habitat connectivity by removing or modifying human barriers to movement in river systems. increasingly, small dams and diversions are being removed for a number of ecological, social, and economic reasons, including the enhancement of longitudinal connectivity for fish migration (stanley and doyle ) . similarly, temporary barriers such as impassable culverts under roadways are being modified to allow for easier recolonization and movement of fishes (roni et al. ) . although this management strategy may promote the adaptive capacity of species to respond to climate change, the removal of such obstructions can simultaneously increase the risk of invasion by nonnative species and associated diseases, which are major threats to ecosystem structure and function. in fact, the conversion of culverts into permanent barriers has been a common management approach to protect isolated native populations from invasive species spreading upstream (fausch et al. ). given the complexities of promoting the movement of native species while concurrently limiting the spread of invasive species, it is unlikely that the removal of barriers to enhance habitat connectivity will be a successful adaptation strategy in all areas for reducing the impacts of climate change on ecosystem function. in addition to restoring or protecting environmental flows and increasing connectivity within stream networks, it may be necessary to protect the species themselves to allow them to move through or persist in more intensely managed landscapes. in aquatic ecosystems, this may involve increasing the size of existing reserves or creating new protected areas that encompass important freshwater resources and allow for native species and assemblages to persist into the future (abell et al. ) . such acquisitions and agreements ideally would capture a large portion of the geographical, ecological, and geophysical range of multiple species, thus increasing opportunities for organisms to adapt and evolve to changing environmental conditions. protected areas have historically been a cornerstone of terrestrial conservationand are discussed in much more detail in that section, below. nonetheless, protected areas will likely play a major role in protecting aquatic species as climate change and areas could easily be prioritized to provide protection for both terrestrial and aquatic systems in a changing climate (abell et al. ). projected increases in air temperatures, combined with reduced snowpack, earlier onset of spring peak flows, and lower summer baseflows, will have direct implications for the thermal regimes of streams and rivers (poff et al. ) . the ecological implications of stream warming are significant. water temperature directly influences the metabolic rates, physiology, and life histories of aquatic species and helps determine rates of important ecological processes such as nutrient cycling and productivity. in general, warming will produce a general shift in species distribution poleward with extinctions and extirpations of cold-water species at lower latitudes and range expansion of warm-water (often invasive species) and cool-water species into higher latitudes. restoring and protecting riparian vegetationthe trees and shrubs that grow along the banks of rivers and streamshas the potential to reduce stream temperatures in certain places. recent modeling suggests that riparian habitat restoration can play an important role in offsetting the effects of climate change. battin et al. ( ) used a series of linked models of climate, land cover, hydrology, and salmon population dynamics to investigate the impacts of altered stream flow and temperature on the effectiveness of proposed habitat restoration efforts designed to recover chinook salmon populations in the pacific northwest of the united states. this study showed that in the absence of habitat restoration, basin-wide spawning populations would decline by an average of - % by . by contrast, they found large increases in juvenile rearing capacity associated with proposed riparian habitat restoration, which limited climate-induced declines to % according to one climate model and increased salmon abundance by % according to another model. however, the model results showed that not all expected climate impacts could be mitigated entirely. in relatively narrow streams, reforestation was predicted to decrease water temperatures by increasing shading, but in wide, main-stem reaches where most chinook salmon spawn, riparian vegetation had a minor effect on water temperature. in addition to restoring riparian areas, targeted protection of forested riparian buffers using fencing to stop livestock from entering the stream and trampling/grazing vegetation or conservation easements to eliminate farming activities may also help to reduce stream temperatures. the many adaptation strategies that have been proposed for addressing climate change in terrestrial systems can be loosely grouped into three categoriesthose that involve protected lands and conservation planning, those that address connectivity, and those that are more species or site specific in nature. protecting land is arguably one of the most effective ways of conserving biodiversity and terrestrial ecosystem services. it is generally assumed that lands that are selected to protect today's species and ecological systems will protect those species and systems into the future. climate change brings this assumption into question. as climates change and species distributions shift, species will move into and out of reserves. changes in species composition, the timing of ecological events, and the rates of ecosystem processes will result in changes in the functioning of ecosystems and the services they provide. many proposed adaptation strategies address these challenges by augmenting current protected areas with additional reserves, parks, and preserves (heller and zavaleta ; lawler ). these strategies range from broad calls for more protected lands to more sophisticated recommendations about how to select new lands or design new reserves to better protect biodiversity and ecosystem services as climates change. a number of strategies have been proposed for determining where to put new protected lands to best address the effects of climate change. some of the simplest strategies involve placing reserves at the poleward or elevational boundaries of the range limits of specific species (peters and darling ) , at major transitions between vegetation formations (halpin ) , or at the core of species environmental distributions (araújo et al. ) . others have proposed placing reserves between existing reservesin the hopes that those new reserves will act as stepping stones allowing species to better move from reserve to reserve. yet another recommendation involves placing reserves across strong environmental gradients. reserves that span many different environments will potentially allow species to shift their distributions without leaving the reserve. in addition to these simple rules of thumb for placing reserves to address climate change, many more sophisticated approaches to selecting areas for new reserves have been proposed. for example, modeled climate-driven shifts in species distributions can be used to select new reserves that will protect biodiversity today and into the future (hannah et al. ; vos et al. ; hole et al. ). although this approach may prove useful for some species and systems, care must be taken in the use of the models developed to project species range shifts. there are many uncertainties associated with the models themselves and the climate projections on which they rely. another proposed approach to building a reserve network that will be resilient to climate change involves protecting environments in lieu of protecting species and systems. this approach has been referred to as 'protecting the ecological stage.' this analogy implies that the species and the ecological systems are players and that the soils and topography of a region form the stage on which they interact and evolve. by protecting a suite of diverse environments, it is assumed that one will protect the areas where new ecological systems can develop as climates change (anderson and ferree ; beier and brost ) . similarly, others have suggested that more resilient reserve networks will be those that include a diversity of current climates (pyke and fischer ) . although climates will change, many of the climate gradients generated by latitudinal and topographic patterns will remain. thus, a set of reserves that protects a diversity of climates today will likely protect a diversity of climates in the future. at least one recent study has applied the concept of protecting environments (topographies, soils, and climate gradients) to a reserve-selection problem (schloss et al. ) . one last approach to building a resilient reserve network in a changing climate involves placing reserves in areas that are likely to serve as climate refugia. refugia are areas that are projected to experience minimal climatic changes and/or have cooler microclimates (saxon et al. ; hansen et al. ; shoo et al. ) . one potential opportunity for climate change adaptation is to consider people as an essential component of species conservation and climate adaptation. while reserve and park systems have a long history of alienating local people, using violence and other coercive measures to separate people and wildlife, in this case, local livelihoods are also threatened by climate change. this provides an opening for a new approach to conservation and a new discourse on the relationship between local people and wildlife. as for freshwater systems, improving the connectivity of the terrestrial landscape will be critical for addressing climate change. in response to historical climatic changes, many species shifted their rangesmoving toward the equator as ice sheets advanced and moving poleward as the ice sheets retreated. today's species will have a much harder time responding to current and impending climatic changes. present-day landscapesunlike the landscapes of the distant pastare fragmented by roads, agricultural fields, and residential development. given that many species will have trouble moving across these human-dominated landscapes, it will be essential to reconnect fragmented landscapes to allow species to respond to shifting climates. not surprisingly, improving landscape connectivity was the most often recommended climate change adaptation strategy in a recent review of the literature (heller and zavaleta ). many of the recommendations for increasing connectivity involve designing corridors that connect current protected lands. traditionally, such corridors have been designed for particular focal species, and they have connected reserves or remaining fragments of habitat. however, corridors that facilitate species ranges shifts and the reshuffling of ecosystems will need to connect what is habitat for a species today with an area that may become habitat in the future. in this way, corridor design will need to evolve to address climate change. one such approach to developing corridors for addressing climate change involves basing the corridors on environmental gradients instead of on the current vegetation (which is often used as a measure of habitat) (beier and brost ) . another approach uses projected changes in climate to orient corridors (ackerly et al. ) . a third method involves using modeled shifts in species distributions to identify potential routes that species would need to take to move from currently suitable climates to places where climates will likely be suitable in the future (williams et al. ; rose and burton ) . these three approaches are based on different assumptions and have different levels of associated uncertainty. in general, the uncertainty increases with the intensity of modeling required to identify the corridor, and thus the first of the three approaches has the least inherent uncertainty and the last of the three has the most. although corridors may be useful for some species, they will not be feasible or effective in all places or for all species. in addition to designing corridors to facilitate species movements, it will likely be necessary to improve the permeability of the landscape in general (franklin and lindenmayer ). increasing landscape permeability will involve changing the way we use and manage the landscape. changes in forest harvest practices from clear-cuts to selective, retention harvests can increase the connectivity of forested landscapes. landscape planning and zoning for residential development can reduce landscape fragmentation. windbreaks, fencerows, and crop choices can be used to increase the connectivity of agricultural lands. at a finer scale, choices of landscaping plants, road placement, and wildlife over-and underpasses can increase landscape permeability. protected lands and connectivity increase species ability to adapt to changing climates at relatively large spatial scales. in general, they address species movements. adaptation strategies are also needed at finer scales. in particular, strategies are needed that address individual species and ecosystems. many of these finer scale approaches tend to involve reducing temperatures or otherwise offsetting the exposure to the changes in climate or climate-driven changes in environmental conditions. for example, it may be possible to reduce temperatures by restoring vegetation to provide shade (wilby et al. ; wilby and perry ) . it may be possible to reduce the impacts of changing fire regimes through forest management practices. fire suppression policies, may, for example, have particularly severe ramifications in a climate in which fires will generally be larger and more frequent. it may also be feasible to provide supplemental water for particularly vulnerable plants or animals in times of drought or during heat waves. similarly, shade shelters could be provided to help some animals weather heat waves in extreme climates. although some of these strategies may prove useful in the short term, many will be neither cost-effective nor long-term solutions. another system-specific approach to addressing climate change involves altering species composition or community structure in a way that allows a system to better cope with climatic changes. for a forested system, this may mean planting new species or planting seeds of the same species that have a different genetic makeup (millar et al. ; glick et al. ). likewise, for grasslands and shrublands, this may mean choosing different species or strains for restoration purposes. moreover, it may mean facilitating the transformation of an ecosystemfor example from a shrubland to a woodland or from a shrubland to a grassland. in many cases, the first species to move into these transitioning systems may be invasive species. facilitating the transition to a new state using species native to the region may, in many cases be preferable to having new systems dominated by invasive species. these extreme cases will require ex situ conservation strategies, including seed banking and captive breeding to ensure the long-term survival of the species. the interface between the land, sea, and air found in coastal areas is a high-energy system subject to storms, winds, and tides. it is also where the majority of protein comes from in many regions of the world. many vital ecosystem services are provided along coastal margins of continents and islands. fringing coral reefs and mangrove forests buffer wave energy, which reduces erosion along the coast. these systems also provide critical habitat for many species, especially nursery areas for provisional services such as fisheries. in many areas, sea-level rise and associated storm surges will make it difficult to protect these systems and the services they provide. nonetheless, there are two main approaches that will likely prove most useful in coastal systems: building resilience through protection and restoration and facilitating the transition to new ecosystems. one of the more beneficial ways, from both an environmental and economic standpoint, to adapt to predicted changes from climate change is by protecting and restoring natural systems that provide multiple ecosystems services. this would include protection of human infrastructure from sea-level rise and increased inundation. protection and restoration of natural buffers such as barrier islands, mangroves, and coral reefs not only require little financial construction and maintenance but also maintain the wealth of ecosystem services associated with these systems, which include nursery and rearing habitat for many commercial seafood stocks such as fish and shrimp, recreational and cultural opportunities for humans, and protection from inundation. some natural protection features such as sea ice cannot be maintained in the face of increasing temperatures. in alaska, the loss of sea ice and melting of permafrost due to warming ocean and air temperatures has exposed the shore to wave energy that used to be absorbed or damped by sea ice or frozen soils. undoubtedly, ecosystem services that depended on that sea ice protection have also been lost. many regions have seen a huge decrease in the area covered by mangroves as coastal areas are drained and mangroves removed for aquaculture. worldwide, it is estimated that more than % of mangrove forests have been destroyed or converted to other uses. on a local scale, many areas have lost all of their mangroves. this loss destroys a wide range of ecosystem services provided by these forests, which range from carbon sequestration to flood protection, spawning, and rearing grounds for many fish and other marine species. restoring mangrove forests results in increased storm surge protection for natural and human coastal systems, provides nursery habitat for many commercially important fisheries, and stores sediments that may damage offshore coral reefs. worldwide, corals exhibit well-documented environmental adaptability over time scales of weeks to thousands of years. corals are actually two creatures that live together in a symbiotic relationship. zooxanthellae are the algal component of corals that provide nutrients generated from photosynthesis to the animal component. different zooxanthellae species and different species of corals in general have differential environmental tolerances. for example, some corals near the arabian peninsula exist under conditions of salinity and temperature that are greater than near-term predicted future average salinities and temperatures resulting from climate change. these adaptive changes likely operate on a decadal time scale. thus, in some cases, it may be possible to identify particular species or zooxanthellae and algal combinations that are more resilient to warming. protecting coral reefs from other stressors such as excess sediment and/or nutrient and pollutant inputs from terrestrial sources and unregulated diving can increase the coral's ability to adapt to increased water temperatures. technological adaptation measures include using sprinklers to create roughened sea surface to decrease light penetration or covering reefs with shade cloth to limit bleaching of corals. as sea levels rise, many coastal systems will need to migrate inland. when there are extensive human uses of coastal space or urban buildup, space for ecosystem recovery and migration of species will not be available. providing space for the migration of biotic elements of the coastal system at the same time as when human populations are forced to move would provide cobenefits for coastal life and human communities. ecosystems will usually adjust to changing salinities and water levels, and the services provided by the system will be maintained where conditions are suitable. for example, in , a subdivision east of houston in baytown, texas, was bought out, the structures removed, and the entire area restored to coastal wetlands. a more extreme example of planned retreat is the maldive islands. their current plan is to exploit the current coastal amenities, primarily beaches and coral reefs; secure capital with which to train younger citizens; and prepare them for relocation to other countries such as india, sri lanka, and australia. in the short term, there are negative consequences to ecosystem services due to commercial and touristic exploitation, but in the long run, by abandoning the islands, the system will be able to come to a new ecological state. in some cases, removing human infrastructure will be a necessary but insufficient means of fostering landward shifts in coastal systems. in such cases, it may be necessary to facilitate the shift in natural systems and stabilize soils by planting more coastal species inland as water levels rise. such actions have been taken as part of the alligator river climate change adaptation project on the north carolina coast in the southeastern united states poulter ) . here we describe some of the adaptation strategies that have been proposed for built environments, agricultural systems, and managed freshwater systems. we explicitly focus on how the most commonly proposed strategies could affect associated ecosystems. the severity of climate change impacts will depend, in large part, on our ability to transform the built environment in response to changing conditions. the following section discusses some of the climate change adaptation strategies that will likely take place in built environments and the effects that these strategies may have on ecosystem functions and services. we briefly discuss human migrations, flood control, sea-level rise, increased water storage, and urban heat-island effects. perhaps the most basic form of climate change adaptation involving the built environment is human migration. vulnerable populations will be forced to abandon their homes, relocate, and rebuild. they will displace resident populations and open up the possibility of conflict in the process. the bulk of climate change-related human migration that is already occurring involves movement from rural areas to cities in developing countries. unplanned urban growth will fuel the expansion of informal urban settlements, and by ,  people, one-third of the world's population, may inhabit urban slums. in both rural and urban environments, proactive planning and resettlement strategies that identify populations at risk and relocate them stand to drastically reduce long-term human hardship stemming from climate change. they can also protect vital ecosystems from the degradation associated with unplanned development. however, resettlement can have profound negative impacts. poorly executed, it can degrade indigenous culture, undermine traditional livelihoods, destroy social networks, and deepen poverty by distancing poor communities from employment opportunities. given these dangers, in some cases, it is preferable to fortify communities against climate change impacts in place. this approach introduces improvements to infrastructure and housing, builds upon the prior efforts of communities, and preserves social networks. in many regions, climate change will result in increased rainfall and flooding. conventional approaches to storm water management rely on defensive hard structures. water runs from impermeable concrete and asphalt surfaces into storm drains, culverts, and concrete embankments. rivers are straightened, channelized, and isolated from their natural floodplains to accommodate human settlement. as flood events become more intense and frequent with climate change, storm water management strategies must evolve. rather than take a defensive stance, they should promote the coexistence of humans, habitat, and water. soft infrastructure or low-impact development strategies, such as permeable paving, green roofs, green walls, rain gardens, bioswales, and constructed wetlands offer a promising alternative to conventional storm water infrastructure. they promote groundwater recharge, reduce flooding, and improve water quality with downstream benefits to aquatic habitats. in areas of severe flood risk, resettling residents on higher ground and reintroducing rivers to their floodplains protects human populations, dissipates flood energy, and creates wetland habitat. through temporal landuse practices such as intermittent farming, floods become an asset rather than a threat, depositing sediments that enrich agricultural lands. by , rising sea levels may displace as many as million people (myers ) . like conventional storm water infrastructure, conventional seawall infrastructure relies on defensive hard structures. as sea levels rise, existing levees will become obsolete. here again, soft-infrastructure strategies offer a promising alternative. graduated transitions from sea to land including manmade offshore islands, expansive shallows, and coastal marshes act as buffers for storm surge better equipped to absorb the impacts of extreme weather events. they provide habitat for marine species and, in coastal cities, public space for recreation when conditions permit. in places like coastal peru and sub-saharan africa, disappearing glaciers and reduced precipitation will increase the frequency, duration, and impacts of drought, undermining food and water security. a number of interventions in the built environment can help communities cope with water scarcity. increasing water storage at scales ranging from dams to urban reservoirs and household rainwater barrels helps bolster water supplies during prolonged dry periods. in coastal areas, desalination plants and, in select localities of south america and africa, fog harvesting may prove to be viable alternative water sources. ecological sanitation technologies such as dry toilets and gray water wetlands conserve potable water for drinking and, as part of a network of integrated ecological infrastructure systems, irrigate and provide nutrients for urban farms and forests. they also help prevent contamination of local water bodies, preserving their viability as sources of potable water and ecological integrity. further impacts of, and opportunities associated with, these various adaptation strategies are discussed in the sections . . . . and . . . . . cities in developed countries are on average - c hotter than the surrounding countryside (un human settlements programme ). higher urban temperatures increase energy use, degrade urban air quality, raise the temperature of urban storm water runoff, and contribute to general discomfort, respiratory illnesses, heat stroke, and other heat-related illnesses. warming in conjunction with increased urbanization will amplify urban heat-island effects and their associated impacts. several strategies can help reduce urban temperatures. at the scale of the site, urban trees with high, dense canopies, strategically planted around buildings, in parking lots, and along streets permit the passage of air, shade heat-absorbing surfaces, and cool the atmosphere through evapotranspiration. they also reduce the temperature of runoff water. green roofs perform a similar function on roof surfaces. buildings and landscapes constructed from materials with high solar reflectance or albedo absorb less heat and reduce urban temperatures. at a larger scale, streets oriented parallel (or up to ) to prevailing winds, permeable networks of public open space, strategically situated low-rise buildings, and gaps between high-rise structures increase the air permeability of urban environments. increased temperatures and changes in precipitation regimes are likely to have negative impacts on global food security by worsening the existing regional disparities between developing and developed countries (easterling et al. ; rosenzweig and tubiello ) . this is supported by the prediction that a moderate warming accompanied with elevated co concentrations is likely to lead to an increase in crop productivity of developed countries in temperate mid and high latitudes, whereas even a slight increase in temperature could damage crops that are already growing at supraoptimal temperatures in countries located in tropical, low latitudes (easterling et al. ; easterling ) . as in other systems, even with significant mitigation efforts, adaptation strategies will be necessary to address the impacts of climate change on agriculture. adaptation strategies for more resilient agricultural systems may include altering the timing and location of cropping, switching crop species and cultivars, selecting livestock breeds, crop rotations, and efficiently using and managing water resources. these types of adaptation strategies have been referred to as autonomous strategies (easterling ) . implementing some of these strategies, such as changing the type of livestock that is grazed or changing crop rotations could have profound effects on ecosystem functioning. however, in general, these types of adaptation strategies will have environmental impacts smaller than more intensive strategies that are designed to transform systems. for many agricultural systems, the magnitude of climatic changes will necessitate more intensive planned adaptation strategies that foster significant transformations of systems or infrastructures (easterling ) . such intensive strategies will be particularly important for sub-saharan africa and south asia where climate will have major impacts on agriculture (lobell et al. ; nelson et al. ). these planned adaptation options include developing new infrastructure (e.g., irrigation infrastructure, transport and storage infrastructure, and efficient water use and reuse technologies), investing in new technical and management options (e.g., crop and livestock improvement, enhancing germplasm), shifts in the distribution of agriculture (e.g., abandoning some areas and farming new lands), and building the capacity to make continuing adjustments. here, we discuss two of these strategiesimproving crop performance and water conservationin more detail. stresses on crops due to extreme heat and pest and pathogen pressures are likely to increase with climate change (deutsch et al. ; battisti and naylor ) . multiple adaptation options are available for improving crop performance in the face of such climate-driven stresses. first, in conjunction with conventional breeding, crop improvement through genetic engineering is seen as an effective adaptation tool in a changing climatealthough public opinion is often divided on the use and consequences of genetically engineered crops (macdiarmid ; jones ) . a second approach involves maintaining crop genetic diversity by, for example, collecting and storing the diversity of current cultivars or local landraces. a third adaptation strategy involves exploiting symbiotic relationships between crops and other organisms. for example, it has been shown that numerous fungal and bacterial endophytes confer stress tolerance to heat, drought, salinity, and pathogen attacks (rodriguez et al. ; rodriguez et al. ), and others promote growth and fix atmospheric nitrogen xin et al. ). other strategies include the application of kaolin particle films to reduce heat stress and photoinhibition and to mitigate pest damage (glenn et al. ; glenn ) as well as the incorporation of biochar into soils to confer numerous agronomic benefits that enhance crop performance and stress tolerance (marris ) . irrigated agriculture accounts for about % of the world's freshwater withdrawals (rosegrant et al. ). with increasing global population, the demand for irrigated agriculture will continue to increase globally because realized crop yields normally increase considerably with irrigation. adaptation strategies that foster more efficient use of water in agriculture will be critical for achieving global food security while conserving water in a changing climate. these strategies will include institutional water management reform, economic incentives for efficient water use, and infrastructure investment for efficient irrigation and water delivery systems (rosegrant et al. ). the 'virtual water' trade through crop products from water-abundant to water-deficit areas should be considered as one of the key adaptation options to climate change (rosegrant et al. ; easterling ) . improving crops for higher water-use efficiency (i.e., greater yield with less water use) should be a goal for genetic, ecological, and agronomic options of adaptation discussed above. alternative strategies of increasing water storage capacity with dams and increasing the number of stream diversions for agriculture will, in contrast, have significant impacts on aquatic ecosystems (see the section . . . . managed aquatic systems). as discussed above, changes in temperature and precipitation are likely to lead to changes in the demand for water. projected climatic changes will likely lead to increased demands for residential use, irrigated agriculture, hydropower production, and carbon capture and storage technologies. in some regions, current water management may not adequately cope with the impacts of changing precipitation regimes and increased evaporation on the reliability of water supply, flood risk, health, agriculture, energy generation, and freshwater ecosystems (palmer ) . adaptation strategies designed to address water management for human uses will either directly or indirectly affect ecosystem functioning and ecosystem services. in addition to the adaptation strategies for addressing water demands for agriculture and residential use (addressed to some extent in sections . . . . and . . . . ), other important adaptation strategies for managed aquatic systems that will impact ecosystem functions and services include increased extraction of groundwater, improving storage capacity by building or modifying dams and reservoirs, desalination of seawater, and expansion of rainwater storage and water transfer (bates et al. ; hirji and davis ) . improving storage capacity by building or modifying dams and reservoirs is likely to be one of the most common regional responses to decreased water availability and increased demand for water. the great utility of running water has resulted in the extensive exploitation of streams and rivers by humans throughout the world, a process greatly facilitated by the construction of hundreds of thousands of dams globally (nilsson et al. ) . by capturing high river flows and releasing the water in a carefully controlled manner, dam managers can deliver steady and dependable water supplies to downstream areas, protect settlements from floods, or generate power. although human manipulation of the world's river flows has provided many societal benefits, it has also caused considerable ecological damage and the loss of important ecosystem services valued by society (baron et al. ) . flow regulation for hydropower and storage, diversion for human use, and extraction of groundwater connected to stream systems often significantly affect the natural seasonal and interannual variation in river flow, commonly referred to as the natural flow regime (poff et al. ) . a natural flow regime is needed to sustain the complex riverine and riparian ecological functions and processes that support native biodiversity and provide myriad ecological services including maintaining water quality, recharging groundwater, and nutrient cycling (naiman et al. ) . facing an ominous future of increasingly severe watersupply shortages in many areas of the world, social planners and government leaders are exploring strategies for ensuring that enough water of sufficient quality is available for use by future generations. human demands for water are expected to increase under a warmer climate, exacerbating current management problems. increasing demands for irrigation and industrial cooling water could conflict with the increasing demands for municipal water supplies resulting from urban growth. climate-induced changes to water availability and increasing human demand for water are likely to prompt the construction or modification of dams and reservoirs to improve storage capacity and enhance water security (wcd ; palmer ). for instance, by storing water in reservoirs, water managers capture high flows during wet years or seasons to supplement water supplies at drier times, thereby maximizing the reliability of water supplies and certain economic benefits each year. for regions where increases in runoff variability or more prolonged drought conditions are associated with climate change, the pressure to build watersupply reservoirs will be great. although the construction of additional dams and reservoirs will undoubtedly provide many societal benefits, a rich body of scientific knowledge has shown that river regulation also causes considerable ecological damage and the loss of important ecosystem services valued by society (baron et al. ; postel and richter ) . river ecosystem health deteriorates when natural flows of water, sediments, and organic materials through a river system are substantially disrupted or modified by human activities (poff et al. ; richter et al. ) . river damming and associated environmental alteration is now widely recognized as a leading cause of declines in freshwater biodiversity globally (bunn and arthington ) . dams have also been implicated in the loss of commercial fisheries in many estuaries and coastal areas, and in the degradation of other natural ecosystem products and services worldwide (wcd ) . in addition, reservoirs increase the loss of water due to evaporation. finally, as mentioned above, reservoirs emit substantial quantities of carbon dioxide and methane into the atmosphere (st. louis et al. ) , and therefore, the creation of additional reservoirs is likely to further promote climate change. in many regions, impending water shortages will also lead to greater interest in desalination as a technique for tapping into the vast and infinitely tempting water supplies of the sea. recent improvements in reverse osmosis technology, coupled with the rising cost and increasing unreliability of traditional water supplies both currently and projected in the future, are making desalination a water-supply option with major plants in operation, in planning, or under consideration in europe, north africa, north america, australia, china, and india. like most large-scale industrial processes, making water has a number of actual or potential impacts on aquatic goods and services (wwf ) . emerging issues include significant cost and pollution emitted by desalination plants and the mostly unknown, but potentially substantially, effects on freshwater and coastal ecosystems. intake pipes can remove larvae and small marine organisms, and discharge from desalination plants can elevate levels of salt and other constituents of seawater such as boron; dead sea life, which consumes oxygen while decomposing; chemicals added to change the composition of the water for processing, as well as reducing contamination and clogging of filters and membranes; corrosion byproducts; and the heat added for or during processing. finally, water manufacturing is an energy-intensive process. although increasing ghg concentrations and the resulting increasing temperatures are global phenomena, the globe will often be an inappropriate scale for developing mitigation and adaptation strategies. human activities generate differential risks and vulnerabilities that vary with location due to differences in social, political, cultural, and economic conditions (beck ) . although, in one sense, all people are vulnerable to catastrophic climate change, at another more fundamental level, the risk of climate change impacts (along with blame and responsibility) are unevenly distributed. we can begin to think of the unequal distribution of the vulnerability to climate change as a scale issue. mitigation and adaptation strategies can be applied at multiple, and often overlapping, scales. at the finest scale, the development of mitigation strategies will depend on local leadership and the attitudes of local populations and individuals toward climate change. at national and international levels, mitigation efforts will be organized through large and formal programs and institutions (e.g., un-redd). although some adaptation strategies will be national and international in nature, much adaptation will take place at finer scales of communities and regions. a hallmark of climate change as a global phenomenon is that leadership and coordination across local, national, and international scales is important for ameliorating its effects. in turn, coordination across scales will entail understanding local climate risks and local and regional understandings of the causes of climate change and the costs and benefits of different mitigation and adaptation strategies. the effectiveness of any mitigation or adaptation strategy (building codes, population planning, dietary changes, etc.) depends on its acceptance in local, regional, and national communities. social impediments to adoption of climate change strategies include ideological outlooks, structural constraints, and cultural barriers. for example, free-market ideologies can impede the ability of governments to invest in alternative energies; agricultural subsidies and corporate monopolies on agricultural inputs may create structural barriers to alternative agriculture; and cultural norms for relative water and energy use, family size, or the flavor and texture of food can inhibit conservation of resources and changes in diet. there is a clear disparity between societies that have contributed the most to ghg emissions and those that will likely suffer the direst consequences of climate change. at the international level, efforts to shift blame from the activities of the rich to the actions of the poor have been used to shirk the responsibility to mitigate climate change and to impede the development of global mitigation strategies. the dispute between the global north and south over who caused climate change and who must now take responsibility for mitigation is by now well known. countries in the global south argue that northern countries have already developed their economies relying on fossil fuel consumption and now, when it is their turn to develop, they are asked to put restrictions on their energy consumption and natural resource use. meanwhile, the united states has used the reluctance of developing countries to cap emissions as an excuse for inaction on its part. mitigation and adaptation activities that do not take globally inequalities in wealth, resources, and adaptive capacity into account, or insist on 'equality' in action in a demonstrably unequal world cannot be effective. addressing population growth clearly plays a key role in both mitigation and adaptation strategies. however, focusing on population size while ignoring global patterns in the consumption of resources and the distribution of wealth will result in failed policies and strategies. the world's human population is approaching  , and a growing population inevitably means greater demand for the numerous goods and services supported by terrestrial and aquatic ecosystems. while the availability of resources has seemingly stretched beyond the direst predictions of several decades ago, the crisis of overconsumptionnamely, of coal and other fossil fuels that produce ghgs and contribute to climate changeis simultaneously becoming clearer. while trends over the past several decades suggest that population growth is a significant driver of co emissions (bongaarts ; dietz and rosa ) , it is also true that urbanization, aging, and changes in household size similarly affect energy use and emissions (mackeller et al. ; cole and neumayer ) . particular point sources of ghg emissions from energy consumption in developed countries, like the internationally managed oil fields of nigeria or emissions from the recent iraq war, which since has released over million metric tons of carbon dioxide, the equivalent of adding million new cars to american roads (reisch and kretzmann ) , should bear at least as much scrutiny as the desires of third world women and men to bear children. an overarching emphasis on population detracts from a nuanced understanding of the multiple drivers of ghg emissions. it will be ineffective to focus on the effects of climate change on ecosystems without considering the concurrent effects on human well-being and vice versa. disparities in climate change risk and human health often overlap, and human health and environmental health are intricately intertwined. although diseases are often approached as singular phenomena, good models exist that demonstrate the interactive effects of multiple morbidity factors on health outcomes and disease trajectories. for example, interactions between acquired immune deficiency syndrome and tuberculosis, or diabetes and severe acute respiratory syndrome, have synergistic impacts on patients that must be understood together. likewise, social attributes, like poverty or exposure to racism, interact with disease conditions to increase the damaging effects of disease. such interactions have the potential to influence the effects of climate change on disease movement and clustering, disease interactions, and the impact of climate change on global health (baer and singer ) . human action and human vulnerability to such interactions between climate, disease, and the environment are structured by social and economic inequality, and thus mitigation and adaptation strategies will need to take these inequalities into account. what is clear from a social and political perspective on mitigation and adaptation is that no one set of mitigation or adaptation strategies will be applicable to all people in all places. suggested approaches to either mitigation or adaptation will involve costs and benefits that are unequally distributed across nations, populations, and societies. there will, for example, always be those who can escape pollution with air filters and private vehicles, use air conditioning to 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decision-making climate change, biodiversity and the urban environment: a critical review based on london statistical downscaling of general circulation model output: a comparison of methods planning for climate change: identifying minimum-dispersal corridors for the cape proteaceae wind power implementation: the nature of public attitudes: equity and fairness instead of 'backyard motives climate change mitigation and transport in developing nations making water -desalination: option or distraction for a thirsty world? world wildlife fund a diazotrophic, indole- -acetic acid-producing endophyte from wild cottonwood we thank the global health and environment faculty fellows at the university of washington as well as thomas hinckley, richard fenske, judith wasserheit, and tikvah weiner for valuable discussions and support. key: cord- -yak s authors: winkler, harald title: towards a theory of just transition: a neo-gramscian understanding of how to shift development pathways to zero poverty and zero carbon date: - - journal: energy res soc sci doi: . /j.erss. . sha: doc_id: cord_uid: yak s as a global community, we need to understand better how a just transition can shift development paths to achieve net zero emissions and eliminate poverty. our past development trajectories have led to high emissions, persistent inequality and a world that is fragmented across multiple contradictions. how can countries shift to development pathways that deliver zero poverty and zero carbon? in developing a theory of just transition, the article begins by reviewing a range of theoretical approaches from different traditions, building in particular on neo-gramscian approaches. it applies and modifies core components of gramsci’s approach, building a neo-gramscian theory of just transitions around concepts of ideology, hegemony, change agents and fundamental conditions. the theory suggests how coalitions of change agents can come together behind a just transition. the coalition needs to gain broader support, establish a new cultural hegemony in support of just transitions and be able to transform the fundamental conditions of the st century. the article briefly considers how this better understanding can be applied to the practice of shifting development pathways. the penultimate section reflects on limitations, including that a fuller development of a theory of just transition will require application for detailed concrete examples and a community effort. together, we might address the multiple challenges of our present conditions to transition to development that enables human flourishing and a healthy planet. what are the fundamental challenges of a just transition? rapid and deep climate action creates winners and losers, yet this action takes place in a world with persistent inequality. just transition is a vision but needs to be more than that. in this article, i advance a theory of how coalitions of change agents can come together behind a just transition in order to transform the fundamental conditions of the st century. we need to understand better how a just transition can shift development paths to achieve net zero emissions and eliminate poverty. a theory of just transitions needs to explain how to shift from high-to low-carbon development paths, while ensuring no one is left behind. the objectives of this article are, firstly, to advance a neo-gramscian theory of a just transition and, secondly, to apply it in aid of a better understanding of shifting development pathways to zero carbon and zero poverty (zpzc). the article builds on neo-gramscian theory as applied earlier applied to climate change (see section ) . the article postulates that a just transition is the ideological element around which a coalition of change agents coalesces. i return to gramsci's core components of ideology, hegemony, material conditions and social forces [ , ] . however, i modify the notion of material conditions for application in the st century, where some conditions are non-material and hence refer to fundamental conditions. that said, there are material bases to non-material conditions, as for example for information technology. combining the modified components and elaborating their relationships is how i develop a renewed explanatory model, a neo-gramscian theory of just transitions. to pursue the second objective, i begin to apply the explanatory model to understanding how to shift development pathways. the shifting of development pathways has a normative direction, towards both zero poverty and zero carbon, signifying development and climate goals. it should be noted that more concrete applications of the explanatory model will require further and collaborative thinking. i aim to advance fundamental understanding in this article, which should ultimately be socially useful. having introduced the problem statement and its objectives, the article proceeds as follows. the remainder of this introduction locates this article in previous theoretical approaches, explains why i choose to build on gramsci's core components, and how i extend neo-gramscian analysis of climate change to focus on just transition. the conceptual structure of section follows core components of gramscian theoryideology, fundamental conditions and change agents, making clear where these are modified. section combines these components and an understanding of their relationships into a renewed explanatory model. it is here that i advance a neo-gramscian theory of just transitions. the section applies the theory to better understanding of how to shift development pathways. the penultimate section presents some limitations of this article, while the concluding section sums up, discusses implications and future research directions. a just transition seeks to address two of the foremost challenges of the st century, development and climate change. the global community has failed to end poverty in all its forms everywhere, as much as it has fallen short of an adequate response to the climate crisisdespite agreeing in on the sustainable development goals [ ] and the paris agreement [ ] on climate change. these chronic crises are now compounded by the coronavirus crisis and the severe socio-economic impacts the response is likely to have. the concept of a just transition creates links between urgent action to address the climate crisis and while the chronic injustices of inequality and poverty. in this article, the focus of the just transition is on directly affected workers and also on broader communities. the global community has pledged that on its "great collective journey … no-one will be left behind" [ : para ] . the implications for this normative statement are that justice must be understood in terms of distributional equity (fair outcomes) and procedural justice [ ] . while the problems are well understood, the agenda on sustainable development has been too unfocused [ ] and action on climate change has been too slow [ ] . given that these crises remain unresolved, we need to reflect why what has been done is inadequate. we need to reflect on our (in)action. in other words, we need to return to theory. in this article, i develop and test a theoretical construct. the first step ("develop") is to advance a neo-gramscian theory of a just transition. i do so by a) considering existing theory and constructs, both briefly considering a broad range of theoretical approaches and building on earlier neo-gramscian theory applied to climate change; b) outlining how the explanatory model is tested; and d) indicating the contribution that this article aims to make. sustainability transitions research sought "to conceptualize and explain how radical changes can occur in the way societal functions are fulfilled" [ ] . the work of this community of practice over the last decade has identified several characteristics of sustainability transitions: multi-dimensionality and co-evolution; multi-actor process; stability and change; long-term process; open-endedness and uncertainty; values, contestation and disagreement; and normative directionality. conceptual frameworks include a multi-level perspective [ ] , strategic niche management [ ] [ ] [ ] and transition management [ , ] . the focus on actors and institutions is cognate with the concept of change agents used in this article, though the latter also draws on a gramscian notion of social forces. pointing to actors and transitions scholars themselves have identified gaps, including: "social inequality, poverty and lack of access to modern services such as sanitation or education in low-income economies might be considered more important than global environmental rationales such as climate change" [ ] . a stronger focus on social forces or changes agents, such as labour, is a gap that the present article aims to fill. transitions can be understood to be driven by focused on corporate, military and political elites, in c. wright mills seminar work [ ] . the power elite in mills' theory is about how elites divide or unite, writing in about us elites. more recently, elite power in low-carbon transitions has been examined in a review drawing on several critical lenses, including "multi-level perspective, michel foucault, antonio gramsci, anthony giddens, karl marx" [ ] . the authors conceptualise power and its different sources, with a specific focus on elites. they conclude that the power relations "are asymmetrical but promisingly unstable", the promise being that just energy futures may be more possible [ ] . while elites are important, a theory of just transitions needs to identify a broader range of change agents. cultural-historical activity theory (chat) seeks to explain the connections between human activity and what people have thought. its roots in a focus on the human mind focuses its analysis at the level of activity. sharing roots in dialectics with marxist approaches, the theory emphasizes the contradiction-driven character of activity, has developed methodologies for formative interventions and studied transformative agency [ ] . the dialectical approach emphasises contradictions among key elements, which are compatible with the theory developed here. more recently, chat has been applied to 'runaway objects' [ ] such as climate change and development connect large numbers of activity systems across national borders [ ] . while i focus on contradictions, which might have led to adopting a chat approach, i focus on just transitions at a larger scale than the individualin countries, aiming to apply an explanatory model across many countries. actor-network theory is a constructivist approach, that explains change in social and material conditions in relation to networks of relationships [ ] . it puts all factors at the same level. network analyses of discourse and advocacy coalitions have been explored in public policy literature [ ] . sabatier developed a framework to think about advocacy coalitions, showing ideas and beliefs are important for their formation [ ] . evidence-based policy making has been analysed mostly in relation to knowledge networks in industrialized countries; however, knowledge production in networks has begun to be studied in the context of climate and development policies in the global south [ ] . actor network theory, network analysis and frameworks for advocacy coalitions are useful elements to develop a theory, but also have limitations. they tend to explain (or even only describe) change ex post. the point of a theory of just transitions is to understand how change may evolve in future. my approach to advancing a theory of just transition is influenced most strongly by antonio gramsci [ ] , both as an inspirational theorist and in his injunction to practice pessimism of the intellect and optimism of the will. gramsci's theory of ideology and cultural hegemony brought an important nuance to marx's thesis that ideas are shaped by our material conditions, an insight that is as true now as when written in in the mid-nineteenth century [ ] . it is a practice by which the ruling class exercises cultural hegemony. to reproduce or challenge cultural hegemony, a coalition of social forces must coalesce around key ideological elements [ ] . neo-gramscian theory was applied to global environmental politics around the turn of the st century, with influential contributions by paterson, newell, levy and egan [ ] [ ] [ ] [ ] . this literature brought a focus on the structural power of capital, gramsci's concept of hegemony and attention to inequality to bear on environmental issues [ ] . the focus for a just transition is more specifically on climate change. neo-gramscian thinking has been applied to the analysis of climate change. some have focused on specific aspects of climate change relating to gender [ ] , corporate strategy in negotiations [ ] , 'petrohegemony' and climate justice [ ] , or certificates from forestry projects [ ] . there is a cognate literature applying neo-gramscian theory to the geopolitics of using information and communication technologies [ ] , the framing of global warming by media [ ] , and education in a globalised world [ ] . examining sustainable energy transformations, stegemann and ossewaarde [ ] argue that includes "populist and post-truth tendencies" in the hegemonic discourse in the european union and even in "environmental populist expressions". within neo-gramscian theories of climate change, just transitions are a particular focus, so far unexplored. others have combined gramsci's concept of hegemony with foucauldian governmentality to better understand climate governance [ , ] . newell has built on contributions to "different strands of literature from neo-gramscian and historical materialist ipe and political economy more broadly" [ ] , focused on the role of the state, globalisation and multilateral institutions, and proposed new research agendas for gramscian global political economy [ ] . as outlined above, neo-gramscian theory has been applied in several ways to environment and climate change. while building on these approaches, this paper develops a neo-gramscian explanatory framework of just transitions. the literature reviewed here has not specifically focused on just transitions. existing studies have not focused on my scope of development and climate, with specific goals of zero poverty and zero carbon. my aim is to focus on a broader set of change agents than the state. the theory of a just transition advanced in this paper builds on the elements of ideology, material conditions and social forces, modifying them for the current context. the theory of a just transition advanced in this paper is neo-gramscian. it uses key elements of neo-gramscian theory. section elaborates ideology, hegemony, fundamental conditions and change agents. these four elements are grounded in the theory of gramsci and neo-gramscian approaches, as discussed in sections . and . above. of the four, one component of the theory is modified and one renamed: gramsci's material conditions are updated to fundamental conditions, and i refer to change agents instead of social forces. the theory presented in section frames the just transition explicitly as an ideological element. the relationships between these components are key to developing an explanatory model, a neo-gramscian theory of a just transition. before outlining how the rest of the article proceeds to develop and apply theory, the approach is located within a paradigm and some uses of methods clarified. the approach taken in this article can be understood as part of a paradigm of critical realism [ ] . it is different in this respect to sustainability transitions research (e.g. as reviewed in köhler [ ] ), which is more in the interpretivist paradigm. my aim is to apply an explanatory model, and i find gramsci and neo-gramscian theoretical constructs compelling. i seek to generate knowledge by updating existing neo-gramscian theory (see section . above) to present a renewable explanatory model, understand how entities (change agents in coalitions) can change structures (fundamental conditions). in this sense, my approach is a critical realist one, while the main theory i build on is gramscian. a note on language is that i use the terms transition, transformation and transformational change, without a sharp distinction. hölscher et al. [ ] attribute the differences between the terms to their use in different research communities. given their approach, i would tend to use transformation, but also follow hölscher and colleagues in treating transition and transformation as not mutually exclusive. the article is focused on a just transition, and so this is the word mostly used. section . sets out ideology and hegemony as applied to the just transition, reflects on fundamental conditions in the st century ( b) and identifies change agents ( c). a neo-gramscian theory of just transitions is presented in section and is applied to thinking how to shift development pathways in section , noting that more granular applications of the theory require further research. in advancing a neo-gramscian theory and applying it to the just transition, i make a nuanced claim to theoretical novelty and its application (section ). future research directions together with limitations of this article are elaborated in section . theory provides analytical tools for understanding and explaining. in the community of practice of which i am part (researchers working on climate change mitigation and sustainable energy development), we use a wide range of analytical toolsbut have no coherent theory. we need a theory of a just transition, in other words a better understanding of how to get from current conditions to human flourishing and a healthy planet. a theory will help to suggest how to shift development paths to zpzc. for the reasons explained in the previous section, i apply neo-gramscian theory to just transition. core elements of this theory are ideology, hegemony, fundamental conditions and change agents. each is elaborated in turn below. ideology in a gramscian sense is the first of the three core elements. the following elaborates what is meant by ideology and a just transition. ideology is used, in a gramscian sense, as more than a system of ideas those that are necessary to bring and hold together an alliance [ ] . ideology is held by a community or groups, most specifically an alliance of change agents. ideology is not judged as true or false, but by its effectiveness in cementing a coalition of change agents. the term is used not in a pejorative sense, as irrational dogma that assumes that certain ideas are in error or dubious. the use of ideology here is broadly consistent with gramsci and neo-gramscian theoretical constructs. ideology is not just in the realm of ideas, but a practice carried out by institutions and individuals. the latter are gramsci's 'organic intellectuals' but not the focus of my analysis. ideology is what drives a 'cultural-social unity through which a multiplicity of dispersed wills, with heterogeneous aims, are welded together with a single aim' [ ] . social forces exercise cultural hegemony, gaining 'consent by means of political and ideological leadership' [ ] , that is by persuasion rather than force. updated for the st century, i would say that ideological elements are the focal point for coalitions. when different change agents coalesce around an ideological element, they are able to challenge hegemony and persuade others of a new vision. i propose a just transition as the ideological element around which a coalition coalesces. not only does ideology bind an alliance together, but it also helps to achieve hegemony, with cultural hegemony being a central concept in gramscian theory. hegemony is closely related to ideology, indeed hegemony is also referred to as "organic ideology" [ ] . a clear vision of a just transition and the values represented are important elements that mobilise support. the just transition is the core element that 'acts as the cement or cohesive force which binds together a bloc of diverse change agents' [ ] . it enables an alliance to achieve hegemony. hegemony is power by persuasion, as distinct from dominance by enforcement or outright conflict. 'a hegemonic class, or part of a class, is one which gains the consent of other classes or social forces through creating and maintaining a system of alliances by political and ideological struggle' [ ] . this contestation takes place in civil society, which is the "sphere in which hegemony is exercised" [ ] . in st century terminology, one worldview replaces another, hence changes in values, norms and ideological elements that are seen as central are important to the process of change that is the just transition. a hegemonic 'bloc' or alliance does not create an ideology anew, but rearticulates ideological elements into a new world view [ ] . the new world view will be contested, a point to which the article will return when explaining the process of gaining broader support (see section . below). but what is meant by just transitions? the concept of a "just transition" emerged in the north american labour movement in the s [ ] . the international labour organisation developed guidelines for a just transition, highlighting the needs of workers, create of decent work and green jobs [ ] . the increasing attention to just transition in climate negotiations resulted the preamble of the paris agreement reflecting "the imperative of a just transition of the workforce and the creation of decent work and quality jobs in accordance with nationally defined development priorities" [ ] . just transitions differ across countries and local context, reflected in a growing literature considering for example, coal in australia and south africa, and land issues in scotland [ ] [ ] [ ] [ ] [ ] . in the south african context, descriptions of a just transition includes a broader economic and social targets, recognizing that fossil-fuel economy transformation impacts will affect across regions and the economy [ ] . in this context, societal issues of poverty and inequality are a priority [ , [ ] [ ] [ ] . the extent of impacts (positive and negative) means that a just transition should engage stakeholders and planning across the economy and society [ ] . while definitions of a just transition vary, they consistently include core elements: investments in low-emission and labour-intensive technologies and sectors; assessment of employment and social impacts [ ] ; affordable access to energy services [ ] ; attention to 'legacy' sectors with retraining of workers; compensation to communities whose livelihoods are at risk in the transition; procedural equity through facilitated dialogue with affected communities and stakeholders [ , ] . 'green jobs' are a key requirement of a just transition [ , ] , but these are not necessarily formal jobs, as the concept has been broadened beyond protection of workers to wider society [ , ] . sustainable livelihoods depend on new ownership models -for example social and community ownership of renewable energy [ ] . such models should form part of local economic diversification plans [ , ] . a recent paper has reviewed the link of just transitions to the unfccc processes and presented recommendations for academic research in this domain [ ] . having reviewed the origins of the concept and its interpretation in different contexts, i turn to just transition as an ideological element. just transition encapsulates the dimensions of both development and climate. a just transition is a process that shifts development pathways to achieve zero poverty and zero carbon. ending poverty and eliminating carbon are chosen as two key goals, each from a broader set. in , the global community agreed the sustainable development goals (sdgs) including that poverty should be zero by [ : sdg goal ] and a long term goal that emissions should be in balance or net zero [ : article . ]. sdg is interpreted as eliminating absolute poverty, when i refer to zero poverty. relative poverty may persist in an unequal world, though sdg is to reduce inequality. the ipcc finds that co emissions needs to be net zero globally around to keep temperature below . • c [ ] , to meet one of the goals of the paris agreement [ : this article focuses on two specific goals, which signify broader concerns of development and climate. the suggestion is not to pursue only two goals (zpzc) but multiple objectives. broader terms such as "human flourishing and a healthy planet" [ ] convey compelling visions but, i would argue, are too broad to mobilise change agents. the shorthand of zpzc, i propose, is powerful in mobilising change agents by outlining specific goals of a just transition. i turn next to the second component, fundamental conditions how can our fundamental conditions be changed, transitioning from high to zero poverty and carbon? fundamental conditions are similar to the gramscian notion of material conditions, yet there are important differences in my theory. and the conditions at the end of the second decade of the st century are different from the pastsome conditions are material, while others are not. the fundamental conditions have been created by past development, with energy based primarily on fossil fuels and leading to high greenhouse gas (ghg) emissions. high-emissions development pathways have also led to high poverty, persistent inequality and other challenges reflected in the sdgs. the development paths which industrialised countries have followed cannot be the model for the future [ , ] . affected communities and workers will not support a low-carbon transition, if it creates more poverty and inequality than the high-carbon development path. elites who have benefited from past development patterns may coopt a low-carbon development [ ] , using and bolstering their hegemony. this is the case globally and especially in countries dependent on fossil fuels for development. it is these fundamental conditions of high emissions, poverty and inequality that create the imperative of just transitions. i postulate that there are multiple contradictions, not a single contradiction as in marx's time, over ownership of the means of production [ ] . there are conflicts between rich and poor; over identities defined by race, gender, sexual orientation; fanned by religious differences; over ownership of technology and information [ ] . the current cultural hegemony around growth has brought about multiple contradictions. many of the contradictions in the st century are materialover resources, class and others. the economies of the world are still characterised by a great divide between the have and the have-nots [ ] [ ] [ ] . income inequality is a persistent condition globally, a fundamental condition in many countries and threatens to widen post-covid- [ ] .thomas pikkety makes a compelling argument that inequality of assets is the condition shaping our world more than income inequality [ ] . yet increasingly, contestation over information and data is increasingly important and furthermore the 'ultimate drivers' of transitions, such as values and needs, identities, knowledge and understanding, power structures and culture, are not material [ ] . ideology can now be shaped by information technology in ways that were not imagined in gramsci's time. the prospects of networked artificial intelligence has potentially large implications on notions of work, and to a new fault-line of inequalitybetween humans and automation [ ] [ ] [ ] . at the same time, planetary boundaries are being reached and breached [ ] . as resources become more scarce, conflicts increaseabout land, food, air, energy, water, desertification, drought and biodiversity [ ] . there are competing and diverging mind-sets on how to relate to the natural environment: exploiting free resources, considering the earth as living or managing to design with nature. a critical condition of the world in the st century is climate change. climate science tells us that these impacts are a fundamental condition of our future world [ ] and moral philosophy affirms that they are a matter of fairness [ ] [ ] [ ] . we are living in an unequal world, fragmenting across multiple contradictions. some conditions are material, others are not. hence, i prefer the term fundamental conditions. a just transition may be the key element for a new cultural hegemony. a coalition of change agents can transform the current fundamental conditions. change agents are social forces that bring about change. change agents come from across the political economy, from civil society, business or government. these agents seek to change fundamental conditions. 'actors' are other organisation, who potentially may become change agents. but first, i examine which change agents might support a just transition, without needing much persuasion? labour unions coined the term just transition and would be key in supporting a just transition. the call for a just transition emerged from the trade union movement, starting in north america and then taken up by the international trade union confederation [ ] and the international labour organisation (ilo) [ , ] . the ilo adopted guidelines for a 'just transition for all towards an environmentally sustainable economy [which] needs to be well managed and contribute to the goals of decent work for all, social inclusion and the eradication of poverty' [ ] . while some labour unions have supported climate action strongly, others have opposed shifts away from fossil-fuel to protect their members' interests. a nuanced understanding of the internal tensions and differences within the labour movement is as important as analysis of the potential for labour to support just transitions. social movements are key change agents, with the ability to change paradigms and the parameters of a debate [ , ] . social movements promoting development have experience of working multiple coalitions, yet with uneven success and needing further strengthening [ ] [ ] [ ] [ ] [ ] . some social movements have been highly effective in mobilisation. examples include the occupy movement highlighting the inequalities of the % across many countries; the treatment action campaign which, through street pressure and legal strategy, made anti-retroviral drugs for hiv-positive people accessible and affordable [ ] . faith-based movements bring important moral perspectives to just transition [ , ] . extinction rebellion, fridaysforfuture and other social movements explicitly in response to the climate crisis [ ] are important change agents. cities are increasingly recognised globally as key actors in climate change and sustainable development. the paris decision makes explicit reference to non-state actors [ ] and sdg goal and new urban agenda highlight the key role of cities as actors implementing more sustainable development [ ] . a global covenant of mayors committed to reductions of urban greenhouse gas emissions of . gt per year by and . gt by [ ] . cities are increasingly organised transnationally through the compact, c , iclei local governments for sustainability, united cities and local governments and other networks. many cities have skilled staff, budgets and institutional capacity for change. further research is needed on how cities might relate to national governments and other change agents in a just transition. among business, there are different and even opposing views on just transitions, which require careful analysis. on the one hand, the renewable energy industry has an interest in just transitions, as the transition would favour its technologies. on the other hand, firms that have built their business models around coal and oil will have to be persuaded to change. haas [ ] suggests that there may be "passive revolution in a gramscian sense", finding that some transnational energy corporations seek to continue dominance the new energy regime in europe. other companies may take a long-term view and develop new business models, or individuals within such companies may defect from the dominant narrative and support the just transition. business alliances have formed about sustainable development and corporate social responsibility [ , ] , with the finance sector increasing promoting climate-related disclosure [ ] . patterns of investment will have to change to shift the trillions [ , ] from high to low emissions and reducing poverty. the united nations (un) brings together governments to address global issues multi-laterally and the unfccc has long aimed to play a "catalytic role" [ ] . collective action among nation-states has led to some remarkable successes, for example on restoring the ozone layer. inter-governmental organisations are key proponents of a just transition, notably through the un general assembly in its adoption of the sdgs [ ] , and the unfccc and its paris agreement [ ] . the broader un system includes undp, unep, iaea, icao, fao and other un agencies focus on development and climate issues within their mandates. the ability of multi-lateral agreements to govern the activities of transnational companies is limited [ ] . however, if the catalytic role of conventions like the unfccc can be strengthened and cooperation with non-state actors improved [ ] , multilateralism can be a core part of building a broad, global alliance for a just transition. the core elements were outlined in section , providing the building blocks of a renewed explanatory model. but how do ideology, change agents and fundamental conditions fit together, in order to develop a neo-gramscian theory of just transition? the relationships between these core elements are elaborated in this section, as well as the broad strategies and specific tactics that shift development pathways to realise a just transition. fig. shows core elements of the theoryideology, change agents and fundamental conditions. applying neo-gramscian theory, the explanatory model advanced here is that an alliance of change agents coalesces around an ideological elementthe just transitionand gains support of others, establishing a new hegemony, and is able to transform these fundamental conditions. combined, these elements and the relationships between them are a renewed explanatory model. fig. includes three propositions. first, change agents coalesce around the just transition as an ideological element. secondly, the alliance gains support from other actors. thirdly, having established hegemony, the alliance with this broader support can transform fundamental conditions, shifting development pathways to achieve zpzc. each of these propositions is elaborated below, together with strategies. a first proposition in fig. above is that an alliance of change agents coalesces around an ideological element, the just transition. support for a just transition is likely to come from labour unions, social movements, non-state actors, some firms and international organisations. these change agents were identified in section . above. how do these change agents coalesce around the just transition? the just transition serves as a unifying principle for an alliance. fig. illustrates change agents coalescing around a just transition. as an ideological element, the just transition pulls change agents together. the alliance is cemented by a common world-viewor what gramsci called an 'organic ideology' [ ] . analogous to gramsci's notion of a 'war of position' [ ] , a wide range of change agents unites around a just transition. the objectives of the just transition are depicted as zpzc, adding concrete goals to the vision. the just transition as an ideological element is also important in norm-setting. the concept of a just transition is broad, combining visions of human flourishing and a healthy planet and sub-elements appealing to a wide range of change agents. the breadth of the vision allows different change agents to support the overall vision. it is a vision addressing both climate and development. change agents actively join the alliance based on their support for a just transition. the alliance is broad as shown in fig. , with examples of various categories of change agents from government, business, labour and civil society. when a social actor joins the alliance supporting a just transition, it is defined as a change agent. actors outside the alliance, shown in fig. above outside the concentric circles are referred to as just 'actors'. within each category of change agent, there are different organisations. different change agents may hold different valuesas long as these include support for a just transition. different members of the alliance may have other concrete objectives, which is not an obstacle as long as all support the concrete objectives of a just transition, zpzc. the alliance can accommodate differences and tensions between goalssome members may be broadly campaigning for development or climate goals. ultimately, the just transition requires cooperation across a very wide range of actors. the breadth of the alliance requires organisation of broad front politics (discussed further in section c below). note that categories are not uniquely placed. for example, firms in the renewable energy industry are fully supportive the alliance, while those based on fossil fuels are likely nothence the former are placed within the concentric circles in fig. and the latter outside. organised labour has been a major change agent and indeed the labour movement coined the term 'just transition', but today many people are unemployed, depend for livelihoods on the informal sector, and artificial intelligence is redefining the future of work. organisations of workers, the unemployed and the informal sector are shown occupying positions both within alliance and also outside. alliances can be built at different spatial scales, from local to global. the predominant focus of just transitions has been at national scale, though there are international linkages and specificity to sub-national contexts. at the national scale, examples of alliances in related but different contexts include miners in the ruhr area of germany [ ] to communities dependent on coal on the highveld in south africa's mpumalanga province [ , ] . an inter-national alliance can coalesce around the concept of a just transition. the international labour organisation has developed guidelines for a just transition that might be applied in different contexts, and generally highlight the needs of the workforce and the creation of decent work and green jobs as part of any just transition [ ] . the "imperative of a just transition" is recognised in the preamble to the paris agreement [ ] . at the cop climate conference in katowice in poland, some heads of state adopted the 'solidarity and just transition silesia declaration' reiterating that these imperatives require "the creation of decent work and quality jobs" (hosg, ). while important in setting norms and building solidarity, action to transform fundamental conditions will take place at local scale. what the material and social basis of such an alliance might be depends on the change agents who lead the processes of just transitions. some might be led by social movements, while others might be initiated by elites. building an alliance requires political, cultural, socio-economic, moral transformational collective leadership by an alliance. forming an 'organic ideology' is practical work, carried out by institutions, with collective leadership by some individualsnot necessarily the leaders of organisations, gramsci's 'organic intellectuals'. such work is carried out by institutions and some individuals, but fundamentally ideology is created by a community or group. the politics of building alliance is critical for collective action on sustainability and development [ ] . this work of applies both to forming an ideology with those who support the just transition, and to persuading others thereby gaining hegemony. a key question is how broader support for a just transition can be built. how does a multi-actor, multi-issue alliance come together in support of a just transition, organising globally for local action? building an alliance of core change agents is a necessary but not sufficient condition for change at the pace and scale required for a just transition. to be able to change fundamental conditions, it is essential that the alliance gains support from other actors. in gramsci's terms, the alliance needs to establish cultural hegemony, building support through persuasion. a second strategy is essential, to gain support, including of those whose worldview would not incline them to support a just transition. otherwise, other actors coalesce around retrogression, defending a narrative of growth. at least some supporters of the currently hegemonic narrative need to change over their support to a just transition. if the alliance can replace the currently hegemonic ideology with a just transition, it can transform fundamental conditions of development and climate. here again, tensions between different goals can be accommodated among different goalsas long as there is broader support for the ideological element of a just transition. the new world-view represented by the just transition will be contested. four tactics are suggested as part of the strategy of building broader support are setting a direction with a meta-narrative of just transition, organising broad front politics, using communications smartly and thinking about instruments to build cooperation. firstly, the meta-narrative of the just transition sets a clear direction, while remaining broad enough to have wide appeal. what is the story that is compelling for more than a relatively small group of environmentalists? in developing the narrative of a just transition, the alliance should appeal to a broad set of moral foundations [ ] . narratives that frame a just transition in terms of a range of ethical arguments, values and mind-sets are important [ , ] . gramsci made it clear that cultural hegemony is not established by force, but is a relation 'of consent by means of political and ideological leadership' [ ] . similarly, the vision of a just transition is not imposed on others; rather, other actors need to be engaged so that at least some of them become change agents. as such, the process of a just transition will be complex and messy, not linear. even when the just transition becomes a widely accepted world view, it will be contested (or in gramscian terms, there will be counter-hegemony). the meta-narrative of a just transition will be contested. there are competing narratives advanced by the fossil fuel industry, agri-business, populist movements and certain presidents that climate change is a hoax and that the benefits of growth will trickle down. hence the just transition must, on the one hand, set out a clear alternative, while it must also have broad appeal. various factors might push those who would resist a just transition (including litigation, moral claims, activism), while push factors might include the potential for human flourishing, and avoiding the risk of economic instability [ ] . secondly, organisation is needed in the form of broad front politics. this is the short answer to the questions 'what types of politics can make the numerous energy and climate policies we discuss achievable?' [ ] . organisation is needed to build the coalition of change agents (see section b above) and broad front politics needs to mobilise those actors who might not strongly support the just transitionbut at least need not to oppose it. in gramsci's terms, this is a 'war of position -'this strategy of building up a broad bloc of varied social forces, unified by a common conception of the world' [ ] . the war of position is distinct from open conflict, the 'war of movement'. the war of position is a political and ideological struggle, in which the alliance builds influence and consent, developing a counter-hegemony to the currently existing. it is conducted across all civil society, not only in the economic sphere [ ] . broad front politics applies to change agents within the concentric circles in fig. above and to gaining support from actors outside the circle. the following elaboration refers to both aspects. in the trump era, noam chomsky has pointed out that individuals live in an 'atomized society lacking the kinds of associations (like unions) that can educate and organise' [ ] . that is a crucial difference between today's despair and the generally hopeful attitudes of many working people under much greater economic duress during the great depression of the s [ ] . without associations like unions or social movements, little is done to educate and organize. what is needed particularly are organisations that play a catalytic role in forming alliances, around multiple issues. broad front politics is needed, able to organise multiple change agents across several contradictions, in pursuit of a just transition. much as in gramsci's time, 'there must be a 'culturalsocial unity through which a multiplicity of dispersed wills, with heterogeneous aims, are welded together with a single aim' [ ] . broad-front politics includes multiple change agents, each organised around another issue but coming together around a just transition. different members of a broad front will be organising climate goals, others for inclusive development and so on. together, they will be organising a 'war of position' and creating a counter-hegemony. some examples of broad front on various issues exist. a broad front has been organised among national governments through the un and then through partnerships; organised sectorally or thematically. cities are increasingly organising transnationally. social movements are often local, but with examples like habitat for humanity or the climate strike showing international organisation for local action; other successes of social movements have been cited above. organising in the past included social relations 'embodied in a great variety of organisations and institutions including churches, political parties, trade unions, the mass media, cultural and voluntary associations' [ ] . each organisation need only contribute to an aspect of the just transition. what is new compared to a broad front in gramsci's times is that a broad front for a just transition must engage multiple issues. while articulating a common and public good, the just transition, the aim of organising is not to fight one other class or group, but to enable cooperation. there is some experience of coalitions across movements supporting labour and environmental issues [ ] and thinking about the role of bridge-builders for energy transitions [ ] . the meta-narrative of just transition is what cements the alliance together. the structure of a broad front is an overall group with sub-groups, pursuing a just transition which everyone can support. persuading those outside of the alliance to support or not oppose the just transition will be helped by a meta-narrative that appeals to broad set of arguments. such narratives should be based on all, or at least several, of five foundations of morality, which haidt identifies as: ) harm/care; ) fairness/reciprocity; ) in-group loyalty; ) authority/ respect; and ) purity/sanctity [ ] . he shows that 'liberals' favour the first two, while conservatives use all five foundations. a just transition needs to be framed around the purity of the atmosphere and of taking right action; support from many groups; authority of leaders at many scales, principles and institutions; leaving no-one behind; and sharing both burdens and benefits of the just transition fairly. this approach is consistent with the argument advanced by scoones, leach and newell [ ] , who argue that local innovation processes are "frequently motivated by a mesh of socio-cultural and livelihood concerns, and understandings of ecology and sustainabilitythat diverge from the narrow notions of 'green' and 'economic benefit' encompassed in most technocentric green economy discourses". the framing of the just transition is significantly broader, but thereby also contains internal tensions. thirdly, in order to draw in a wide range of actors, specific messages about the just transition are communicated to different audiences. the overall vision of a just transition remains the same, but it is articulated differently to various audiences. different actors will 'hear' the narrative of a just transition in different ways, relating them to their existing world-views [ ] . the types of information and feedback that are most effective in influencing actors in support of a just transition need to be considered, similar to a question in energy research [ ] . persuasion to introduce behavioural change is more effective if it addresses motivation at individual levels and structural transformation. klinsky [ ] among others has argued that more attention should be paid to the central importance of psychology in relation to equity and climate change. perceptions of justice matter in negotiations among countries, in that equity will only enable ambition "if parties, despite different views, 'perceive each other as being members of the same moral community'. psychological positions on fairness are shaped by underlying ideas about what the problem is, which in turn relate to interests, interpretations of the geopolitical context, and perceived power" [ ] . analytical challenges are complex, including many theoretical approaches, often focused on specific decisions, varying in formality and often not explaining change clearly [ ] . nevertheless, behavioural change is salient in individual agency and wider systems of practice [ ] , and an important part of developing communication tactics. the alliance connects to the specific issues of different sub-groups and to actors outside the alliance. given that the just transition combines a wider range of objectives, addressing multiple contradictions, it is only necessary that actors support the vision of a just transition. they may also support other objectives, not shared by other members of the alliance. it is here that tailored messages can be developed. as long as the actors find a common interest in the just transition, this is sufficient. fourthly, instruments to build cooperation should be further explored. cooperation between countries through multi-lateralism needs to be complemented by transnational organisation of cities, social movements and other change agents. development and climate change require collective solutionsyet in the latter, a key problem is the risk of free riders. some existing instruments might benefit from altruistic punishment, where actors punish others who are free riding, though it is costly for those punishing and yields no material gain. it has been shown that 'cooperation flourishes if altruistic punishment is possible, and breaks down if it is ruled out' [ ] . we need to construct systems that increase cooperation, take into account different moral arguments. if these cannot be integrated into a meta-narrative, instruments like altruistic punishment may increase cooperation. the previous sections have explained how an alliance coalesces around the ideological element, the just transition, and then gains broader support. with this support, the just transition becomes hegemonicit is common cause and common sense. the cultural hegemony of a just transition would mean that there is broad support to pursue a vision a vision of human flourishing and a healthy planet, goals of zero carbon and zero poverty, through inclusive and equitable development, as signified in fig. . establishing a counter-hegemony enables the transformation of fundamental conditions. so, consider the situation illustrated in fig. , in which an alliance has coalesced around the vision of a just transition and, having gained sufficient support, is able to transform fundament conditions. fig. shows the alliance coalesced around a just transition and the objectives of zero poverty and zero carbon (zpzc; on the left-hand side). the dotted line on the right-hand side indicates that the alliance has established a new cultural hegemony, with sufficient support from other actors. yet the question remains: how is the just transition to be achieved? achieving the objectives of the just transition requires shifting development pathways. it is by shifting development paths from the past and current direction to ones that can achieve zpzc. fig. shows illustrative paths from a situation of high poverty and carbon to zpzc. zero poverty is achieved by inclusive development. equitable development aims at reduced inequality. climate-resilient development avoids the worst impacts of climate, leading to loss and damage and makes possible a future world of zero impacts. development becomes lower-carbon and around mid-century reaches zero carbon globally. zero poverty and inequality lead to human flourishing. these development pathways are shown by the solid lines converging on the circle illustrating zero poverty and carbonas well as low impacts (aided by zero carbon) and reduced inequality (in the absence of poverty, though that does not imply zero inequality). the pathways indicate a different quality of development to the previous pathways shown in dotted lines. in high-level overview, fig. illustrates how a focus on development paths opens up a broad range of policies and actions, enabling transformational change across multiple objectives. each of the paths can be further unpacked; for examples, inclusive development could be the goal of a national development plan, a dedicated strategy for poverty reduction and specific to a sector, for example, eliminating energy poverty. the lowest dotted line in fig. , as another example, illustrates that the past pathways, based on fossil fuels for energy development and agri-business leading to rapid deforestation was high carbon. an energy development pathway needs to shift to renewable energy sources and transform patterns of land use, if we are to reach zero carbon. transforming such fundamental conditions requires fair sharing of energy and natural resources, agriculture sustaining livelihoods and decentralised power. shifting development pathways is not only a techno-economic exercise. many of these changes relate to the "ultimate drivers" [ ] (see section . above). the mind-shift to 'living well with less' (rather than more) is illustrated for energyusing less energy, more efficiently, mostly renewables leads to a good life for all. the energy system transitions to decentralised power, both technically for electricity systems and politically with energy democracy. shifts development pathways are determined by many decisions of multiple actors, but this does not mean they cannot be managed. major socio-technical transitions in the past have been unintentional, such as the transition from coal to oil. by contrast, many social changes have been purposefulfrom the abolition of slavery to women's suffrage to decolonial movements, to name a few. such transitions were not managed by governments, but entailed profound shifts in visions of what furthermore, scoones [ ] has argued that "transformations cannot be managed or controlled, but must draw on an unruly politics, involving diverse knowledges and multiple actors". habib points out that movements can rapidly shift the parameters of what is possible, though others need to sustain the gains through policy and institutions [ ] . a just transition requires adaptive management [ ] , which is not likely to be in the hands of any single change agent, and may be guided more or less by the state, in different contexts. further research is needed to understand how to shift development pathways. the ipcc framed six high-level enabling conditions that can be applied to shifts in development paths: governance, behaviour and lifestyles, innovation, enhancing institutional capacities, policy and finance [ ] . shifting development paths requires longer time horizons, systems thinking, adaptive management, and integrated decision making in community planning [ ] . this article proposes a neo-gramscian theory of just transitions. i have elaborated core components of ideology, hegemony, change agents and transforming fundamental conditions, combined them into an explanatory model and applied this to a better understanding of how to shift development pathways to zero carbon and zero poverty. this section qualifies this claim by acknowledging limitations, as well as pointing to areas that would benefit from further thinking. the first of the two objectives of this article, as set out in the introduction, is to advance a neo-gramscian theory of a just transition. the article is not the first to apply gramsci and neo-gramscian theory to environment and climate change (see section . ). i build on existing theoretical constructs, applying them to the just transition as an ideological element, and modified components which i call change agents (social forces) and fundamental rather than material conditions (as elaborated in section ). change agents coalesce around the concept of a just transition, and establish a new cultural hegemony. combining these elements and their relationships does provide a renewed explanatory model, a new conceptual framework. while i have presented such a theoretical framework in section , i make a limited claim to theoretical novelty, neither entirely creating new, synthesizing multiple nor or fully testing existing theory [ ] . the article thus claims a modest but also precise contribution in advancing existing neo-gramscian theoryits application to just transitions. even then, in writing a single paper, i can at best provide a theory of just transitions for further discussion in the community of practice. a theory that makes sense only to me is no use at all. the fuller development of a theory of just transitions will require a community effort. the second objective of this article was to apply the theory to our understanding of shifting development pathways. this objective has been pursued in section , yet achieved at an abstract level, illustrating how shifting development pathways to be more inclusive, equitable, climate-resilient and low-carbon can achieve zpzc. further research is needed to apply the theory in particular contexts, each with their specific political, social, economic and environmental context. given the understanding of the coalition of change agents, with broader support, driving such a change, i argue that this is a non-trivial application. this seems to me consistent with the theoretical nature of this article, and adequate for a single article. yet the limitation is clear, only a few examples are offered, without providing in-depth case studies. further research is needed to understand more concretely what shifting development pathways entails. applying the explanatory model to more granular case studies would be a useful direction for further research. this article proposes a theory of just transitions, in order to tackle the challenges of development and climate. a just transition is needed to shift from past development paths that brought us to a world of high carbon, poverty, inequality and impacts and carbon, to ones that achieve zero poverty and carbon. the article posits a just transition as an ideological element. building on neo-gramscian theory, this ideological element acts as a unifying vision, around which an alliance of change agents coalesces. the st century is characterized by multiple contradictions, some of which around material interest and others non-material, hence i modify gramsci's material to fundamental conditions. i explain the relationships between these elementsideology, change agents, hegemony and fundamental conditionsand advance a neo-gramscian theory of just transitions. the implications of the theory are that just transitions require coalitions of change agents coalescing around an ideological element -the just transition. the meta-narrative of a just transition brings together a range of change agents into alliances, which might initially include cities, social movements, some firms and countries, un agencies and others. the meta-narrative has broad appeal, allowing multiple organisations organising on multiple issues to uniting behind a just transition. a just transition requires organising broad front politics and finding ways to cooperate with others. this establishes a new world-view, or in neo-gramscian terms, cultural hegemony. yet there is contestation over just transitions and potential for counter-hegemony. the programme of a just transition alliance would focus on shifting development paths. the quality of development becomes inclusive, equitable, climate resilient and low carbon. this points to a just transition providing a basis for a new social contract, ensuring human flourishing and a healthy planet. while the objectives of this article are theoretical, i hope it will contribute to make difference in the world, across many countries and communities. good theory should help us reflect on our practice, and transform our world for the better. in this spirit, i will seek to engage many other intellectual, in the academy and gramsci's organic intellectuals. together, we might address the multiple challenges of our present conditions to transition to development that enables human flourishing and a healthy planet. the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. gramsci's political thought selections from the prison notebooks transforming our world: the agenda for sustainable development. a/ res/ / (sustainable development goals -sdgs) annex to decision /cp. , document fccc/cp/ / /add. , conceptualizations of justice in climate policy hot air or comprehensive progress? a critical assessment of the sdgs emissions: world has four times the work comment ontologies, socio-technical transitions (to sustainability), and the multi-level perspective human choice and climate change regime shifts to sustainability through processes of 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and comparing behavioural theories in models of social-ecological systems climate-relevant behavioral spillover and the potential contribution of social practice theory altruistic punishment in humans the adaptive challenge of climate change global warming of . • c. an ipcc special report on the impacts of global warming of . • c above pre-industrial levels and related global greenhouse gas emission pathways, in the context of strengthening the global response to the threat of climate change triggering transformative change: a development path approach to climate response in communities thanks to the heinrich boell foundation for support of my time, and to the rockefeller foundation bellagio centre for a wonderful space in which to reflect. i wish to thank janet small for reading of earlier drafts and encouragement, and anonymous reviewers for their valuable comments. all remaining errors are mine. key: cord- -cuatqy u authors: wei, maryann title: social distancing and lockdown – an introvert’s paradise? an empirical investigation on the association between introversion and the psychological impact of covid -related circumstantial changes date: - - journal: front psychol doi: . /fpsyg. . sha: doc_id: cord_uid: cuatqy u the coronavirus disease (covid ) pandemic has effected the implementation of social distancing and lockdown measures across the globe, and the psychological impact of associated life changes is experienced more severely by some individuals than others. anecdotal evidence points to a common belief among the general public that introverts are faring better than their extraverted counterparts to this end. however, the claim lacks empirical research, and seems counterintuitive when the broader literature on the association between introversion and mental health is considered. the current study investigated whether the psychological impact of covid -related circumstantial changes was moderated by introversion, based on outcome measures across psychosocial, cognitive, and affective domains. the role of several demographic factors in determining covid -related mental health symptoms was also examined. one hundred and fourteen individuals ( usa residents) completed measures of introversion, and reported on the extent to which they experienced loneliness, anxiety, depression and cognitive impairments as a function of covid -related circumstantial changes. results showed that introversion predicted more severe loneliness, anxiety, and depression experienced as a function of covid -related circumstantial changes, but not cognitive impairments. among the range of demographic factors examined (age, gender, living condition, recent unemployment), living with others (vs. living alone) predicted more severe covid -related mental health symptoms. however, these effects were only observed on outcome measures pertaining to anxiety and cognitive impairments, but not loneliness and depression. current findings have implications for both consumers and disseminators of information on popular internet hubs. current findings also highlight the possibility that living with others (close human affiliation) may have protective and detrimental effects on different domains of mental health during the covid pandemic. the introversion-extraversion dimension is central to leading trait theories of human personality in psychology (e.g., myers, ; cattell, ; eysenck, ; hathaway, ; mccrae and costa, , although exact conceptualisations vary between theories), and the construct is ubiquitous in both academic and popular literature. commonly described in dichotomic terms, introverts and extraverts are often differentiated by the sources they draw energy from (internal vs. external, respectively). adjectives traditionally associated with introversion include "inhibited, " "reserved, " and "undemonstrative, " while those associated with extraversion include "outgoing, " "friendly, " and "enthusiastic" (eysenck, ) . the coronavirus disease (covid ) pandemic has effected the implementation of social distancing and lockdown measures across the globe, and the psychological impact of associated life changes is experienced more severely by some individuals than others (williams et al., ) . anecdotal evidence points to a common belief among the general public that introverts are faring better than their extraverted counterparts to this end. for example, a "how to survive social distancing as an extravert" guide on a popular psychology website begins with the following statements: "for introverts, being stuck at home without social interaction for long periods of time really isn't the worst thing at all. they are accustomed to this time spent alone and feel energized and recharged by it. when it comes to extroverts, the idea of social distancing can feel like somewhat of a death sentence" (personality growth, ) . in articles published on other widely-frequented non-psychology websites, introversion has been championed as an asset for thriving in covid -related social isolation (e.g., bloomberg, ; reuters, ; the conversation, ) . such beliefs are exemplified in the influx of user-generated pictorial content (more colloquially known as "memes") across the internet with similar sentiments (see supplementary material for exemplars (data sheet )). on that grounds that introverts prefer less stimulating environments (myers, ; cattell, ; eysenck, ; hathaway, ; mccrae and costa, ) , the assumption that introverts experience the psychological impact of covid related circumstantial changes less severely than extraverts seems plausible. however, the claim lacks empirical research, and there are several lines of work in light of which the claim appears counterintuitive. first, introversion has been linked to personality traits associated with the tendency to experience more intense emotions and more difficulties in regulating these emotions, namely the "feeling" dimension of the myer-briggs type indicator (janowsky, ) and neuroticism, respectively (janowsky, ; jylha et al., ; fadda and scalas, ) . additionally, and possibly resultantly, introversion has also been associated with more psychological problems in general (janowsky, ; jylha et al., ; fadda and scalas, ) , and adjustment problems in particular. specifically, studies have demonstrated that introverts struggle more than extraverts in adjusting to life events which entail changes in day-to-day life, including shifts between educational institutes (bauer and liang, ; davidson et al., ) , job relocation (pinder, ) , and retirement (löckenhoff et al., ; robinson et al., ) . although increased amounts of time alone should in theory be welcome by introverts, these findings raise questions on whether introverts necessarily have an advantage over their extraverted counterparts in adapting to covid related circumstantial changes. additionally, the psychological impact of covid -related circumstantial changes (and mental health in general) has psychosocial, cognitive, and affective aspects, which in turn represent functional domains which may be differentially moderated by personality traits (segel-karpas and lachman, ) . the primary aim of this study was to examine whether the psychological impact of covid -related circumstantial changes is moderated by introversion, based on outcome measures across psychosocial, cognitive, and affective domains. a second aim was to examine the unique role of several other demographic factors (which were also considered as control variables in fulfilling the primary aim) in determining covid related mental health symptoms. between late april and early may , a call for participants for a study on the psychological impact of covid was placed on the sub-reddit r/samplesize, an online platform designated to connect researchers and research volunteers. based on previous research (shatz, ; jamnik and lane, ) and the current author's own experience, recruitment using this platform reliably produces quality data from adult individuals dominantly residing in the united states of america (usa). the latter demographic profile seems appropriate for the current research, given the high incidence of covid in the usa and the strictness of lockdown/social distancing measures which ensued. one hundred and fourteen individuals responded to the call for participants (mean age = . , sd = . ; female). sixty two respondents were located in the usa. the other respondents were distributed across the following countries, including united kingdom (n = ), canada (n = ), australia (n = ), germany (n = ). usa and non-usa residents were compared on all outcome variables (described below) to identify cases where the current sample could not be considered as a whole. introversion-extraversion was measured as a continuous dimension using the introversion scale developed by richmond and mccroskey ( ) . this scale was developed based on the extraversion subscale in the eysenck personality questionnaire (eysenck et al., ) . to illustrate, items such as "can you usually let yourself go and enjoy yourself at a lively party?" and "do you tend to keep in the background on social occasions?" in the eysenck personality questionnaire have counterparts in richmond and mccroskey's introversion scale in "can you usually let yourself go and have a good time at a party?" and "are you inclined to keep in the background on social occasions?, " respectively . the introversion scale consists of such statements. respondents indicate whether each statement applied to them on a -point scale ranging from strongly disagree ( ) to strongly agree ( ). six statements serve as distractors and are not scored. alpha reliability estimates were above. in the initial validation study by richmond and mccroskey ( ) , and closely matched in the current study (cronbach's alpha = . ). scores range between and , with higher scores indicating higher introversion, and lower scores indicating lower introversion (i.e., higher extraversion). the following demographics were measured as predictor variables of interest: age, gender, living condition (alone/with others), recent unemployment due to covid (no/yes). the psychological impact of covid -related circumstantial changes was measured with a battery of established questionnaires, with instructions modified to elicit mental health ratings directly associated with the implementation of social distancing and lockdown measures. that is, instead of reporting on mental health symptoms based on a given retrospective timeframe, participants were asked to provide ratings as a function of covid -related circumstantial changes. the exact phrase of instructions participants received is detailed in context below. functional aspects in the psychosocial, cognitive, and affective domains were measured, with the affective domain further broken down into depressive and anxious sub-domains. participants completed the dejong gierveld loneliness scale (de jong gierveld and van tilburg, ) to provide an indicator of loneliness as a function of covid -related circumstantial changes. participants responded "no", "more or less, " or "yes" to six statements, headed by the question "how true are these statements for you, following the implementation of covid social distancing and lockdown measures?" on negativelyworded statements (e.g., "i miss having people around me"), "more or less" and "yes" responses are scored as while "no" responses are scored as . on positively-worded statements (e.g., "there are enough people i can trust completely"), "more or less" and "no" responses are scored as while "yes" responses are scored as . scores are summed across items (range - ), where higher scores indicate higher loneliness. reliability and validity of the scale has been demonstrated across the lifespan (de jong gierveld and van tilburg, ). cronbach's alpha for the de jong gierveld loneliness scale as presented in the current sample was slightly below the conventional acceptable benchmark of . at . ; however, cronbach's alphas bordering on . were observed in the initial validation study (de jong gierveld and van tilburg, ) , so that internal consistency estimates in a slightly lower tier are likely normative given the few number of items in the scale (tavakol and dennick, ) . cognitive impairments associated with covid -related circumstantial changes were assessed with the cognitive failures questionnaire (cfq; (broadbent et al., ) , a item inventory of self-reported day-to-day slips and errors in cognition. instructions were phrased as follows: "the following questions are about minor mistakes which everyone makes from time to time, but some of which happen more often than others. we would like to know how often these things have happened to you, following the implementation of covid social distancing and lockdown measures." respondents indicated on a scale ranging from (never) to (very often) how often they experience certain incidents (e.g., "do you find you forget what you came to the shops to buy?"). scores (range - ) are summed across all items, where higher scores indicate more extreme cognitive impairments. in the initial pilot study, internal consistency of . was demonstrated (broadbent et al., ) . cronbach's alpha for the cfq as presented in the current sample was . . depression. depressive symptoms associated with covid related circumstantial changes were assessed with the patient health questionnaire [phq- ; (kroenke and spitzer, ) ]. instructions were phrased as follows: "to what extent (frequency) have you experienced these symptoms, following the implementation of covid social distancing and lockdown measures?" participants report the frequency with which they experience nine depressive symptoms on a -point scale ranging from "not at all" ( ) to "nearly everyday" ( ) (e.g., little interest or pleasure in doing things). scores are summed across the nine items (range - ), where higher scores indicate higher depression severity. the phq- has been validated not only as a useful tool to recognize clinical depression but also subthreshold depressive symptoms in the general population (martin et al., ) . cronbach's alpha for the phq- as presented in the current sample was . . anxiety. anxious symptoms associated with covid related circumstantial changes were assessed with the generalized anxiety disorder screener (gad- ; (spitzer et al., ) . the response format for the gad- is identical to that of the phq- . instructions for the gad- were also identical to that which were presently used for the phq- . scores are summed across seven items (i.e., seven symptoms of anxiety; e.g., not being able to stop or control worrying), where higher scores indicate higher anxiety severity (range - ). similar to the phq- , the gad- has been demonstrated as a reliable and valid measure of anxiety in the general population (löwe et al., ) . cronbach's alpha for the gad- as presented in the current sample was . . all analyses described as follows (including the generation of descriptives) were processed with spss (version ). to evaluate whether introversion moderates the psychological impact of covid -related circumstantial changes, scores on the introversion scale were used to predict each outcome variable listed above. hierarchical regression analyses were used, with demographic factors entered in the first step as control variables. the predictive value of each demographic factor across the range of outcome variables was also of research interest (pertaining to the second aim). where significant differences were observed on outcome variables between usa and non-usa residents, regression analyses were performed separately for the two groups. for comprehensiveness, the two groups were also compared on all predictor variables. table gives means and correlations between all study variables for the full sample. usa and non-usa residents did not differ on any of the predictor variables (age, gender, living condition, recent unemployment, and introversion). however, pertaining to outcome variables, usa and non-usa residents differed on the psychosocial domain. specifically, usa residents reported experiencing higher loneliness as a function of covid related circumstantial changes compared to non-usa residents (m = . vs. m = . on the dejong gierveld loneliness scale, respectively), t( ) = . , p = . . thus, regression analyses predicting loneliness were performed separately for usa and non-usa residents. table gives standardized β coefficients for predictor variables and associated model statistics in hierarchical regression analyses predicting the psychological impact of covid related circumstantial changes, across psychosocial, cognitive, and affective domains. after controlling for age, gender, living condition and recent unemployment, higher introversion (higher introversion scale scores) uniquely predicted higher depression (phq- ) and anxiety (gad- ) experienced as a function of covid -related circumstantial changes, β = . , t = . , p = . and β = . , t = . , p = . , respectively. higher introversion also uniquely predicted loneliness (dejong gierveld loneliness scale) experienced as a function of covid -related circumstantial changes after controlling for demographic factors, although this effect was unique to usa residents (β = . , t = . , p = . ). introversion did not predict cognitive impairments (cfq) related to covid circumstantial changes after controlling for demographic variables (β = . , t = . , p = . ). in a model including introversion, recent unemployment predicted higher loneliness experienced as a function of covid -related circumstantial changes only for non-usa residents (β = . , t = . , p = . ). interestingly, after including introversion in the model, living with others (vs. alone) was associated with more severe cognitive impairments and anxiety experienced as a function of covid -related circumstantial changes (β = . , t = . , p = . and β = . , t = . , p = . , respectively). it is worth noting that the living condition did not have predictive value for loneliness and depressive symptoms experienced as a function of covid -related circumstantial changes (see table ). this study examined whether the psychological impact of covid -related circumstantial changes was moderated by introversion, based on outcome measures across psychosocial, cognitive, and affective domains. as a second aim of the current study, the role of several other demographic factors in determining covid -related mental health symptoms was also examined. overall, higher introversion (i.e., lower extraversion) was associated with higher loneliness, depression and anxiety experienced as a function of covid -related circumstantial changes. the finding that introverts experience the psychosocial and affective impact of social distancing and lockdown measures more severely than their extraverted counterparts converges and deviates from previous literature in several ways. first, the finding is in line with previous studies demonstrating that introversion is associated with more psychological problems in general (janowsky, ; jylha et al., ; fadda and scalas, ) , and adjustment problems specifically (pinder, ; bauer and liang, ; löckenhoff et al., ; robinson et al., ; davidson et al., ) , however, this finding appears to be in disagreement with the notion that introversion is associated with a preference for less stimulating environments (myers, ; cattell, ; eysenck, ; hathaway, ; mccrae and costa, ) such as that created in everyday life following the implementation of social distancing and lockdown measures. in turn, this assumption has fuelled the lay belief that introverts are coping better during the covid pandemic compared to extraverts (detailed in section "introduction"). current findings may be best understood without considering the two lines of thought as mutually exclusive. introversion has been linked to decreased help-seeking behavior (swickert et al., ; atik and yalçin, ; kakhnovets, ) , which may in part explain higher psychological problems among introverts at baseline (janowsky, ; jylha et al., ; fadda and scalas, ) . when experiencing negative emotions, introverts are similarly more likely turn inwardly to cope (shapiro and alexander, ) . while introspective behaviors can facilitate emotional self-regulation, such habits can also function as a double-edged sword in perpetuating internalization (bowker and rubin, ) , rumination (verhaeghen et al., ; cohen and ferrari, ) , and worry (philippi and koenigs, ) -key cognitive underpinnings of loneliness, depression, and anxiety, respectively (beck, ; newman et al., ; ypsilanti, ) . such an account of why individuals higher on introversion might experience the psychosocial and affective impact of covid -related circumstantial changes more severely is corroborated by other aspects of present findings. specifically, cognitive impairments experienced as a function of social distancing and lockdown measures were not moderated by introversion, suggesting that cognitive function and activity remains intact across the introversion-extraversion dimension through covid -related circumstantial changes. current findings are in keeping with previous research demonstrating that functional domains of mental health are differentially moderated by personality traits (segel-karpas and lachman, ) , and highlight the particular relevance of evaluating domain-specific effects in research on the association between introversion and mental health. crucially, these findings have implications for both consumers and disseminators of information on popular internet hubs -specifically, to keep in view that the notion of introverts thriving under lockdown and social distancing conditions may not necessarily be empirically supported. mental health professionals dealing with covid -related psychological issues should also be aware that introverts may risk being erroneously left out of the mental health system. one aspect of present observations is worth noting before proceeding to discuss findings pertaining to the second aim of the current study. namely, the psychosocial impact of covid related circumstantial changes (loneliness) was predicted by both introversion and demographic factors (specifically, recent unemployment), but these effects were unique to usa and non-usa residents, respectively. there are several possible explanations for this observation including: ( ) predictors of covid -related loneliness differ qualitatively at different levels of loneliness severity, given usa residents reported higher loneliness as a function of covid -related circumstantial changes in the current sample, and ( ) the psychosocial impact of covid -related circumstantial changes is predicted by qualitatively different factors in different cultures. while not within the scope of the present study's aims, these speculations represent testable hypotheses which may be of interest in future research. besides recent unemployment, other demographic predictors examined were age, gender, and living condition. only living condition made a unique contribution to covid -related mental health symptoms after accounting for introversion. specifically, living with others (vs. living alone) was associated with experiencing more cognitive impairments and anxiety as a function of covid -related circumstantial changes. adjacently, it was observed that covid -related loneliness and depressive symptoms were not predicted by living condition. interpreted together, it is possible that close human affiliation serves as a protective buffer against social disconnectedness and low mood during the covid pandemic, but works in the opposite direction for clarity of thought and keeping calm. further information on household dynamics would have helped in the development of this speculation, but was not obtained in the present study. other limitations of the current study include its crosssectional nature, so that pre-covid mental health issues may have been conflated with covid -related mental health symptoms as presently assessed. on a related note, the current study assumes that presently used outcome measures (worded with reference to covid social distancing and lockdown measures) captured psychological health as shaped specifically by social orders placed as preventative measures against covid . however, responses on these measures may also reflect psychological health as impacted by the globalscale pandemic more generally, so that responses may not be tied solely to increased amounts of solitary time. further, demographic variables were considered only in broad strokes in the present study. accounting for a wider range of demographic variables, including but not limited to income, would allow for a clearer picture of the association between introversion and the psychological impact of covid -related circumstantial changes to be drawn. next, the participant count in each non-usa country was small in the present sample, so that non-usa respondents had to be collapsed in a single "non-usa residents" group. although the covid outbreak is considered a global pandemic, there may still be subtle differences in the covid impact between countries. more targeted and selective recruitment according to location/residence should be considered in future research. finally, given present interests in multiple outcome variables, the current study would have benefited in terms of statistical power from a larger sample size. the datasets presented in this study can be found in online repositories. the names of the repository/repositories and accession number(s) can be found in the article/ supplementary material. ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. the patients/participants provided their written informed consent to participate in this study. the author confirms being the sole contributor of this work and has approved it for publication. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fpsyg. . /full#supplementary-material help-seeking attitudes of university students: the role of personality traits and demographic factors the effect of personality and precollege characteristics on first-year activities and academic performance the evolution of the cognitive model of depression and its neurobiological correlates for introverts, quarantine can be a liberation self-consciousness, friendship quality, and adolescent internalizing problems the cognitive failures questionnaire (cfq) and its correlates the scientific analysis of personality take some time to think this over: the relation between rumination, indecision, and creativity introversion and medical student education: challenges for both 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cognitive functioning: the role of personality the experience of introversion: an integration of phenomenological, empirical, and jungian approaches fast, free, and targeted: reddit as a source for recruiting participants online a brief measure for assessing generalized anxiety disorder: the gad- extraversion, social support processes, and stress making sense of cronbach's alpha personalities that thrive in isolation and what we can all learn from time alone why we sing the blues: the relation between self-reflective rumination, mood, and creativity public perceptions and experiences of social distancing and social isolation during the covid- pandemic: a uk-based focus group study. medrxiv lonely but avoidant-the unfortunate juxtaposition of loneliness and self-disgust the author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © wei. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- - ebve k authors: rohr, jason r.; dobson, andrew p.; johnson, pieter t.j.; kilpatrick, a. marm; paull, sara h.; raffel, thomas r.; ruiz-moreno, diego; thomas, matthew b. title: frontiers in climate change–disease research date: - - journal: trends ecol evol doi: . /j.tree. . . sha: doc_id: cord_uid: ebve k the notion that climate change will generally increase human and wildlife diseases has garnered considerable public attention, but remains controversial and seems inconsistent with the expectation that climate change will also cause parasite extinctions. in this review, we highlight the frontiers in climate change–infectious disease research by reviewing knowledge gaps that make this controversy difficult to resolve. we suggest that forecasts of climate-change impacts on disease can be improved by more interdisciplinary collaborations, better linking of data and models, addressing confounding variables and context dependencies, and applying metabolic theory to host–parasite systems with consideration of community-level interactions and functional traits. finally, although we emphasize host–parasite interactions, we also highlight the applicability of these points to climate-change effects on species interactions in general. the notion that climate change will generally increase human and wildlife diseases has garnered considerable public attention, but remains controversial and seems inconsistent with the expectation that climate change will also cause parasite extinctions. in this review, we highlight the frontiers in climate change-infectious disease research by reviewing knowledge gaps that make this controversy difficult to resolve. we suggest that forecasts of climate-change impacts on disease can be improved by more interdisciplinary collaborations, better linking of data and models, addressing confounding variables and context dependencies, and applying metabolic theory to host-parasite systems with consideration of community-level interactions and functional traits. finally, although we emphasize host-parasite interactions, we also highlight the applicability of these points to climate-change effects on species interactions in general. the climate change-disease controversy global climate change and the unprecedented rate of infectious disease emergence represent two of the most formidable ecological problems of our time [ ] [ ] [ ] [ ] [ ] . several high-profile papers assert that climate change will increase the global distribution and prevalence of infectious diseases to the detriment of human health, biodiversity and ecosystem services (see glossary), which has placed climate change-disease interactions at the center of scientific, political and public agendas [ ] [ ] [ ] . indeed, there is compelling evidence that climate affects many diseases, including malaria, cholera, dengue and plague in humans [ ] [ ] [ ] [ ] , bluetongue in livestock [ ] and diseases of amphibians, turtles and corals [ , [ ] [ ] [ ] . however, the notion that climate change will generally increase diseases has been challenged recently in several papers demanding greater rigor and a better appreciation of the complexity of climate change-disease interactions [ , [ ] [ ] [ ] [ ] . these papers emphasized the presence of potentially confounding factors in many climate change-disease studies, calling into question whether climate change will, in fact, cause widespread increases in human and wildlife diseases. moreover, papers published on the absence of disease are scarce relative to those on its presence and, thus, there is likely to be a publication bias against climate preventing disease outbreaks. this controversy surrounding climate change-disease interactions underscores the need for a clearly defined research agenda. here, we outline key gaps in data, theory and scale that point to the frontiers in climate change-infectious disease research. we hope that this will help resolve this controversy, synthesize knowledge and advance understanding. we emphasize the interdisciplinary nature of the problem, encouraging collaborations among epidemiologists, disease ecologists, climatologists, modelers, geographical information system (gis) specialists, sociologists, economists and policy and management practitioners. amplifying host: a host that contributes positively to pathogen transmission, as opposed to a diluting host whose presence causes an overall decrease in transmission. complex life-cycle: a parasite life-cycle requiring more than one host species. dilution effect: a decrease in disease risk owing to an increase in host species diversity. direct transmission or life-cycle: a parasite life-cycle requiring only one host species. ecosystem service: processes and characteristics of ecosystems that benefit humans. emerging disease: a disease that is increasing in incidence or in its host or geographic range. ensemble modeling: an approach that integrates the forecasts of several climate change models. free-living stage: a stage of a parasite that lives outside of its host or hosts. herd immunity: the resistance of a whole group of hosts to an infectious agent, owing to the resistance to infection of a proportion of the group members. metabolic theory: describes how the rate at which organisms take up, transform and expend energy and materials (i.e. metabolic rate) controls ecological processes at all levels of organization, from individuals to the biosphere. prevalence: the proportion of hosts infected with a given parasite. secondary extinction: an extinction caused by the extinction of another species. corresponding author: rohr, j.r. (jasonrohr@gmail.com) gaps in data, models and their integration null models some of the controversy surrounding the effects of climate change on disease stems from questionable null models that can lead to erroneous conclusions. for example, although researchers frequently assume that pathogens will experience range expansions as they move pole-ward, tropical range contractions might also occur [ ] . it is presently unclear whether range shifts, contractions, or expansions are most likely and, thus, a neutral hypothesis of no change in the geographic ranges might be the most defensible null expectation [ , ] . similarly, the need to shift distributions pole-ward or to higher altitudes as the planet warms, coupled with species variation in dispersal abilities, has stimulated the hypothesis that climate change will cause phenological mismatch between parasites and their hosts [ ] . singer and parmesan [ ] , however, recently pointed out that evidence in support of this hypothesis is based on the null assumption of perfect synchrony, despite phenological mismatch being common before anthropogenic climate change, at least for some insect-host plant interactions. clearly, historical baseline data are needed to generate appropriate null models and test climate change-disease hypotheses properly. multiple variables, confounded variables and context dependencies predicting the impact of climate change on disease requires determining the net impact of numerous effects, including those that have opposing directions. a wellstudied example is the effect of temperature on the transmission of vector-borne pathogens, such as malaria. at cooler temperatures (e.g. c), an increase in temperature is expected to increase not only biting rates, parasite replication within mosquitoes and mosquito development, but also mosquito mortality, making the net effect of increasing temperatures difficult to determine. mathematical models provide a powerful tool for integrating these complex interactions, but model validation requires welldesigned laboratory experiments (box ) and field data sets that are long and detailed enough to enable fitting of the relevant parameters. disease control efforts can also make it challenging to determine effects of climate change on disease. for instance, if climate change causes range shifts of parasites from tropical to temperate countries, this might result in an overall reduction, rather than increase, in human diseases because temperate countries often have superior health infrastructures [ ] . regardless of the outcome of climate change on diseases, integrating control efforts into projections should improve predictions of future disease risk for humans and wildlife. furthermore, given that control measures could obscure increases in transmission, incorporating control measures into models could reveal underlying increases in disease risk that might otherwise be missed. similar to disease control measures, intrinsic factors, such as temporal variation in herd immunity, pathogen spread and parasite evolution, can co-vary with changing climate [ ] . intrinsic factors can give rise to oscillations in disease whose frequencies might differ from those of extrinsic drivers, making it difficult to identify the contributions of each to temporal population patterns [ ] . for instance, the effects of climate on cholera dynamics became more evident after controlling for cycles in temporary immunity because climate has fewer impacts on cholera when a large fraction of humans are resistant to the bacterium [ ] . as another example, effects of el niñ o southern oscillation (enso) events and climatic variability on disease-related amphibian declines were only revealed after controlling for a multidecadal pattern in extinctions that was probably caused by the spread of the pathogenic chytrid fungus [ ] . observational studies can identify the best climatic predictors of disease-related response variables, but manipulative experiments are crucial for testing whether climatic factors truly have causal relationships with disease. here, we present some common issues with climate-change experiments and suggest improvements that should enhance the quality of data obtained from future studies. issue: climate-change researchers commonly treat samples within a single environmental chamber as independent replicates of temperature (e.g. [ ] ) (i.e. pseudoreplication), which can confound temperature with anything else that might differ among chambers (e.g. light, humidity or air circulation). have true and adequate replication of temperature treatments, for instance by building a large number of independent incubators out of styrofoam, heat tape and thermostats. replicate the effect of temperature in time (i.e. temporal blocks). place multiple experimental units within each incubator and analyze the data using appropriately nested mixed-effects models that treat the chamber as the level of replication for testing temperature effects (e.g. [ ] ). such analyses require a minimum of four chambers to compare two temperature treatments. including more than two levels of temperature and treating temperature as a continuous predictor. this can enable the detection of nonlinearities and is required to provide functional relationships for integrative and/or predictive models [ ] . conduct meta-analyses of independent tests of temperature effects [ ] . issue: in many experiments, all individual organisms are initially held at a single temperature and then a subset are transferred to higher or lower temperatures and experimentally infected with a pathogen. in this design, temperature is confounded with the magnitude of the temperature shift that occurs at the start of the experiment, making it unclear which is driving any observed effect of the temperature treatment. improvement: adequately acclimate study organisms to the temperature of interest before applying treatments. issue: field experiments often provide more ecologically relevant data than do laboratory experiments, but it can be challenging to manipulate climate in the field. improvements: use creative ideas for manipulating climate in field experiments. heating coils or continuous co input chambers can be used to replicate climate or climate-associated treatments. increase temperatures via the greenhouse effect by enclosing small, open-top plots in clear plastic [ ] . trends in ecology and evolution june , vol. , no. unconsidered components of climate are also potential confounders of disease-climate interactions. many hosts and pathogens are influenced by the interactive effects of multiple abiotic and climatic factors, such as moisture, temperature and co [ , ] . imagine the hypothetical scenario in which a region experiences increases in temperature and precipitation that have opposite effects of a similar magnitude on a disease. a univariate analysis might erroneously suggest that the host-pathogen system was insensitive to the climate change. despite the obvious need to understand how climate components interact to affect disease, there is a paucity of studies that do so. also problematic is the likelihood that diseases are affected by interactions between climate and other natural enemies [ , ] or environmental changes (e.g. land-use change) [ , ] . nonlinear effects can generate important and surprising climate impacts on host-pathogen interactions. for instance, the fitness of most organisms decreases in either direction away from the optimal climate (although more complex nonlinear functions can also occur). hence, changes in climate should often generate nonlinear effects on fitness, which contrasts with the frequent expectation that there will be consistent increases or decreases in host or parasite fitness with climate change. these expectations are probably only justified for small climatic shifts that do not cross the optima of an organism. however, most studies have insufficient variation in climate to detect nonlinearities and few generate reliable parameter estimates for modeling (box ). one example of a nonlinearity is given by threshold responses, which transpire when large shifts in variable response (e.g. transmission intensity) occur over narrow windows of change in a crucial parameter, such as temperature [ ] [ ] [ ] . climatic threshold responses have been suggested to be important in biodiversity losses [ ] and human, coral and plant disease outbreaks [ , , ] . for instance, evidence suggests that coupling between cholera dynamics and climate is transient, occurring only during strong enso events. this observation is consistent with a threshold response, whereby climate is only an important driver of cholera during climatic extremes [ ] . threshold responses are difficult to capture with standard linear statistical models and challenges associated with stochasticity, finite population sizes, time-lags and covariates present additional obstacles to detecting thresholds [ ] . more sophisticated statistical approaches that allow for nonlinearities [ ] , as well as other techniques, such as scale-dependent correlation analysis [ ] , the significant zero crossings (sizer) model [ ] , or models that allow for flexible treatment of regime shifts [ ] , might be necessary to detect climatic threshold effects on disease. improved data and data-model integration this review of null models, confounding variables, context dependencies and nonlinearities in climate-disease interactions underscores the need for: (i) better data on baseline interactions and intrinsic and extrinsic factors affecting disease; (ii) long-term data sets that can effectively parse out how variation is explained by different factors [ , ] (e.g. control efforts, host immunity); (iii) improved use and development of statistical and mathematical models to reveal adequately relationships between climate and disease dynamics, ideally in conjunction with a more thorough integration of field and experimental data (box ); and (iv) the need to consider both climate change and alternative hypotheses as drivers of disease (using information theoretic approaches; e.g. [ , , ] ) ( figure ). ideally, both experiments and models should take into account the substantial uncertainty associated with climate projections, for instance by using model-averaging or ensemble-based approaches [ ] . whenever possible, model assumptions should be made explicit and models should be effectively validated [ ] . improving data collection and modeling efforts will require collaborations among epidemiologists, disease ecologists, statisticians, modelers and gis specialists. furthermore, judicious decision-making will require effectively communicating scientific results to sociologists, economists and policy and management practitioners, who must carefully weigh scientific findings, economic costs and public perceptions. as data on climate change-disease interactions accumulate, the time is ripe for the maturation of predictive theories on climate change-disease interactions. we suggest three areas of theoretical development ( figure ): (i) theory to predict the outcomes of specific host-parasite interactions as a function of climate; (ii) theory to predict where on the earth climate change will have the greatest impact; and (iii) theory to predict which host-parasite systems might be most sensitive to climate change (box ). the metabolic theory of ecology has been useful in describing biological patterns from organismal to macroecological levels [ ] . although metabolic theory is often too coarse to predict accurately outcomes of fine-scale differences among metabolic rates of organisms, it captures broad variation among organisms that differ substantially in size [ ] . given that most parasites are orders of magnitude smaller than their hosts, metabolic theory might be useful in predicting and understanding the outcome of climate-dependent host-parasite interactions [ ] . this should be especially true for parasites with free-living stages and for poikilothermic hosts, whose body temperatures fluctuate with environmental temperatures [ ] . as an example, global climate change is expected to increase climatic variability [ , ] and metabolic theory offers predictions for how host-parasite interactions might respond to this climate change. first, owing to their faster metabolisms, parasites should acclimate to temperature shifts more quickly than will their hosts [ ] , perhaps providing them with a temporary advantage in host-parasite interactions. second, smaller organisms have fewer cells and processes requiring adjustment following temperature shifts and, thus, generally withstand greater temperature extremes than do larger organisms [ ] . finally, owing to their shorter generation times, parasites opinion trends in ecology and evolution june , vol. , no. should evolve more quickly than their hosts to changes in climate. nevertheless, most research on climate change and disease has neglected evolutionary processes, despite evidence highlighting the importance of evolution in disease processes on ecological timescales [ ] and in mitigating against the impacts of climate change [ ] . in support of these predictions derived from metabolic theory, recent empirical and theoretical evidence suggests that increased variability in temperature can alter hostparasite interactions. for instance, temperature variability appears to be important in amphibian-chytrid fungal interactions [ , , ] (figure c ), avian influenza outbreaks [ ] and malaria epidemics in the east african highlands [ ] . additionally, diurnal fluctuations around low mean temperatures accelerated the growth of plasmodium parasites (the causative agent of malaria) relative to an equivalent constant mean temperature (figure a) , whereas fluctuations around high mean temperatures slowed parasite growth (figure b) [ , ] . why temperature shifts sometimes benefit the pathogen and other times the host remains unclear, but further research on the impacts of climatic means versus variances will be needed if one is to predict accurately the impacts of climate change. locations where climate change will have the greatest impact identifying the geographic regions of the earth that are most vulnerable to climate change will greatly assist in targeting disease management and monitoring efforts. there are two general schools of thought on determining where climate change will have the greatest impacts. the more traditional notion is that organisms in temperate and polar regions will be most affected by climate change because temperatures are increasing disproportionately in these regions relative to the tropics [ , , ] . recent work, however, suggests that tropical hosts and parasites might be as impacted by increasing temperatures as those at higher latitudes, despite the smaller increases in tropical temperatures [ , ] . because tropical climates are less variable, tropical organisms are adapted to much narrower temperature ranges than are temperate and arctic species and, thus, are expected to be more sensitive to small changes in climate [ ] . additionally, because metabolic rate increases exponentially with temperature, organisms in the warmer tropics experience a greater change in metabolism with each unit increase in temperature than do organisms in temperate and polar better theory • can metabolic theory predict climate effects on parasitism in different locations and systems? • can functional traits and control measures be used to predict climate-induced changes in range, incidence, or severity of parasitic diseases (fig. dilutio tank treatm chem conce bd categ quant a y y a c. n n . c a n n a a n n a c. y y . c a y y a a y y a better data and models • gather higher-quality data over longer time periods • conduct improved experiments to help inform predictive models (box ) • incorporate new factors into models: means and variances of interacting climate components, justified null models, uncertainties, nonlinearities, disease control measures (box ), intrinsic factors, and alternative hypotheses for disease dynamics regions [ ] . indeed, when considering both recent global warming and the exponential relationship between metabolic rate and temperature, dillon et al. [ ] estimated that organisms in tropical and northern temperate zones are experiencing the largest absolute increase in metabolic rates and, thus, are being impacted most by climate change. although metabolic theory has the potential to help predict host-parasite outcomes and geographic regions where organisms might be most affected by climate change, we suspect that several issues will need to be addressed before its predictive abilities can be validated. first, it is unclear whether parasites will follow the same metabolic 'rules' as box . a risk matrix for predicting host-parasite systems sensitive to climate change determining which host-parasite systems are most sensitive to climate change will also help to target management and monitoring efforts. parasites with poikilothermic hosts, vectors and free-living stages, or that live at high latitudes or elevations, have greater exposure to variable climatic conditions and, thus, might be more likely to respond directly to changes in climate than will parasites with endothermic hosts and direct transmission [ ] . although these intrinsic properties of host-pathogen systems might determine 'fundamental' sensitivity to direct effects of climate change, the 'realized' effects of climate change will be determined by behavioral adjustments (e.g. microclimate selection by vectors), extrinsic adaptation and evolution by hosts, vectors and pathogens, as well as disease control measures. hence, we suggest that a functional, trait-based approach, which addresses direct sensitivity to climatic factors, coupled with an understanding of control measures and the other confounding factors mentioned in the section ''gaps in data, models and their integration'' might prove valuable for determining the overall significance of climate change for different diseases. this risk matrix results in six general disease categories, where overall risk is the product of direct sensitivity to climate and management difficulty ( figure i) . some diseases, such as those restricted to high elevations or latitudes or that prefer cooler temperatures, might experience range contractions with climate change (categories , ). for example, several fungal entomopathogens of insects are expected to decline [ ] (category ). similarly, white pine blister rust, cronartium ribicola, which costs more to control than any other conifer disease, is expected to decrease if conditions get warmer and drier [ ] (category ). indeed, many pests of crops are expected to decrease under warmer and drier conditions [ ] . category diseases show limited direct responses to climate and have good options for control. an example is measles, which is directly transmitted and has a highly effective vaccine. category diseases are also relatively insensitive to direct effects of climate change, but have less effective mitigation measures. this category might apply to numerous wildlife and zoonotic viruses for which options for management or control are limited (e.g. sars corona virus, hendra virus, nipah virus and ebola virus). other diseases are more directly sensitive to climate-change impacts, but might be countered by effective control measures (category ). for example, malaria is sensitive to climate change but high capacity exists for mitigation in developed regions, such as europe and the usa [ , ] , although it may be a category disease in other regions where resources are more limited [ , ] . category diseases, which are both sensitive to climate and difficult to control, would also include many wildlife diseases, such as chytridiomycosis in frogs [ , ] and various diseases of coral [ , ] . this risk matrix emphasizes the direct sensitivity of hosts and parasites to climate change, but host-parasite systems can also be affected by climate through more subtle indirect mechanisms. it is therefore important to quantify how climate change modulate hostparasite interactions both directly and indirectly. direct impact of climatic change free-living organisms. second, the metabolic approach does not yet explicitly incorporate species interactions, such as parasitism [ ] , and accounting for these interactions often improves predictions of climate-change impacts [ ] . third, the emphasis of metabolic theory has been on effects of mean temperature, but changes in other climatic components, such as precipitation and climatic variability, also could impact species interactions (figure ), especially for parasites with life stages outside the host. finally, understanding of how abiotic factors influence host immunity remains in its infancy [ , ] , but will probably have an important role in predicting the outcome of host-parasite interactions (e.g. [ ] ). until more of these knowledge gaps are filled, it will remain unclear whether climate change will have the biggest impact on host-parasite interactions at mid-high latitudes, in the tropics, or at difficult-topredict locations scattered throughout the world (figure ). addressing these gaps will require collaborations among physiologists, immunologists, community ecologists, climatologists and modelers. gaps in scale: a community-and biodiversity-based perspective contemporary research has uncovered the importance of community dynamics to parasite transmission and vice versa [ , , ] and how biodiversity buffers communities against both disease [ ] [ ] [ ] [ ] and climate change [ ] . nevertheless, most disease research has emphasized single host-single parasite interactions [ ] . thus, under-standing of climate change impacts at the scale of whole communities of hosts and parasites remains early in development. in particular, there is an apparent paradox, at the level of communities, which has not been explicitly mentioned in the climate change-disease literature. evidence is mounting that climate change will reduce biodiversity [ , ] , including parasite diversity [ ] . indeed, parasites might be more sensitive to secondary extinctions than might nonparasitic species [ ] . this expected loss of parasite diversity, however, seems to be at odds with the notion that climate change will generally increase diseases [ ] [ ] [ ] . to shed light on this apparent paradox, researchers must understand the patterns of climate-induced parasite declines and the dilution effect, the hypothesis that biodiversity generally reduces wildlife and human diseases [ ] [ ] [ ] [ ] (figure ). this will only occur with collaborations among epidemiologists, theoreticians and community ecologists. climate-driven patterns in declines of parasite species if climate change causes parasite extinctions rather than just range shifts [ ] , the probable non-random nature of these declines [ ] could influence disease severity. for instance, relative to generalist parasites, parasites that specialize on one or a few hosts should be more likely to go extinct as their hosts decline [ ] . furthermore, we predict that climate change will cause more extinctions of parasites with complex life-cycles than of those with direct trends in ecology and evolution june , vol. , no. transmission, because there is a greater chance that at least one of their necessary host species will go extinct with climate change. we also expect a greater fraction of human parasites to go extinct in tropical than temperate regions because a higher percentage of tropical human diseases specialize on a vector species ( % tropical versus % temperate) and/or a wild animal reservoir ( % tropical versus % temperate) [ ] . although parasite extinctions might reduce wildlife and zoonotic diseases, the severity of the remaining diseases could increase or decrease. if, for instance, rare or less pathogenic parasites go extinct more so than abundant or highly virulent parasites, then the impact of parasite extinctions on overall disease incidence and severity might be small. however, the loss of many rare parasite species could be substantial. furthermore, range shifts could expose hosts to novel parasites, which might lead to more severe disease than in disease-endemic areas. this is a concern for potential climate-induced range shifts of human malaria [ ] . in addition, generalist parasites, which might be more likely to persist with climate change (see above), can be more challenging to control because they can be maintained by multiple host species and can therefore persist with higher virulence to a subset of their host species relative to specialist parasites [ ] . increased temperatures might also increase the frequency and intensity of transmission by lengthening the 'growing season' of parasites that survive climate change [ ] . however, the same changes might sometimes drive decreases in transmission if host immunity is enhanced at higher temperatures or when temperatures exceed the optimum for parasite transmission. finally, global warming is generally increasing temperature minima more than maxima and this might be more likely to move temperatures for parasite and vector performance towards their optima than beyond it [ ] . the severity of disease is also likely to be altered by climate-driven changes to host composition. in some host-parasite systems, the most abundant and resilient species are also hosts that amplify transmission, whereas other species might decrease disease risk [ ] . in some of these systems, such 'amplifying' hosts increase in abundance as the density of less resilient, 'diluting' hosts decline [ ] . if extinctions caused by climate change are biased towards these rarer hosts, as we suggest, and these are indeed disease-diluting hosts, climate change might reduce the disease-buffering capacity of biodiversity and increase prevalence and severity of diseases that persist with climate change [ ] [ ] [ ] . however, the relative contributions of individual species to transmission are poorly known for most pathogens and, thus, the significance of any loss in host species remains uncertain. accounting: determining net effects most importantly, the net effect of any anthropogenic factor on disease requires careful accounting [ , ] . researchers must balance the loss of parasites against the loss of the buffering capacity of biodiversity, changes in disease severity, impacts of emerging co-infections (e.g. effects of hiv emergence on malaria [ , ] ) and the ability of humans to enact control measures. regardless of what this accounting reveals, researchers would be remiss to ignore impending changes to parasite, host and non-host diversity when forecasting the effects of climate change on host-parasite interactions. understanding climate change-disease interactions is a formidable problem because of its interdisciplinary nature and the complexities of hosts, parasites and their interactions with the multiple factors that can co-vary with climate change. effective forecasting of climate-change impacts on disease will require filling the many gaps in data, theory and scale (figure ). although this 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of a pesticide on amphibians? partitioning effects on exposure and susceptibility to pollution knowing when to draw the line: designing more informative ecological experiments climate warming strengthens indirect interactions in an old-field food web climate change and forest diseases we thank mercedes pascual for comments on this paper and the ecological society of america for sponsoring the symposium on climate change and disease that helped develop the ideas presented here. key: cord- -hvrly e authors: stanton, robert; to, quyen g.; khalesi, saman; williams, susan l.; alley, stephanie j.; thwaite, tanya l.; fenning, andrew s.; vandelanotte, corneel title: depression, anxiety and stress during covid- : associations with changes in physical activity, sleep, tobacco and alcohol use in australian adults date: - - journal: int j environ res public health doi: . /ijerph sha: doc_id: cord_uid: hvrly e the novel coronavirus (covid- ) has enforced dramatic changes to daily living including economic and health impacts. evidence for the impact of these changes on our physical and mental health and health behaviors is limited. we examined the associations between psychological distress and changes in selected health behaviors since the onset of covid- in australia. an online survey was distributed in april and included measures of depression, anxiety, stress, physical activity, sleep, alcohol intake and cigarette smoking. the survey was completed by adults (mean age . ± . years, % female). negative change was reported for physical activity ( . %), sleep ( . %), alcohol ( . %) and smoking ( . %) since the onset of the covid- pandemic. significantly higher scores in one or more psychological distress states were found for females, and those not in a relationship, in the lowest income category, aged – years, or with a chronic illness. negative changes in physical activity, sleep, smoking and alcohol intake were associated with higher depression, anxiety and stress symptoms. health-promotion strategies directed at adopting or maintaining positive health-related behaviors should be utilized to address increases in psychological distress during the pandemic. ongoing evaluation of the impact of lifestyle changes associated with the pandemic is needed. first reported in november , the novel coronavirus (covid- ) has resulted in a global health emergency. as of june , the virus has claimed more than , lives globally and infected more than . million people. the scale of the pandemic has resulted in worldwide concern, not only for the loss of life but also the social and economic impacts. there is significant concern over how the changes in the ways that people normally engage in everyday activities impact their health and well-being. this is especially relevant for those in self-isolation or quarantine, where feelings of depression, fear, guilt, and anger may manifest [ ] . in australia, similar to other countries, social distancing, travel bans, the cancellation of sporting and other mass participation events, and changes to work practices have dramatically affected daily life. the partial lockdown procedures implemented by the australian government to protect citizens and reduce the spread of the virus forced the closure of many businesses in late march saw unemployment levels rise to . %, more than three times higher than the pre-covid- predicted unemployment rate [ ] . the impact of these changes likely comes at significant personal cost, including the onset, or worsening, of mental health issues. to address the psychological distress experienced by australians in the current pandemic, more than aud million has been committed to the development and delivery of mental health and well-being support services in australia. multiple calls to ensure the preparedness of psychological services have been presented [ ] [ ] [ ] ; however, the uptake and immediate and long-term impacts of these services are unclear. the covid- pandemic may also lead to adverse changes in health behaviors, such as physical activity, smoking, alcohol use and sleep. with the enactment of social isolation and physical distancing restrictions in march , the usual places to be physically active, such as gyms and outdoor recreation facilities, were no longer accessible. although some people may have sufficient autonomous regulation of physical activity to pursue alternate activities (e.g., online fitness classes, other home-based physical activities), others may reduce their physical activity due to the lack of social support available or concerns for contracting the virus in an outdoor environment. on the other hand, those forced to work from home may have spent less time commuting, and may have seized the opportunity to create new physical activity habits. alternatively, since exercise was one of few legitimate reasons for being able to leave the home some people may have developed a walking or cycling habit as a reason to escape being housebound. as many studies have demonstrated strong positive associations between physical activity and lower psychological distress [ , ] , the commencement or continuation of physical activity during the pandemic will likely aid in reducing psychological distress. however, some concern has been expressed regarding increased risk of respiratory illness in those engaged in high-and very-high intensity exercise due to the potential for reduced immune response [ ] . in contrast to health-promoting behavior such as physical activity, some people may manage social isolation and any pandemic-related psychological distress by commencing or increasing adverse health behaviors such as smoking or alcohol use. since covid- is an acute respiratory illness, commencement or continuation of tobacco use during the covid- pandemic may lead to the worsening of outcomes for those infected with the virus [ ] . indeed, early indications suggest the proportion of current and former smokers is higher among those with severe disease and among those admitted to intensive care and requiring ventilation [ , ] . harmful intake of alcohol leads to neuroadaptations that exacerbate alcohol cravings during times of stress [ ] . hence, social isolation, coupled with changes in employment status or uncertainty about the future may trigger an increase in alcohol intake for susceptible individuals [ ] . the combined effect of changes in lifestyle behaviors; confinement to the home through government restrictions in travel; and elevated depression, anxiety and stress associated with the current covid- pandemic, may have significant negative impacts on sleep [ ] . this has been especially evident in healthcare workers, who may be required to work longer shifts in highly stressful environments [ , ] . poorer sleep quality has been associated with higher levels of depression, stress, and anxiety [ ] . maintaining sleep quality is important in strengthening immunity [ ] , hence any sleep disturbances subsequent to covid- -pandemic-induced stress, may increase susceptibility to infection, or compromise recovery in the case of infection [ ] . there is currently limited research regarding psychological distress subsequent to the covid- pandemic. two recent studies from china reported high levels of psychological distress during the initial stages of the pandemic [ , ] ; however, the association between psychological distress and health behaviors remains unclear. early evidence during the covid- outbreak suggests positive associations between increased physical activity and physical health and inverse associations between sedentary behavior and physical and mental health outcomes [ ] . a more detailed exploration of health behaviors during stages of the covid- pandemic may help direct future public health messaging to promote positive behaviors and guard against uptake or the worsening of negative behaviors in order to maintain community well-being and mental health. therefore, the present study aims to examine associations between depression, anxiety and stress and changes in health behaviors, including physical activity, sleep, smoking and alcohol use subsequent to the onset of covid- and the implementation of social isolation rules in australia. an anonymous online survey was hosted on the survey platform qualtrics and distributed using social media sources (facebook and twitter) and via institutional sources including email and public marketing. eligible participants included all australian adults aged years and over. ethical approval was granted by central queensland university's human research ethics committee (approval number ). data collection occurred between and april . at the time of survey distribution, australia was in the midst of significant personal distancing, partial lockdown and travel restrictions. social distancing measures included keeping a minimum . meters between people, a ban on any public gatherings, a limit of no more than five people at personal gatherings such as weddings and funerals, and no person was allowed to meet with more than one other person outside of their own household. lockdown restrictions also included the closure of restaurants and bars, many retail stores, and restricted access to outdoor parks. most schools were closed, with students advised to study from home while being supported by online learning platforms and materials. university campuses limited or ceased face-to-face teaching and transitioned to online learning, with most clinical placements, residential schools, and simulations postponed. inter-and intra-state travel was banned, and travel within towns and cities was only permitted for essential work/workers, or to access essential services such as medical or health care, or to shop for groceries. existing covid- surveys from china, the united kingdom and germany were reviewed to inform development of the present survey. in addition to demographic information, the survey included questions examining chronic health conditions; depression, anxiety and stress; and physical and health behaviors such as physical activity, sleep, smoking and alcohol consumption. the current paper only reports on measures included in the survey associated with the study aim. demographic characteristics included age, gender, marital status, educational attainment, income and chronic disease status. psychological distress was assessed using the well-established -item depression, anxiety and stress scale (dass ) [ ] . seven items for each component were scored on a -point likert scale ranging from (did not apply to me at all) to (applied to me very much, or most of the time). scores for depression, anxiety and stress items were summed with valid scores ranging from - for each component. symptom severity was scored according standard cut-points [ ] . physical activity was assessed using the active australia survey (aas) which comprises eight items assessing frequency and duration of walking, moderate and vigorous leisure physical activities, and vigorous gardening over the past seven days. total physical activity was calculated according to the aas guidelines, where total minutes of physical activity = minutes of walking + minutes of moderate activity + (minutes of vigorous activity × ) [ ] . a single item asked participants to report their change in physical activity since the onset of covid- , with six response options ranging from (i am much more physically active than usual) to (i have ceased physical activity altogether). sleep was assessed using two items. first, participants were asked how many hours, on average, they slept per night prior to the onset of the covid- pandemic (sleep quantity). second, participants indicated the effect of the covid- pandemic on current sleep quality using the question, "since the onset of the covid- pandemic, i...". five response options ranged from "am sleeping much better than usual" to "am sleeping much worse than usual". smoking behavior was assessed by asking whether respondents consumed cigarettes or other tobacco products prior to the onset of covid- . change in smoking behavior was examined using a single item with ten response options ranging from "since the onset of the covid- pandemic, i... smoke much more than usual", to "have not smoked (i am a non-smoker)". current alcohol use was examined using the first item of the alcohol use disorder identification test consumption (audit-c) [ ] , which asks how often alcohol is consumed. response options were "never", "monthly or less", " - times a month", " - times a week", and " or more times a week". changes in alcohol consumption was assessed using a single self-report question: "since the onset of the covid- pandemic i . . . ", with the following response options: "drink much more than usual", "drink a little more than usual", "drink about the same as usual", "drink less than usual", "drink much less than usual", "intend to reduce my drinking", "intend to cease drinking", or "have ceased drinking altogether". sas v . (sas institute inc., lane cove, australia) was used for the analysis. the descriptive statistics, including frequencies and percentages, were generated for categorical variables; means and standard deviations (sd) were generated for continuous variables. depression, anxiety and stress scores were compared based on participant's sociodemographic and health status using non-parametric analysis of wilcoxon rank-sum, the kruskal-wallis test and spearman's correlation. the responses for each behavior, i.e., physical activity, sleep, smoking and alcohol use, were recoded into negative change (− ), no change ( ), or positive change (+ ) for separate analyses of changes in each behavior. a multiple lifestyle behavior index [ ] was created by summing the scores of the four behavior change items to reflect a composite health behavior change score, ranged from - to + . the average composite health behavior change scores and sd were presented separately for each level of depression, anxiety and stress. linear regression was used to test associations between composite health behavior change score and depression, anxiety and stress. crude estimates and estimates adjusted for age, years of education, gender, marital status, household income and chronic disease status were reported with % confidence intervals (ci). logistic regression was used to test whether negative changes in individual behavior change items were associated with depression, anxiety and stress. crude odds ratios (or) (model ) and ors adjusted for age, years of education, gender, marital status, household income and chronic disease status (model ) with % ci were reported. all p-values were two-sided and considered significant if less than . . the sociodemographic and health characteristics of the study sample are presented in table . in total, people (mean age . ± . years, female) completed the survey. most (n = , . %) were married or in a relationship, and almost half (n = , . %) reported having at least one chronic health condition. the average score for depression was . ± . ; anxiety, . ± . ; and stress, . ± . . the average physical activity of participants was . minutes/week, but almost half (n = , . %) reported a reduction in physical activity since the onset of the covid- pandemic. the average sleep duration reported prior to the onset of covid- was . ± . h per night, with half (n = , . %) reporting no change in sleep quality since covid- . most (n = , . %) were non-smokers, and the majority (n = , . %) reported no change in smoking since the onset of covid- . almost one-quarter (n = , . %) reported consuming alcohol on four or more occasions per week, and just over half (n = , . %) reported no change in alcohol consumption. the depression, anxiety and stress scores in relation to different sociodemographic and health characteristics are presented in table . no significant differences were found between males and females for depression and anxiety; however, females had significantly higher stress scores compared to males. younger individuals ( - years) had significantly higher depression, anxiety and stress scores compared to their older counterparts. similarly, those who were not in a relationship had significantly higher depression, anxiety and stress scores compared to other categories of relationship status. the spearman's correlation showed a significant negative association between years of education (recorded as a continuous variable) and scores for depression, but not for the anxiety or stress scores. those in the lowest income category had significantly higher depression scores compared to higher income categories; however, no difference was observed between different weekly household incomes and anxiety and stress. respondents who had been diagnosed with a chronic illness reported significantly higher depression, anxiety and stress scores, compared to those without chronic illness. diagnosed with a chronic disease the mean changes in composite health behavior score, stratified by depression, anxiety and stress severity, are presented in table . for depression, anxiety and stress, the number of people in each symptom severity category decreased as the symptom severity increased, except for the categories of extremely severe depression and anxiety. for depression, anxiety and stress, the mean composite health behavior change score decreased as the symptom severity increased, except for the categories of extremely severe anxiety and stress. associations between depression, anxiety and stress severity and negative change in behavior are outlined in table . since adjustment for age, years of education, gender, marital status, household income and chronic disease status did not impact associations, only adjusted or's are presented. participants who reported a negative change in physical activity were more likely to have higher depression (adjusted or = . , % ci = . , . ), anxiety (adjusted or = . , % ci = . , . ), and stress (adjusted or = . , % ci= . , . ) symptoms. those who reported a negative change in sleep were more likely to have higher depression (adjusted or = . , % ci = . , . ), anxiety (adjusted or = . , % ci = . , . ), and stress (adjusted or = . , % ci = . , . ) symptoms. for those who reported a negative change in smoking, they were more likely to have higher depression (adjusted or = . , % ci = . , . ), anxiety (adjusted or = . , % ci = . , . ), and stress (adjusted or = . , % ci = . , . ) symptoms. similarly, those who reported a negative change in alcohol intake were more likely to have higher depression (adjusted or = . , % ci = . , . ), anxiety (adjusted or = . , % ci = . , . ), and stress (adjusted or = . , % ci = . , . ) symptoms. the results were consistent for composite change scores. there was a decrease of . ( % ci = − . , − . ), . ( % ci = − . , − . ), and . ( % ci = − . , − . ) points in composite change score for every point increase in depression, anxiety and stress. the present study examined the association between depression, anxiety and stress and the change in health behaviors of physical activity, sleep, smoking and alcohol use subsequent to the onset of covid- , as individual health behaviors and as a health behavior change index composite score. the main findings were that all aspects of psychological distress (depression, anxiety and stress) were significantly associated with changes in health behavior, both independently and as a composite score. numerous studies have examined the association between a range of health behaviors and psychological distress factors. for example, rebar and colleagues reported significant inverse associations between physical activity participation and depression and anxiety levels in their meta-analysis [ ] . previous work has reported significant positive associations between smoking, and depression [ ] , but not between smoking cessation and reductions in depression or anxiety [ ] . large-scale studies also demonstrate a significant association between alcohol misuse and psychological distress [ ] . taken together, the findings of previous work suggest variability in the associations between lifestyle behaviors and depression, anxiety and stress that appear to depend on the nature of the behavior under investigation. the present study also demonstrated that, as the severity of depression increased, the composite health behavior change score worsened. that is, those with normal levels of depression symptoms reported a small negative change (− . points), while for those with extremely severe symptoms, the change in composite health behavior change score was more than three times greater (− . ). for anxiety and stress, as symptom severity increased from normal to severe, so did negative changes in composite health behavior change score. linear regression showed a significant association between increased depression, anxiety and stress, and negative changes in composite health behavior change scores. logistic regression showed that, compared to no change or positive change, a negative change in all behaviors was associated with a significantly greater likelihood of increased depression, anxiety and stress. a number of reports suggest covid- is likely to have significant impacts on psychological distress [ , ] ; however, the data from the present study suggest that the mean scores for depression, anxiety and stress are mostly within the normal to mild range. moreover, the mean scores for depression and stress were only slightly elevated when compared to normative data for australian adults, and anxiety the scores were marginally lower [ ] . viewed another way, more than % of all respondents reported psychological distress within the normal range, and less than % reported severe to extremely severe scores. the mean scores for depression, anxiety and stress in the present study are all substantially lower than those reported in italy. mazza and colleagues [ ] reported mean depression, anxiety and stress scores of . , . , and . , respectively, compared to . , . , and . , respectively, in the present study. these differences may be accounted for in the timing of data collection as data from italy were collected in mid-march, differences in government responses to the pandemic, and differences in the severity of impact on the population. the prevalence of moderate to severe depression in the present study ( . %) is comparable to that reported in china ( . %) [ ] ; however, the prevalence of moderate to severe anxiety is markedly less in this study ( . %) compared to that in china ( . %). in contrast, the prevalence of moderate to severe stress reported in this study ( . %) is almost double that reported in china ( . %). the timing of data collection may account for some of these differences since the data from china were collected from residents in cities during late january-early february, one day after the world health organization declared a public health emergency. in contrast, the data for the present study were collected in early to mid-april when significant travel and social distancing restrictions were already in place. it is possible that the low prevalence of depression may also be a result of government investment in mental health support services. the lower anxiety scores in the present study may be attributed to respondents being somewhat accustomed to changes in social contact, whereas the higher stress levels may be attributed to the uncertainly about the future, particularly regarding job losses and economic stress. the total average physical activity was . ± . min/week. this is similar to recent australian bureau of statistics data based on the active australia survey, showing that australians aged and over reported min of daily activity, or min per week on average [ ] , but substantially less than the peak of min of activity per week reported by alley and colleagues using the same measure [ ] . physical activity guidelines for australian adults suggest they should accumulate - min of moderate intensity physical activity, - min of vigorous intensity physical activity, or an equivalent combination of both, per week. however, here we report total physical activity, not moderate or moderate-to-vigorous activity. the aas is known to overreport physical activity participation, but actigraphy is not practical in large samples [ ] . therefore, our data may be an over-representation of actual physical activity performed. almost half of our respondents ( . %) reported a negative change (reduction) in physical activity since the onset of the covid- pandemic, but about % also reported a positive change. this is important to note, since there has been considerable emphasis in the media on the importance of maintaining physical activity for physical and mental health benefits [ , ] . our data suggests these recommendations may have been ineffective for most people, but not all. these data are hard to interpret as there has been a visible increase in people using walking paths all over the country, as well as a strong increase in registrations to the , steps australia program [ ] . it may be that the extra people who are walking are predominantly those who were already active (e.g., gym and sports club members) prior to the covid- onset, but had to undertake different activities at different locations due to the closure of exercise and sporting facilities. the reported overall decline in physical activity is likely a consequence of social distancing, travel restrictions, the closure of usual exercise venues, or unwillingness to change previous exercise habits. nonetheless, given the psychological distress responses to covid- , [ ] and the established benefits of physical activity on psychological distress [ , ] , additional strategies to promote physical activity are needed. prior to the covid- pandemic, mean sleep duration was . ± . h, which meets the guidelines of - h for adults [ ] and aligns with a recent national study of australian adults [ ] . although half ( . %) of all respondents reported no change in sleep quality since the onset of the covid- pandemic, . % reported a negative change. this is unsurprising given the potential for psychological distress during a global pandemic, change in exercise behaviors, and employment and relationship concerns. a number of recommendations have been made to address poor sleep during covid- , including maintaining a regular sleep routine, taking time for self-reflection, limiting exposure to covid- -related news, and getting regular exercise during daylight hours [ ] . apart from these covid- -specific recommendations, most principles mirror those recommended for good sleep hygiene in usual circumstances. only % of survey respondents were smokers. this is less than the % prevalence of smoking recently reported among australian adults [ ] . overwhelmingly, respondents have not changed their smoking behavior, with almost % reporting no change or a positive change (reduction) in smoking status since the onset of the covid- pandemic. among smokers, . % (n = ) report a positive change (reduction), . % (n = ) report no change, while . % (n = ) report a negative change (increase) in smoking behavior. since covid- is a respiratory illness, and smokers are more susceptible to respiratory tract infections, there is significant potential for adverse events in this population. early evidence from china suggests either a significant association, [ ] or at least a trend [ ] toward smoking being associated with poor prognosis in covid- cases. to date, there has been limited attention in the media to smoking cessation programs or adverse risk associated with smoking. although more research is needed, health promotion efforts directed at educating the population regarding the risks for smokers during the covid- pandemic are needed. these may include higher exposure to passive smoking during periods of lockdown or relapse-preventions strategies targeting those who have recently ceased smoking. almost three quarters of respondents reported no change or a positive change (reduction) in alcohol use since the onset of covid- . a reduction in alcohol use might be driven by closures to licensed establishments such as bars and clubs and temporary restrictions on alcohol purchases. in contrast, around one quarter of respondents reported a negative change (increase) in alcohol consumption. this is consistent with research by australia's foundation for alcohol research and education [ ] reporting that % of australians increased alcohol purchases since the onset of covid- and % were drinking more than usual. worryingly, this report suggests that almost % of adults are drinking more to cope with psychological distress [ ] . concerns such as these have prompted the australian government to invest more than aud million into drug-and alcohol-related services to combat the risk of substance abuse and related harms, such as domestic violence, due to the pandemic. to the best of our knowledge, this is the first published study to report associations between health behaviors and psychological distress in australian adults during the covid- pandemic. one published study from europe reported that reductions in physical activity and increased sedentary behaviors during lockdown were associated with negative changes in physical and mental health [ ] . moreover, a number of reports have highlighted the need for rapid and comprehensive responses to increasing mental health needs during covid- [ , ] ; however, it is expected this support will need to be maintained for some years to come given the magnitude of the covid- pandemic. there are a number of strengths of the present study, including the inclusion of multiple health behaviors, a large sample size, and the timing of data collection relative to lockdown restrictions in australia. however, there are also some limitations to consider. firstly, all data are self-reported meaning responses are subject to recall bias. secondly, data are cross-sectional and therefore causality cannot be inferred. thirdly, participants in the present study are older compared to other studies examining health behaviors such as sleep [ ] , and thus the generalizability to other populations needs to be confirmed. additionally, longitudinal data are needed to observe changes over time to assess the impact of changes in social restrictions. finally, our sample was recruited conveniently and therefore the results may not be generalizable to populations with different characteristics. in conclusion, our data suggests negative changes in health behaviors are associated with increased psychological distress in australian adults during the covid- pandemic. effective health promotion strategies directed at adopting or maintain positive health-related behaviors such as targeted social media messaging and balanced media reporting, should be used to reduce the acute and chronic increases in psychological distress during these unprecedented times. ongoing evaluation of the impact of lockdown rules and social distancing (associated with the pandemic) on health behaviors is necessary to inform these targeted health promotion strategies. the psychological impact of quarantine and how to reduce it: rapid review of the evidence psychological interventions for people affected by the covid- epidemic the emotional impact of coronavirus -ncov (new voronavirus disease) the mental health consequences of covid- and physical distancing: the need for prevention and early intervention role of physical activity and sedentary behavior in the mental health of preschoolers, children and adolescents: a systematic review and meta-analysis the evidence for physical activity in the management of major mental illnesses: a concise overview to 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sales and use during covid- ; foundation for the enemy who sealed the world: effects quarantine due to the covid- on sleep quality, anxiety, and psychological distress in the italian population this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the research received no external funding. the authors declare no conflict of interest. key: cord- -qkoc ky authors: dietz, thomas title: political events and public views on climate change date: - - journal: clim change doi: . /s - - - sha: doc_id: cord_uid: qkoc ky nan grown more substantial over time. over the last decade, while democrats, independents, and even liberal to moderate republicans have increasingly accepted that global warming is happening and that it is worrisome, conservative republicans have become less likely to see climate change as real and a cause for worry (ballew et al. ). this phenomenon is strongest in the usa and stronger in anglophone countries in general (hornsey et al. ). the ideological split on climate change was foreshadowed as much as years ago. earth day in mobilized perhaps , events at colleges, universities, and communities and was one of the largest social movement actions in us history (dietz ). it appears that earth day increased environmental concern among liberals and moderates, but it had no impact on conservatives (johnson and schwadel ) . most members of the public do not come to their views on climate change through a careful reading of the scientific literature. humans routinely use a variety of cognitive shortcuts to make decisions quickly and efficiently. this is sometimes referred to as system i thinking, in contrast to system ii thinking that deploys careful weighing of evidence, costs, benefits, and risks, essentially the "rational actor model" (kahneman ) . in system i, we consider the sources of new information, and are more likely to accept information from sources we perceive as similar to us. we are more likely to accept new information if it is congruent with our existing views. when presented with facts, we may think about the implications of those facts for policy and reject facts that might lead to policies we do not like. so our values, our existing beliefs, and our social networks substantially shape how our beliefs evolve. in turn, as we form social networks from which we derive information, we tend to link to people who think the way we do and strongly avoid those who think differently (henry and vollan ; jasny et al. ). an extensive research literature examining these processes spans the social sciences. unfortunately, a number of different terms have emerged to label what are roughly the same phenomena. in particular, the process by which we let our assessment of facts be influenced by values, prior beliefs, policy preferences, and by how we feel about a source of information is variously called biased assimilation, hot cognition, or motivated reasoning. ongoing research offers some subtle distinctions across these concepts (bolsen and palm ; druckman and mcgrath ) . here, i will use the oldest of the terms, biased assimilation, to describe the general phenomena. we are all susceptible to these "heuristics and biases." in most day-to-day situations, these shortcuts work well and save a great deal of time and cognitive effort. indeed, the ability to learn from others, the basis of biased assimilation, seems to be at the heart of human ecology and cultural evolution (henrich ) . we also have mechanisms to overcome the biases. we use the scientific method specifically to counteract these tendencies in assessing new information (elliott ) . indeed, some disciplines deploy double, rather than single, blind peer review to ensure that the prestige of authors or other author characteristics does not bias assessment of the quality of a paper. unfortunately, these shortcuts can be manipulated by those who want to shape our thinking (cialdini (cialdini [ ). climate denial is a case in point-public views have been influenced by ongoing campaigns to discredit the scientific consensus and block action, exploiting our cognitive shortcuts (givens et al. ; mccright ; mccright et al. ) . the history of such campaigns stretches back at least to arguments about the introduction of tetraethyl lead as a gasoline additive, and have been mounted around acid precipitation, ozone depletion, and most recently the covid- pandemic (michaels ) . because of biased assimilation, the views of prominent people, such as presidential candidates, can have a substantial influence on public opinion. research on the bases for support for and opposition to trump's candidacy is ongoing, but it appears that immigration and racial issues were important (algara and hale ; hooghe and dassonneville ; setzler and yanus ; wright and esses ). members of the public who aligned with him on these or any of a variety of other issues, or supported him simply because they identify as republicans, could then be expected to also align with his views on climate change. while environmental issues were not central in the presidential campaign, trump clearly signaled his skepticism about action on climate policy, support for the coal industry, and opposition to environmental regulation (bomberg ; rosenbaum ) . how then did trump's election influence public views on climate change? hahnel et al. and zawadzki et al. offer important insights on the impact of trump's presidency by considering the social psychological processes that shape public views. hahnel et al. conducted a panel study, re-interviewing the same respondents roughly month before and weeks after the election. post-election, both republicans and democrats held more positive feelings toward the republican party than they had before the election, although the effect was much stronger for republicans than for democrats. republicans perhaps felt vindicated while, as the authors note, democrats may have been reducing cognitive dissonance. certainly, many political commentators at the time suggested that president trump would be more moderate that candidate trump had been, so perhaps the shift in views among democrats reflected an effort to reduce discomfort about the incoming administration. these changes in positive feelings toward the republican party in turn shifted views about climate change. at least since the reagan administration, the republican party has tended to oppose environmental regulation, and climate change denial has become almost universal among republican leadership (dietz ; fisher et al. ) . increased positive feeling toward a party where climate denial is commonplace led to reduced concern with climate change. the impact was stronger for republicans than for democrats, so the effect of trump's election in the short run was to widen the ideological split on climate change. as the authors note, their data is capturing the effect of the election itself, not the actions of the trump administration. zawadzki et al. were not able to re-interview the same respondents, but they have data from four time points: the day before the election, days after trump took office and began to issue executive orders, days into his presidency (which is often seen as a benchmark in a new administration), and days after trump began the process of withdrawing the usa from the paris climate agreement, the st day of his presidency. so while hahnel et al. are capturing the impact of the election per se, zawadzki et al. provide insights into the impact of trump's early actions as president and the reaction to them, such as the march for science (april , ) and the people's climate mobilization (april , ), both of which occurred just before trump's th day in office. they find that once trump was in office and his views on climate, the environment, and government regulation were instantiated in policy actions, the effect was to some degree the opposite of what hahnel et al. found when looking immediately after the election. trump supporters still became less likely to accept the reality of climate change but non-supporters became more likely to believe that climate change is real, anthropogenic, and a serious threat. the two papers use somewhat different measures of climate views, but i believe the results are consistent. the immediate effects of the national election were better feelings toward the victors and more resonance with their views. but once policies emerged that clarify what the new administration would do, supporters became less accepting of the scientific consensus while opponents became more accepting. both of these effects tend to increase polarization. similar effects seem to follow from gubernatorial elections (meyer ) . once trump announced that the usa was withdrawing from the paris treaty, climate beliefs among his opponents became stronger. perhaps surprisingly, denial of the scientific consensus declined among his supporters, who moved back to climate beliefs roughly equivalent to their pre-election views. the move among opponents might be seen as a defensive posture. given a president who seems hostile to environmental policy, opponents strengthen their pro-environmental position. a similar pattern has been observed around the passage of us federal environmental law, where environmental legislation is most likely to be passed when a democratic congress faces a republican president-the legislative action seems intended to counter possible actions by the president (steele ). hahnel et al. suggest that trump's supporters may have shifted views because trump's speech on the paris treaty acknowledges the reality of climate change. alternatively, withdrawal from the paris accords and related reneging on commitments to reduce greenhouse gas emissions removed the policy threat that comes with climate mitigation. that in turn may have reduced the need of mitigation policy opponents to reject the scientific consensus. both papers use the presidential election as a natural experiment to examine the processes that can shift public views on climate change. they not only show the election's effect on climate denial but also explore the mechanisms by which that happened. hahnel et al. analysis is consistent with a process that has been extensively examined in policy networks-we tend to shift our views to make them more in alignment with those we view positively. in policy systems, actors tend to affiliate with those with similar views, avoid those with opposing views, and accept information mostly from those who are like-minded and that are resonant with our prior views. these effects can be very strong and make it difficult to address complex issues such as climate change (henry and vollan ; henry et al. ). zawadzki et al. examine the processes that may lead to, or block, public action on climate change. much of the literature on pro-environmental behavior demonstrates the importance of what zawadzki et al. refer to as "moral sentiments," often called altruism in other literatures. people take action because they are concerned with the well-being of other humans and other species, and because they feel a personal obligation to act on problems like climate change (steg ) . their analysis shows that believing either that climate change is anthropogenic, and/or that it is serious, increases the moral sentiment that one should act. that in turn leads to a greater likelihood of being willing to reduce energy consumption and support climate policy. trump's presidency tended to reduce these climate beliefs among his supporters while increasing them among his opponents. those changes in beliefs, acting through changes in moral sentiments, led to increased willingness to take action among trump opponents and decreased willingness among supporters. how can we encourage social change that leads to a just and sustainable global society? how can the gap between scientific assessment of climate risks and climate action be closed? social change comes about through the interplay of actions by the public, by political elites, and by activists who work to influence both elites and the public. the last century has seen massive social and policy change around the environment and civil rights, for example (dietz ; silver ) . understanding how these processes unfold, particularly in the face of urgent sustainability challenges, must be a high priority for social science research. these two papers demonstrate the potential of linking the analysis of large-scale events, such as elections, to individual-level processes. unfortunately, while conceptual frameworks and research methodologies exist to undertake that challenge, resources to support such efforts are sadly lacking. the environmental social sciences receive very modest funding relative to their potential contribution (overland and sovacool ) . one result is that the kind of long-term, consistent community data sets needed to untangle causal effects and to link the micro and the macro are seldom available. one can imagine the state of climate science if funding over recent decades had precluded the production of high-quality time series data and allowed only episodic observations scattered unsystematically across time and space. yet, that is exactly the situation faced by the social sciences. nonetheless, a great deal of progress is being made. in particular, a rapidly evolving literature helps us to understand the individual-level processes that lead to agreement with scientific consensus. the literature also makes clear what leads to action by individuals in their roles as both consumers and citizens. our understanding is increasingly integrated across multiple disciplines and perspectives, although most research is still grounded in western, educated, industrial, rich democracies (nielsen et al. ; steg ; steg et al. ) . hahnel et al. and zawadzki et al. take that literature in an important new direction. their analyses are solidly grounded in, and make contributions to, our understanding of the social psychological processes that shape climate beliefs and willingness to take action on climate change. but they go a step further in linking those individual-level models to large-scale political events, in this case the election of a republican president aligned with climate denial and opposed to climate policy. understanding how these large-scale events shape individual views, including the mechanisms involved, is critically important but the neccessary analyses are rarely done, in part because of the lack of time series data noted above. we need more analyses of this sort, as well as analyses that trace causal influence in the opposite direction, from citizens and consumers to large-scale social change. the processes by which changes in individual beliefs, norms, and actions influence businesses and government are complex and fraught with sharp power differentials between the average citizen or consumer and the powerful individuals and organizations that shape policy and the economy (dietz and whitley ) . but individuals do have an impact, and we need a better understanding of how it unfolds. work on this issue may be most advanced around how consumer choices have impacts not only on direct consumption of energy in the household but also impacts on the supply chain (gilligan and vandenbergh ; stern and dietz ) . social movement scholars have examined how mobilization can influence policy (fisher et al. ) . but more work is needed to understand how social movement support by individuals influences largerscale social change to parallel needed work on how large-scale changes influence individuals. of course, readers of climactic change will ask how these results, and the literature on which they build, can help close the gap between current actions on climate change and what is needed to reduce risks to an acceptable level. certainly, we are learning how to communicate science more effectively (moser ; u.s. national academies of science ). but, since decisions always involve both facts and values, even if the public and policy makers agreed with the scientific consensus on the magnitude, impacts, and risks of climate change, there is still considerable room for debate based on value differences and inequities entrained in climate mitigation and adaptation. nearly a century ago, john dewey noted the difficulty of resolving problems that are grounded in science and that involve value conflicts (dewey ). he called for linking scientific assessment with deliberation by interested and affected parties. the us national academies of science has argued repeatedly that such linked analysis and deliberation can help build trust in science and mutual understanding even in the face of value differences (national research council ; u. s. national research council ; u.s. national research council ) . we know a great deal about how to structure successful analytic deliberative processes at the local and regional level (bidwell ; stern ) . climate change challenges us to develop processes that can work at the national and global levels (bächtiger et al. ). as we try to move in that direction, work such as that of hahnel et al. and zawadzki et al. is crucially important in providing the scientific underpinning that links large-scale processes and events to change in the views and actions of individuals. the distorting effects of racial animus on proximity voting in the elections oxford handbook of deliberative democracy climate change in the american mind: data, tools, and trends thinking through participation in renewable energy decisions motivated reasoning and political decision making environmental politics in the trump era: an early assessment influence: the psychology of persuasionrevised edition edn. harpercollins, new york dewey j ( ) the public and its problems earth day: fifty years of continuity and change in 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women vote for donald trump? change doesn't usually come this fast fivethirtyeight com an analysis of u.s. federal environmental legislation in the nineteenth, twentieth and beginning twenty-first centuries, with emphasis on presidential party and political majorities in congress values, norms, and intrinsic motivation to act pro-environmentally what drives energy consumers?: engaging people in a sustainable energy transition design principles for governing risks from emerging technologies a broader social science research agenda on sustainability: nongovernmental influences on climate footprints public participation in environmental assessment and decision making communicating science effectively: a research agenda understanding risk: informing decisions in a democratic society it's security, stupid! voters' perceptions of immigrants as a security risk predicted support for donald trump in the us presidential election translating climate beliefs into action in a changing political landscape climatic change in press publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord- -zab s n authors: gareau, brian j.; huang, xioarui; pisani gareau, tara; didonato, sandra title: the strength of green ties: massachusetts cranberry grower social networks and effects on climate change attitudes and action date: - - journal: clim change doi: . /s - - - sha: doc_id: cord_uid: zab s n the cranberry, a commodity of social, cultural, and economic importance to new england, is under threat due to climatic change in this region of the united states. yet, previous research reveals that cranberry growers have mixed attitudes about the anthropogenic roots of climate change, with many being skeptical. building on the researchers’ analysis of the personal and ecological conditions that affect climate change attitudes among cranberry growers, this paper examines the effect that key actors in the growers’ social networks have on those attitudes. through statistical analysis of survey data and content analysis of two important cranberry newsletters, the paper finds that cranberry growers’ perceived importance of two key cranberry growing institutions, the “sociopolitically focused” cape cod cranberry growers’ association and the “technically focused” university of massachusetts cranberry station, as well as connections to other cranberry growers, is associated in nuanced ways with growers’ climate change attitudes. drawing on the sociological theory of “social capital,” the paper examines how these social ties to key actors/institutions may result in greater threat perception or worry about climate change. it then considers how “green ties,” if harnessed and supported by these important actors in the cranberry grower network, might significantly mitigate climate change in the future. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. introduction later, warmer summers will occasion berry scalding, injuring the fruit and causing poorer color and therefore marketability; hotter summer temperatures will also lead to vine and flower stress along with potential die-off of pollinating insects such as honey and native bees that are critical to cranberry flower pollination. estimated increases in intense rain events in the summer will lead to higher levels of fruit rot, lower levels of budding (because of flowers lost to rain or hail), lower pollination levels, and less bee activity . given the significant threat that climate change poses for their crop, it is important to discern how prepared cranberry growers are to recognize and respond to the danger. research results reported in gareau et al. ( ) demonstrate that there is a complex relationship between the personal and ecological conditions of cranberry growing and attitudes toward climate change among massachusetts cranberry growers. some conditions (discussed more thoroughly below) appear to create spaces in which actions on climate change might emerge, such as among growers who think weather is an important problem facing the cranberry industry. the search for connections between attitudes and certain conditions among growers, and whether certain social networks foster concern and action against climatic change, is a central question in this paper. in this, our research draws from the literature on grower attitudes toward climate change and literature focusing on the effects that powerful actors in social networks can have on attitudes and actions of people in the network. are there ways in which social networks among cranberry growers are arranged that allow for climate change to be seen as a concern? using a survey disseminated to the entire massachusetts cranberry grower community, examining the newsletters of two key cranberry organizations, and drawing from our decades of combined knowledge of the massachusetts cranberry growing industry, this paper seeks to answer this question. literature review . grower attitudes toward climate change scholarship in the social sciences aims to understand the attitudes that growers in the united states hold toward climate change, and particularly, whether certain social and economic conditions might connect to greater levels of awareness and concern. while grower attitudes vary across regions and circumstances, most studies report high levels of awareness of environmental changes and extreme weather events that affect farming, yet acknowledgement of its anthropogenic causes is less common (chatrchyan et al. ) . even in the organic sector, where it might be assumed that growers hold environmental values above their conventional counterparts (fitzmaurice and gareau ; jackson et al. ) , attitudes toward climate change can be unexpected. research shows that conservative white male growers tend to be less concerned about climate change and are less likely than other social groups to believe it is occurring, which is consistent with the broader trend among u.s. citizens (liu et al. ; mccright and dunlap ; running et al. ) . in short, there is no particular sector or grower group that appears more attuned than another to the threat of climatic change and the need to act. since, on the whole, u.s. growers are less concerned about climate change and more skeptical of climate change science than average u.s. citizens, it is particularly important to frame local weather events and other conditions in a broader context that triggers interest among growers (chatrchyan et al. ) . discussing the increased likelihood of extreme weather events and temperature variability and their effects on particular sites due to climatic change will help with mitigation and the adoption of climate-resistant strategies. in other words, framing the issue in a way that makes sense to the daily lives of growers will increase the likelihood of implementing climate adaptation strategies. the notion of framing discussions of climatic change in ways that are sensible to the local context suggests the importance of achieving trust and rapport among the stakeholders involved in the framing process as well as creating communal conditions that foster ) an awareness of climatic change as a serious threat to local ways of life, and ) the adoption of climate mitigation and adaptation strategies specific to the local context. if growers are involved in social networks that include experts that they trust, whose knowledge of farming and other matters they respect, the more likely they will be to listen to and incorporate the experts' knowledge in their own decision-making. this establishment of trust is often described in sociology as the development of social capital. social capital refers to the persistent, long-term connections that exist between social actors in a network, leading to acts of reciprocity, the sharing of norms, and collective action to resolve social and ecological problems (ostrom ) . social capital "has explanatory power specifically in the area of collective action for environmental management" (adger : ) . from responding to specific ecological disasters to gathering resources to cope with longstanding economic hardship brought on by environmental degradation, these social connections are consistently identified as important for garnering support to work toward improvements in social welfare. as a theoretical frame, social capital "provides an explanation for how individuals use their relationships to other actors in societies for their own and for the collective good" (adger : ) . social capital has been used to understand how innovations have emerged in smallscale farming scenarios worldwide (lowitt et al. ) . it is often used to conceptualize how social ties among actors may make up for poorly designed development practices in the global south, or to make up for the fragility of weak institutions among impoverished peoples and communities (fafchamps ; lowitt et al. ; van rijn et al. ). in such cases, the connections among actors are seen as providing decentralized planning in lieu of the policy implementation that can be enforced by a powerful centralized body such as a local or regional governmental institution (bodin and crona ; crona and hubacek ) . when examining the role of social capital in the adoption of farming adaptation methods (e.g., conservation technologies, services, programs), it becomes clear that the results reflect disparities among the factors leading to successful implementation. this could be attributed to various causes, such as the use of different methods and models when collecting and analyzing data, or the location/region of the study. some variables are found to be rather universally significant, however, such as the presence of extension services and farming associations (see deressa et al. ; takemura et al. ; hoang et al. ) . while climate attitudes among growers, such as worry and threat perception, are important, this paper also examines how social networks might contribute to the implementation of climate adaptive cranberry growing practices. grower associations make up a form of social capital found to be highly significant; the diffusion of new technologies and practices is typically linked to membership in agricultural organizations (yaméogo et al. ) . for example, in a synthesis report covering over years of research on growers' adoption of conservation agriculture, involvement in private and governmental associations was found to be an important social tie, the most consistently positive variable (knowler and bradshaw ) . as adger ( ) argues, the achievement of adaptation strategies must be based on social capital understandings of trust, reciprocity, and shared norms in order to alter perceptions of climate change as being inconsequential. while the majority of studies have focused on the positive effects of harnessing social capital, there are negative aspects associated with it as well, including the swaying of deliberations on environmental policy choices in global agreements due to uneven access to powerful actors via vertical capital (gareau ) and uneven access to natural resources among networked groups (ballet et al. ). as such, social capital can create shared beliefs and a knowledge production platform that undermines what ethicists describe as the "common societal good" (van deth ; hollenbach ). the combination of positive and negative aspects of social capital highlights the importance of recognizing that these social ties of influence are situated in the personal, communal, and ecological conditions of the grower (gareau ; gareau et al. ; white et al. ) . the ties that allow for social capital to be constructed are enmeshed in the communal conditions of growers, in that "communal conditions provide the social framework in which actors operate, where norms of sharing advice, equipment, access to water in adjacent wetlands, and other resources can be created to allow livelihoods to persist even in difficult situations" . here, growers' views of climate change might depend in part on the cultural fabric of the cranberry community, where the ideas of powerful actors are able to shape, frame, and influence attitudes and thereby encourage certain actions (cf. lucier and gareau ; olson and gareau ) . our concern in this paper is whether ties to certain social actors have produced any level uniform "belief" among the overall cranberry grower community regarding what the scientific community understands to be a fact: anthropogenic climate change (national research council ) . more specifically, can the social ties that connect important social actors in a network work to induce a high level of concern about the threat of climatic change among growers? does a grower's social network have an effect on their attitudes and the kinds of practices they engage in? here, we are looking for social connections that might constitute "green ties," ties that encourage growers to perceive the threat of climatic change as palpable, in the hope that such awareness will translate into collective action on the local level through adoption of mitigation and adaptation techniques (adger ) . in this, our study joins bourdieusian scholarship on social capital, which focuses on the perceptions of the importance (or influence) of actors, processes, or mechanisms in a network, perceptions that provide insights into the makeup of social ties in a network (e.g. bourdieu ; sanginga et al. ; pretty ; jones et al. ). if such an attitude among this social group has emerged, we can say that the communal conditions of cranberry production are right for collective action on the local level. conversely, if low concern about the threat of climatic change is evident, local engagement with its solutions is unlikely. work on social capital stresses the influence that certain powerful actors have on the social networks in which they are embedded. those with influence can shape what is deemed important to the network of actors and influence the communal conditions of production of those operating in that network. we apply this theoretical frame to the case of cranberry production in massachusetts. in massachusetts, cranberry growers are connected to a range of organizations that exist to support and sustain the cranberry industry. the result is a tightly knit community in which production and livelihood are intimately fused together. organizations with important social ties to cranberry growers include the university of massachusetts (umass) extension program and the umass cranberry station in particular, the cape cod cranberry growers' association (cccga), the cranberry cooperative ocean spray, and other cranberry handlers. also making up the communal conditions of production are cranberry growers' involvement with one another through family ties, grower meetings, the annual cape cod cranberry harvest festival, their pest control advisors, and similar connections. the social links that growers develop with one another provide powerful ways of sharing knowledge and other forms of support among themselves. yet, social capital research points to the importance of the social ties made through connections to influential actors outside the personal relations of individuals, whether these are state or non-state organizations, in establishing certain "ways of knowing" within the social network (adger ) . do certain social ties in the social network of the cranberry growers' community foster greater concern for climate change among them than among other socially networked growers? do some social network connections increase recognition of the threat that scientifically predicted climate change poses to the cranberry grower community? if so, does that recognition lead to collective action in the form of specific management practices to mitigate climate change? while to our knowledge no study has been conducted on the communal/social networks of massachusetts cranberry growers, previous research reveals how cranberry growers' attitudes toward climate change are tied to their personal and socio-ecological context. gareau et al. ( ) find that massachusetts cranberry growers' attitudes toward climate change are not only affected by their personal conditions but are also contextualized by the ecological and communal conditions of cranberry production. with regard to personal conditions, the authors find that cranberry growers who identify as women and those who have college degrees are more likely to view climate change as a threat and to worry about it. with regard to ecological conditions, cranberry growers who report weather in general as an important condition are more likely to see climate change as a threat. however, attitudes toward climate change are more varied when growers single out individual weather conditions for consideration. for example, identifying insufficient chill hours during winter (a climatic change in new england that is particularly damaging to cranberry production) is positively associated with the likelihood of seeing climate change as a threat and worrying about it. the fact that there is little growers can do to compensate for the insufficient chill hours may well account for this view. growers can regularly irrigate their cranberry bog, which helps reduce the risk of frost when cranberry plants break dormancy too early during an insufficiently or inconsistently cold winter, but this is a laborious and water-intensive operation and does not solve all the problems caused by insufficient chill hours. gareau et al. ( ) argue that to cope with the warming winter temperatures, cranberry growers will need to increasingly rely on their communal networks (i.e., communal conditions of production) and their sponsorship of such measures as the development of new cranberry varieties with higher frost tolerance and requiring fewer chill hours. influential actors might tap growers with higher levels of climate awareness to increase awareness among the cranberry grower community. on the other hand, gareau et al. ( ) find that perceptions of water scarcity and competition for water are not associated with climate change attitudes among cranberry growers, perhaps because of the growers' long history of collectively coping with the variable precipitation levels that are typical in new england (playfair ) . in any case, cranberry bogs are highly resistant to drought and flooding, so it is quite possible that cranberry growers would take longer to identify changes in these ecological conditions as problematic. gareau et al. ( ) also find that control of certain ecological conditions of production is associated with climate change attitudes: an increase in the acreage of wetland habitat owned by the cranberry grower increases the odds of worrying about climate change, while an increase in upland habitat owned reduces those odds. gareau et al. ( ) speculate that this may be because ownership of wetland habitat prompts cranberry growers to be more attuned to the impacts of climatic change on the water resources in these habitats. contrastingly, ownership of dry upland habitat, by allowing for other forms of agricultural production and other money-generating activities such as selling sand and gravel, may offer a protection against, and reduce attention to, the climate impact on cranberry production. while gareau et al.'s ( ) work shows the connections between climate change attitudes and personal and ecological conditions among massachusetts cranberry growers, it remains unknown whether certain networks of actors create higher levels of climate awareness among community members. are there certain socially networked actors who contribute to higher awareness levels among cranberry growers? in other words, are certain communal conditions of production-influenced by the social ties of particular actors-leading some cranberry growers more than others to greater awareness and/or concern, and are some growers engaging in practices to mitigate the effects of a warming climate? based on several years conducting social and ecological research in and around the cranberry industry, conducting in-depth interviews with cranberry growers, cranberry extension agents, and other members of the cranberry community, the authors determined that it would be useful to study the scientific community and institutional actors operating to provide scientific and other forms of support to the cranberry industry in massachusetts and their influence on the attitudes of cranberry growers. these actors, functioning as advisors on scientific matters, on practical issues such as pest outbreaks, and on the political, economic, and legislative issues concerning the industry, communicate their knowledge about the cranberry industry to growers in three important ways: ) personal communication with growers, ) dissemination of updates and reports via monthly newsletters, and ), more distantly, through public statements given in the local media by the leaders of these organizations. as an extension agency mandated to help cranberry growers thrive in their industry, the umass cranberry station communicates with growers at the personal level, through annual and seasonally held conferences and workshops, and through updates provided in their monthly newsletter, the cranberry station newsletter. the cccga is a growers' association also created to assist cranberry growers in their production, but they provide information more focused on the broad grower environment and "business" sides of the industry, giving updates on the state of the industry, political and regulatory updates, and the like through their monthly newsletter, bogside. while ocean spray and other cranberry handlers are also institutions that likely impact cranberry growers' knowledge frames, we limited our focus to the umass cranberry station and cccga. we did this because ) both groups operate outside the industry per se, meaning their interest is not first and foremost advancing profits and sales, but instead offering agricultural information relevant to cranberry growing; and ) both groups publish monthly newsletters addressed to growers-affording us a second method with which to understand how social ties to these two organizations might be significant in shaping climate attitudes among their readership. the use of this "mixed methods" approach helps add validity to the findings that are achieved in one analysis by allowing for results to be compared to those of a different, yet complementary, method of inquiry (hesse-biber ). in order to examine the effect of important social ties among actors in the cranberry grower network, we employ two primary research methods: statistical survey analysis and content analysis of monthly newsletters. in , two of the authors (gareau and pisani gareau) solicited all of the massachusetts cranberry growers to participate in a survey. grower contact information was provided by the umass cranberry station, and cccga sent the solicitation to its members. growers were either emailed the solicitation or saw it posted as an advertisement in the cccga monthly newsletter, bogside, throughout the year. the researchers mailed hard copies of the solicitation, along with the survey and postage-paid return envelopes, to all growers on the cccga mailing list who had not returned the survey. grower contact information was not released, in order to protect the privacy of cccga members. the online consent form explained the nature of the study prior to answering questions, and a hard copy of the consent form was provided as part of the survey form. the research complies with ethical practices and was approved by the boston college institutional review board (protocol number . . e) prior to disseminating the survey. the researchers received responses out of roughly cranberry growers, or % of the grower population, making this is an adequate sample size with a sampling error of ± to ± % at the % confidence level (salant ) for this particular group. (see gareau et al. for further details.) as reported in gareau et al. ( ) , "the sample reflects a large portion of total cranberry acreage in massachusetts (over %), and roughly % of upland habitat owned by cranberry growers" (p. ). it is important to note that none of the authors are affiliated with cccga or the umass cranberry station. actors at both the umass cranberry station and cccga use monthly newsletters to communicate with cranberry growers and disseminate information and views on issues pertinent to growers, with the goal of helping them in their production pursuits. as we discuss below, each of these organizations has a mandate and mission that both governs the content of the newsletters and shapes how issues are framed and disseminated. examining the newsletters will therefore reveal how influential knowledge passes via this social tie to the cranberry growing network. if the newsletter of an influential actor discusses climate change and associated issues that impact cranberry growing, for instance, we would anticipate the cranberry growers in that network will have higher levels of concern about climate change. finally, we also search for references to climate change in media stories featuring key actors in these two organizations, and again, we examine the framing used to present them. while the frequency of social interactions among growers and the cccga and umass cranberry station is imprecise in the survey data, our understanding of the industry as researchers gives us confidence to consider the connections between these organizations and the growers influential, based on the fact that the growers identify their connections to these organization as important. growers have ample opportunities besides the monthly newsletter to interact with the umass cranberry station throughout the year at workshops and field days, requested visits to growers' bogs, and the annual meeting to review the cranberry harvest. for cccga, in addition to the monthly newsletter, growers receive announcements about issues of concern. for example, since publishing the april issues of bogside, the cccga has issued two "grower news alert" updates on covid- to help growers navigate the federal paycheck protection program (ppp) and the economic disaster injury loan (eidl) program, as well as posting links for webinars to help growers through the pandemic. therefore, while exact frequency data are lacking, we accept that connections to these organizations are important when growers say as much; and if a pattern of attitudes among growers emerges in concert with their expressed importance, the results will be instructive. first, we lay out the statistical analysis of the survey. we conducted statistical analyses using data gathered from the survey of cranberry growers. we model two sets of dependent variables. the first set includes two variables on attitudes toward global warming: global warming threat and global warming worry. both variables are used in gareau et al. ( ) to capture cranberry growers' global warming attitudes, and are also consistent with measures used by chong ( ), dunlap et al. ( ), and mccright et al. ( ) to study climate change attitudes in the united states, among many other studies. global warming threat is a binary variable operationalized using cranberry growers' responses to the survey item "do you think that global warming will pose a serious threat to you or your way of life in your lifetime?" ( = yes, = no). global warming worry is an ordinal variable capturing growers' responses to the survey item "please tell us how much you personally worry about global warming" (a to scale: "not worry at all" to "worry a great deal"). the second set of dependent variables includes three variables on the management practices growers implemented in cranberry production that are related to climate change adaptation and mitigation. the first variable, management practices count, measures the number of management practices that cranberry growers reported to have implemented in their bogs in the past - years. in the survey, we asked growers: "which of the following management practices have you implemented on your bog in the last - years?" growers were to select all that applied from a list of practices we provided, including integrated pest management, sanding, late water, "smart" irrigation and/or frost alarm, organic amendments, conservation or creation of pollinator habitat, and other. respondents who chose "other" were invited to elaborate on their responses in the survey; we then recoded these into three additional categories: water recovery, other , and other . "water recovery" was supplied by several respondents as an answer in the "other" category; "other " and "other " denote idiosyncratic explanations of the "other" category that are unique to specific respondents, which we recoded in this way to account for them in management practices count. we also model one dependent variable on a specific management practice, pollinator habitat conservation, which is a binary variable capturing whether growers have implemented pollinator habitat conservation in the past to years ( = yes, = no). cranberries require animal pollination to set fruit (armstrong ) . with global findings of biomass and biodiversity loss amongst insects and other pollinators due to environmental degradation and climate change, and with continued threats of parasitism and disease among honey bees (apis mellifera l.) commercially managed for agricultural pollination, this management practice is essential for the continued sustainability of cranberry production. the third variable, management practices importance, captures growers' responses to the survey item, "on a scale of to , have some or all of these management practices become more important over the past to years?" (a to scale: not important to very important). only three respondents chose " " (not important) or " " (minimally important). therefore, we elected to recode the responses into three ordered categories: somewhat important or less (including minimally important and not important), important, and very important. in the survey, we asked growers to indicate whether each of the three actors potentially tied to their social networks-the cccga, umass extension, and other cranberry growers-had been "important" to their cranberry operation from to present. while not the focus of this paper, in-depth interviews with cranberry growers reveal that these three actors are important for a variety of reasons related to gaining knowledge on cranberry growing techniques, practices, pest outbreaks, and the like. for example, one grower told us that growers would often convene at the umass cranberry station in wareham, ma, to collaborate on addressing common problems such as weed control and share their latest inventions. (the industry is so small, growers often make their own equipment and share the designs.) again illustrating the importance of both fellow growers and the umass cranberry station, another grower told us how they and their father "frequently went to the umass cranberry experiment station for advice, camaraderie, and conversation. and if growers had a problem, the umass station would send people to the bogs to help the growers free of charge" (pers. comm.). interviews with growers at the cranberry harvest festival, held in wareham, ma, describe the cccga as an important actor that promotes both the tradition and modernization of cranberry growing. in particular, growers expressed their appreciation for the cccga exhibit "rhapsody in red: years of massachusetts cranberries," and how the organization worked to promote the shared relationships among cranberry farms in the wider community. we coded growers' responses into three dummy variables: view cccga as important, view umass extension as important, and view other growers as important ( = yes, = no). we also include additional independent variables on growers' ecological and personal conditions of cranberry production that our prior study found to be associated with growers' climate change attitudes . table details the operationalization and summary statistics of each variable. we estimate a baseline regression model for each dependent variable that only includes three key independent variables on social networks, and we then estimate additional models that control for other independent variables. we estimate logistic regression models for the two binary dependent variables: global warming threat and pollinator habitat conservation. for ordinal dependent variables-global warming worry and management practices importance-we estimate generalized ordered logit regression models. this modeling technique estimates a single coefficient for each independent variable that meets the proportional odds assumption; for each independent variable that violates the assumption, this technique estimates multiple coefficients, one for each cut point of the ordinal dependent variable. this modeling technique is more parsimonious than multinomial models and allows for more flexibility than proportional odds models (williams ) . for management practices count, we estimate poisson models because likelihood ratio tests do not indicate overdispersion. in models that include the important control variables that we identified in our prior study , the degrees of freedom are limited due to the small sample size (n = ). furthermore, we are interested in identifying the aspects of global warming attitudes and management practices that are associated with the three social networks of interest, as well as in the aspects where such associations are absent. therefore, we elect to use a more lenient alpha level of . for tests of statistical significance in order to reduce the risk of type ii errors (i.e., the nonrejection of a false null hypothesis). we also allow the sample size to vary across models according to the missing data for different combinations of independent variables in each model. we gathered all available issues of the umass's cranberry station newsletter and cccga's bogside that have been published within the last decade and imported all issues into the nvivo qualitative data analysis package for analysis. in order to guide our analysis of each organization's respective newsletter, we first examined the mission statements as found on their websites. in both organizations, actors publish newsletter content in line with the values and priorities expressed both in their mission statements and on their webpages. we performed content analysis in two stages. first we did a "conventional" analysis by reading through all issues, coding loosely for common themes, and creating word clouds to illustrate the most common topics of discussion (hsieh and shannon ). these findings were then compared with the priorities outlined in each organization's mission statement. second, we performed a more specific "summative content analysis," which allows for an indepth examination of a topic already determined to be of importance, in this case, climate change (hsieh and shannon ). the summative content analysis that we employed is focused on the framing and context within which climate change is presented. framing, "involves the intentional or inadvertent crafting of a message to describe an issue, place responsibility, and/or suggest solutions" (mossler et al. : ) . to examine the framing of climate change references in the newsletters we first searched for the phrases "climate change" and "global warming"; we considered any use of one of these phrases as a reference to climate change. we also searched for the term "climate" alone and qualitatively determined whether the instance was a reference to climate change specifically or to climate in general. the climate change references were then examined in combination with the "broader meanings present in the data" that were identified during the conventional analysis (hsieh and shannon : ) . in this way, a summative content analysis focused on the framing of climate change can take in the "underlying" and "contextual" meanings that surround climate discourse in these newsletters with specific attention to how climate change is described as an issue, who is responsible for mitigation, and what types of solutions are to be implemented (mossler et al. ). around half of the respondents ( out of ) view all three social ties (to the cccga, to umass extension, and to other cranberry growers) as important. only three respondents out of did not view any of the three as important (see supplementary table ) . among the three social networks, we find that the growers who view the cccga as important are more likely to consider global warming as a serious threat to their way of life during their lifetimes, compared to the growers who do not consider it an important network, as shown in table , below. this finding holds true even after we control for the variables on personal and ecological conditions of cranberry production that were found in our earlier study to be significant predictors of the perception of global warming as a threat (gareau et al. : - ). according to model , when cranberry growers who do not view the cccga as an important network are compared to growers who do value the cccga, the odds of the latter group considering global warming a serious threat is about times higher (i.e., . − = . ) than the former group (a = . ). this positive association is also observed for a smaller sample (n = ) in model that includes other relevant independent variables, albeit at a different statistical significance level (a = . ). in both models and , the odds ratios for view cccga as important are notably larger than those of other significant predictors. this might be because it is a binary variable rather than an ordinal or continuous variable like the other significant predictors (with the exception of female, which is also binary and has the second largest odds ratio in model ). whether a grower values the connection with umass extension or with other cranberry growers, however, is not associated with the odds of this grower considering global warming a serious threat. we also find a positive association between viewing other cranberry growers and umass extension as important actors in their social networks and the degree to which growers worry about global warming (see table ). specifically, model suggests that the odds of being more worried about global warming are . times greater (i.e., . - = . ) for growers who view other cranberry growers as an important network compared to growers who do not value other growers as much, holding other independent variables constant (a = . ). similarly, the odds of being more worried about global warming for growers who view umass extension as an important network are about . times greater (i.e. . - = . ) compared to growers who do not share this view (a = . ). next, we examine the relationship between the perceived importance of the ties to these three social network actors and the implementation of various climate adaptive management practices in cranberry production. the results are presented in table . we do not find a significant association between the perceived importance of any of the three social actors and the number of management practices implemented. moreover, whether a grower views the cccga, umass extension, or other growers as an important network tie is not associated with ) whether this grower implements conservation or creation of pollinator habitat or ) the degree to which a grower thinks management practices are important to their cranberry production. most control variables, which are significant predictors of global warming attitude, are not associated with climate adaptive management practices. the only exception is the perceived importance of weather-related events, which is positively related to the perceived importance of management practices. for the generalized ordered logit regression models of global warming worry, the dependent variable is an ordinal variable coded in this way: ( ) not worry at all, ( ) worry a little bit, ( ) worry a fair amount, ( ) worry a great deal. for variables that violate the proportional odds assumption: a odds of worrying at least a little bit rather than not worrying at all, b odds of worrying at least a fair amount rather than worrying a little bit or less, c odds of worrying a great deal rather than worrying a fair amount or less * p < . ** p < . *** p < . (two-tailed tests) we acquired newsletter issues: issues of bogside and issues of the cranberry station newsletter. the bogside sample spans nearly four and a half years (from july to november ) and the cranberry station newsletter spans nearly years (from march to december ). the most frequent words found in each sample are depicted in the illustrative word clouds in fig. . the size of each word is positively correlated with the count of stemmed versions present in the two newsletter samples. (a full list of the top words in each sample is given in supplementary table ). the mission of the umass extension cranberry station is "maintaining and enhancing the economic viability of the massachusetts cranberry industry through research and outreach and serving the public welfare by supporting economic development and the protection of the for each of the three dependent variables, we have also estimated additional unreported models with different subsets of control variables, and found substantively similar results for the generalized ordered logit regression models of management practice importance, the dependent variable is an ordinal variable coded in this way: ( ) somewhat important or less, ( ) important, ( ) very important. for variables that violate the proportional odds assumption: a odds of considering management practices as important or very important rather than somewhat important or less, b odds of considering management practices as very important rather than important, somewhat important, or less *p < . **p < . ***p < . (two-tailed tests) environment" (ag.umass.edu/cranberry/cranberry-station). the first priority listed is "the economic viability of the massachusetts cranberry industry"; the second priority, "serving the public welfare," is split into "supporting economic development" and "the protection of the environment." we therefore would expect the newsletters to reflect three priorities: economic success for the cranberry industry, the importance of economic development more generally, and environmental protection. consistent with this emphasis on economic viability, the cranberry station newsletter largely discusses specific management-related issues and solutions that contribute to cranberry yield and economic viability. the top words in the cranberry station newsletter sample confirm this; "using" is the number one word, while "applicators," "managing," and "control" are all included in the top . common topics discussed in this newsletter are fruit keepingquality forecasts, updates on new trainings, information on pollination, pesticide application, frost management, and other specific problems affecting crop yields. the first priority in the mission statement, economic success in the cranberry industry, is the theme most clearly present in the data. the concerns and practices that are mentioned in this newsletterincluding issues like climate change, for example-are therefore likely to be framed in ways that support this goal. the mission of the cccga is "to work to enhance the sustainability and profitability of cccga members" (https://www.cranberries.org/about). this statement differs from the umass statement in its focus on serving the cranberry grower community specifically, rather than on economic growth or the public welfare in the abstract. of the four management-related words in the umass top word list, "using" alone also occurs among the top words of the bogside sample. bogside newsletters instead regularly include a number of social, historical, political, economic, and management-related sections-for example, the cranberry marketing committee's "updates from the cmc"; "notes from the hill," which highlights agriculturerelated news from the state capitol; and "harvesting the past: a historical look at the cranberry industry," which reprints stories of the early cranberry industry first published in "cranberries" magazine. various advertisements for products and upcoming events like meetings and festivals are often present as well. bogside, therefore, is likely to include content which appeals to the grower's whole life, to a wide arrangement of communal conditions. the "about" page on the cccga website provides further confirmation, saying that the association "assists growers in solving everyday problems, offering assistance in regulatory compliance, sponsoring professional development seminars and organizing association activities…" (https://www.cranberries.org/about). issues like climate change, therefore, we might also expect to be discussed in the context of how they can affect a grower's life-and their surrounding community-in a multitude of ways. both newsletters together contain fewer than references to climate change during the sample periods examined (bogside, months; cranberry station newsletter, months). (for a full list of specific references, see supplementary table .) there are climate change references in each newsletter sample, for a total of . six of these mention climate change as a topic of discussion at an in-person event without further context, indicating that both the umass cranberry station and the cccga hire speakers who address how climate change is impacting the cranberry industry. just this information, however, does not provide the context necessary for analysis-how climate change is framed and discussed. in the cranberry station newsletter, after accounting for the in-person event references, five of the remaining eight frame climate change as an issue that exacerbates a specific cranberry-related problem that should be addressed with management practices or agricultural inputs. the december cranberry station newsletter considers how to manage poison ivy, "a big winner in the climate change game since it responds positively to the increased co in the atmosphere" (p. ). the article suggests using callisto ® , an herbicide. similarly, the november and february issues of the cranberry station newsletter mention climate change for its role in exacerbating the conditions that lead to fruit rot. specifically mentioned is a "national document listing research priorities…towards the development of new and improved fruit rot management strategies" (cranberry station newsletter, nov. : and feb. : ) . once again, climate change is framed as an issue to be addressed with practical management strategies. even so, climate change is listed as a "low" priority issue rather than a cause for alarm (cranberry station newsletter nov. : and feb. : ) . the two references found in the december issue of the cranberry station newsletter follow a similar pattern; both references are framed in the context of water management, where the focus is on recommending strategies such as heat mapping and late water treatments. while bogside also discusses climate change as a problem for cranberry production, the issue is framed more broadly, as a problem that requires engagement with complex environmental and social processes, not just agricultural inputs and management strategies. bogside may explains that climate change is contributing to declines in native and commercially managed honey bee populations (apis mellifera l.) that are vital for cranberry pollination (bogside, may : ) . this reference is made in the context of a specific cranberry-related topic-ensuring adequate pollination-but does not go on to provide a management or application-based solution. instead, the problem discussed, the global pollinator decline, is presented as partially caused by agricultural applications and pesticides, not fixed with them. the bogside march and april issues also discuss climate change in the context of a complex socio-environmental problem. these issues include discussion of the massachusetts global warming solutions act (gwsa) (bogside, march : and bogside, april : ). gwsa was signed into law in , "making massachusetts one of the first states in the nation to move forward with a comprehensive regulatory program to address climate change" ("global warming solutions act"). including this reference demonstrates that the cccga recognizes the political relevance of climate change action, and these examples thereby contribute to a message that climate change is a complex threat to both the cranberry industry and its growers. the most recent occurrences of the term climate change in the sample are in the bogside issues of july , september/october , and november . these references not only discuss climate change as a complex socio-environmental problem, but also emphasize the magnitude of the problem. bogside july contains an article written by the umass cranberry station that lists climate change as one of the top three "drivers of change" in the cranberry industry (p. ). moreover, these "drivers of change" are identified as extremely complex issues that require help and insight not only from grower management and natural science, but from " academic disciplines" (bogside, july : ). similarly, in bogside september/october , climate change is mentioned in an appeal to the growers to support funding for a year study on fruit quality. the cccga stresses that these fruit quality concerns will take collaboration among the whole industry to address: "this will be a collaborative effort of industry, growers and researchers to collectively work towards developing solutions to a problem that is only getting more difficult with climate change and marketplace demands for high quality fruit" (bogside, september/ october : ). bogside november continues this trend by including a quote from senator marc r. pacheco (district of first plymouth and bristol). in this issue, the senator expresses his pleasure that cranberry operations in his district received "over $ , though the massachusetts cranberry bog renovation enhancement grant program" (p. ). but he also states, "as the worst effects of climate change continue to emerge, our agricultural sector's need for infrastructure upgrades will only intensify" (bogside, november : ). this example highlights both the increasing alarm that climate change is prompting and its complex interconnection with the political realm and the resources of the government. the november bogside issue includes a "special fundraising appeal," in which the cccga highlights both the economic constraints on addressing fruit quality and the importance of recognizing that climate change will only continue to affect the cranberry industry in more adverse ways in years to come: "we recognize the financial situation of our growers and that the timing of this fundraising effort is challenging at best. this will be a collaborative effort of industry, growers, and researchers to collectively work towards developing solutions to a problem that is only getting more difficult with climate change and marketplace demands for high quality fruit" (p. ). once again, this bogside item emphasizes that climate change must be addressed with collective action among industry, growers, and researchers, and not only through management strategies. a third climate change mention in the november bogside issue refers to the subject in similar fashion. the author of the article, brian wick, executive director of the cccga, notes that by using renewable energy, like solar, the cranberry industry can support climate mitigation practices like lessening dependence on fossil fuels. but he then goes on to say that not only cranberry growers, but also the wider community, must support these practices, and he expresses his frustration that this is often not the case. in this study we examine how social ties to certain actors in their social networks affect cranberry growers' climate change attitudes and management practices in farming operations. cranberry growers in massachusetts, an essential farming community in the american and new england social, cultural, and ecological landscape, face a multitude of threats to their industry and livelihood in a world increasingly defined by global climatic change. we find that the perceived importance of the cccga, umass cranberry station, and fellow cranberry growers in the eyes of the growers we studied is associated in nuanced ways with their climate change attitudes. when these connections amplify threat perception or worry, they can be considered "green ties," that is, ties with the potential to lead to collective action to combat climate change at the local level. survey results indicate that the majority of growers view the grower's association (i.e., the cccga), extension service (i.e., the umass extension), and fellow cranberry growers as important actors in their social network. only growers who view the cccga as important, however, are associated with higher odds of considering global warming a threat to their way of life. viewing umass extension and a network of other growers as important, on the other hand, is associated with higher odds of feeling worried about climate change. like many growers in the united states, cranberry growers are somewhat predisposed to treat challenges related to weather conditions as an integral part of their everyday practices rather than as abnormalities to be attributed to anthropogenic climate change. consequently, although cranberry growers might be concerned or worried about weather conditions, they are less likely to view them as an existential threat (see gareau et al. ) . those who value the cccga, however, overcome this predisposition. as our statistical analysis illuminates, growers who identify the cccga as an important actor in their social network are more likely to acknowledge that they believe climate change is a "threat" to their "way of life." one possible reason for this may be that the cccga disseminates information that contributes to and encourages communal conditions conducive to considering climate change a threat, as our content analysis of the newsletter bogside reveals. if a grower identifies the cccga as important to their network, this can reasonably be considered an indication of a grower's trust in this organization as a source of important information. as supported by our content analysis of bogside, the cccga frames climate change as an important issue in wider environmental and communal conditions and provides a rather consistent and clear picture of the threat created by global climate change. even if growers may feel threatened by the impacts that climate change poses for certain aspects of their farming operations, this does not necessarily translate into feeling "worried" about its effects. one possible reason for this is a disposition in which a person acknowledges that climate change is a serious threat, but believes that it will, in time, be effectively addressed by human innovation. this sentiment was expressed by a longtime key actor in the cranberry community, former director of the umass cranberry station, dr. carolyn demoranville, in a article in the christian science monitor. demoranville referenced climate change while discussing the umass cranberry station's efforts to create cranberry hybrids. she warned that these developments on fruit type might not be adequate to deal with the possible future effects of climate change, but she also expressed optimism that human developments will address the problem: "if we're talking about numerous degrees of difference in years, that may be beyond working with the tools we have. but presuming that somebody decides to finally start doing something about climate change, then i think we would still have the tools…to deal with the amount of change that's predicted in the less-drastic models" (azios ) . this existential threat, in contrast, is less often highlighted in the cranberry station newsletter, which focuses on immediate, practical issues threatening cranberry production, such as pests and weeds, rather than broader concerns related to political, economic, or global climatic conditions. this helps explain why the perceived importance of umass extension is not associated with growers' recognition of climate change as a threat to their way of life. this is not to say that the cccga is more concerned about climate change. the umass cranberry station, for example, authored a powerful article about climate change in july , but it had it published in cccga's bogside. if a grower were only to read the cranberry station newsletter, therefore, they would not necessarily receive this message. the cccga, in short, seems to be the more powerful social actor with regard to promoting "green ties" within the growers' social network. through bogside, growers gain access to a knowledge frame that conveys the global scientific community's position that climate change is a threat. the cccga transmits its knowledge frame among actors in the cranberry community, creating social conditions in which concern for climate change among growers is evident. the umass cranberry station generates a different knowledge frame in which worry over climate change is expressed among its networked growers, though this tie does not generate the recognition of climate change as an existential threat. the difference between "worry" and "threat to the way of life" might have something to do with the different ways these powerful actors influence the cranberry growing community. it is noteworthy that the social ties among cranberry growers that foster communal conditions supporting worry about climate change parallel the ties created by the umass cranberry station. although growers may feel threatened by the impacts that climate change can have on certain aspects of their growing operations, this does not necessarily translate into feelings that climate change threatens their "way of life" or that they feel "worried" about it. social ties among growers, therefore, might lead to different interpretations of climate change depending on the specific dispositions of the growers in their network. for example, the "personal conditions" of gender and age of other growers with whom they are connected were found to be significant predictors of climate change attitudes (see gareau et al. ) . this analysis also sought to discover whether certain social networks are associated with the adoption of management practices that address climate change. we find that climate change attitudes are, to various degrees, associated with cranberry growers' ties to important social actors (i.e., ties to the cccga, umass cranberry station, and other cranberry growers), yet none of the perceived importance of these powerful actors is associated with the actual implementation of climate adaptive management practices. this finding suggests that both the cccga and umass could refine and strengthen their outreach to growers regarding climate change mitigation and adaptation strategies. for example, although both newsletters discuss many different management techniques, including those that could be considered climate adaptive strategies, none of the references to climate change found in these samples emphasizes these types of practices. it is important to acknowledge the limitations of our findings on a couple of levels. for one, the statistical analyses were conducted based on survey responses, which is a relatively small sample. as noted in the methods section, however, our sample accounts for roughly one quarter of the population of cranberry growers in massachusetts, which makes it an adequate sample for statistical inference about the population (salant ) . nonetheless, the power of hypothesis testing is limited by the small sample size, especially in models that include additional control variables. we partially mitigated this issue by using an alpha level of . and, when applicable, more parsimonious models, such as the generalized ordered logit regression models, over other alternatives. we are also limited to trusting the growers when they report to us that certain actors are indeed important to them. moreover, it is challenging to determine in our survey data the strength of the social interactions between the cranberry growers and these three social actors. whether growers in the survey attended the many umass cranberry station meetings, thoroughly read each and every monthly bogside or cranberry station newsletter issue, or routinely engaged in activities with fellow growers is not directly captured in the survey. our findings, however, suggest that the connection between growers and the cccga is strong enough to create a pattern of influence on climate change perceptions among growers connected to this powerful actor, which is also supported by participant observation and in-depth interviews not reported in detail in this paper. second, allowing growers to self-report the importance of certain actors creates a potential for endogeneity and might impose a limitation on the research findings concerning network ties. we are confident, though, that self-reported importance is worth discussing, mainly because it is widely understood among growers and researchers that cccga, umass cranberry station, and grower networks are all important actors in this tightly knit, growing community. as a unique growing community of only or so in number, cranberry growers rely heavily on their networks to remain viable in this struggling industry. our findings suggest that connections to cccga are raising awareness among growers about the connections between those struggles and climate change. finally, there are limitations to the ways we introduce management practices to the growers. by limiting the count of management practices to activities undertaken in the past - years, the measure systematically excludes individuals who may have taken certain long-term actions more than years ago. we focused on this time span so that the survey results matched the time frame of the newsletters included in our content analysis, which extends to years in the cranberry station newsletter. social networks are clearly an integral aspect of understanding cranberry grower attitudes and actions toward climate change. as the example of demoranville's comment in the christian science monitor cited earlier suggests, the cccga and umass cranberry station's official communications are also augmented by the climate discussions that powerful actors in these networks have in the mass media. a november usa today article titled "global warming targets thanksgiving," for another example, quotes brian wick, the current cccga executive director, saying, "the growers are at the point where there's concern that the climate is changing. we are seeing these sort of extremes in weather" (montgomery ) . or again, a cape cod times article titled "climate change: heat may drive cranberry industry north," which cites both demoranville and wick; demoranville expresses concern for recent patterns of "big rain events with long dry periods in between," and says, "eventually, if it got hot enough, you would come to a time when you couldn't grow cranberries" (fraser ). however, the article reports, neither demoranville "nor wick see that happening anytime soon" (fraser ) . these examples show that there are key actors in these social networks who are willing to discuss climate change as a threat, although, as with our newsletter findings, they do not necessarily advocate for immediate collective action. in sum, identifying the cccga as important is associated with greater odds of perceiving climate change as a threat, and identifying the umass cranberry station as important is associated with greater odds of worrying about climate change. this illuminates a need for these social actors to work together more closely to foster more powerful green ties bridging their disparate connections to climate attitudes. although these organizations already collaborate to an extent (for example, bogside often includes a section written by the umass cranberry station), our findings highlight that these two organizations represent two distinct fields of knowledge in the cranberry industry. the technical focus of the umass cranberry station seems to complement the socioeconomic and sociopolitical focus of the cccga. however, when it comes to encouraging growers to consider climate change as a serious threat to their way of life-a threat calling for immediate action-their differing knowledge frames for now seem to prevent a deeper connection and interdependence with one another that could more consistently and forcefully convey what needs to be done about the impact of climate change on the cranberry industry. climate change needs to be framed as a problem that is vast and socio-environmental (givens et al. ; huang ) , but also one with many tangible effects on specific, local processes (adger ) . by avoiding both social and natural reductionist thinking, this framing enables an understanding of climate change that allows the severity and complexity of the problem to be expressed, and acted upon (white et al. 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hansen, alana; hanson-easey, scott; xiang, jianjun; cameron, scott; liu, qiyong; liu, xiaobo; sun, yehuan; weinstein, philip; han, gil-soo; bi, peng title: china's capacity of hospitals to deal with infectious diseases in the context of climate change date: - - journal: soc sci med doi: . /j.socscimed. . . sha: doc_id: cord_uid: bbbq ylr objectives: infectious diseases are a major cause of morbidity and mortality in china. the capacity of hospitals to deal with the challenge from emerging and re-emerging infectious diseases due to climate change is of great importance to population health. this study aimed to explore the capacity of hospitals in china to deal with such challenges. methods: a cross-sectional questionnaire survey was utilized to gauge information regarding capacity of hospitals to deal with infectious diseases in the context of climate change among clinical professionals whose roles pertained to infectious disease diagnosis, treatment and management in anhui province of china. descriptive analysis and logistic regression analysis were performed on the data. results: more than % of participants believed climate change would have an adverse influence on population health and infectious disease control in china. most indicated that their hospitals were well prepared for emerging infectious diseases at present, and they considered that logistical support in hospitals (e.g. administrative and maintenance services) should be strengthened for future capacity building. the majority of participants suggested that effective prevention and control measures, more interdisciplinary collaborations, more funding in rural areas for health care, and improved access to facilities enabling online reporting of infectious diseases, were extremely important strategies in building capacity to curb the population health impact of emerging and re-emerging infectious diseases due to climate change in china. conclusions: clinical professionals recognized that climate change will likely increase the transmission of infectious diseases. although rural health care and hospitals’ logistical support need to be improved, most professionals believed their hospitals to be capable of dealing with emerging diseases. they thought that interdisciplinary and cross-regional collaborations, together with necessary resource support (e.g. improved facilities for rural health care) would be important control strategies. currently there are roughly million cases of reported infectious diseases in china, which result in approximately , deaths annually (national health and family planning commission of the prc, ) . previous studies have found the incidence of infectious diseases has been sharply reduced due to great improvements in health care services (hipgrave, ) . however, the emergence and re-emergence of some climate-sensitive diseases, such as malaria, dengue and hemorrhagic fever with renal syndrome (hfrs) have occurred in recent decades in china (huang et al., ; xiang et al., ; zhang et al., b) . malaria was a serious public health problem during the s with an incidence of approximately per , population . in , the incidence was only per , with about , cases reported . however, malaria cases in china increased to , in (zhou et al., ) dengue cases were reported prior to , but frequent outbreaks have occurred during the last few decades in china (lai et al., ) . hfrs, a serious zoonotic disease caused by hantaviruses, can be transmitted to humans by contact with infected rodents or their excreta . the disease is frequently reported in china where roughly % of the world's cases occur (xiao et al., ) . although the incidence of hfrs significantly declined during the s, there has been an increasing trend since (xiao et al., ; zhang et al., ) . the possible reasons for the emergence and re-emergence of these infectious diseases may be linked to climate change, population movement, rapid urbanization and increased surveillance efforts (tong et al., ; wu et al., ) . it is widely known that the global average combined land and ocean surface temperatures have increased by . °c over the period from to (intergovernmental panel on climate change, ). the intergovernmental panel on climate change (ipcc) indicates that the global average surface temperature will increase by . - . °c by - compared to - , and other climatic variations, such as changes in rainfall patterns and relative humidity will also occur (intergovernmental panel on climate change, ). climate change has, and will continue to, impact the transmission of vector and rodent-borne diseases by affecting the growth and development of the vectors or hosts, shortening the incubation period of the pathogens within the vectors, and impacting human behaviour (e.g. more time spent outdoors) (world health organization, ). the projected temperature increase and change in rainfall patterns may bring about an increase in cases of infectious diseases such as malaria, dengue and hfrs zhang et al., a; zhou et al., ) . in china, annual average land surface air temperature has increased by . - . °c over the past years, and is projected to increase by . - . °c by as compared to (china national development and reform commission, ) . extreme climate events, such as extremes in temperature, drought in north china, and flooding in south china, may become more frequent and intensive (china national development and reform commission, ) . the variations in temperature, rainfall, humidity and extreme events posed by climate change could facilitate infectious disease transmission and result in the possible increase in cases of infectious diseases in china (tong et al., ) . in the chinese health care system, both the clinical health sector and preventive medicine (public health system) play important roles in the protection of population health. in the clinical health system, hospitals provide patient diagnosis, treatment and management. in the public health system, the centers for disease control and prevention (cdc) work to protect and improve public health and safety, and focus on disease prevention, control and surveillance; and cases of notifiable diseases are reported by clinical professionals to the local cdc (he, ) . therefore, hospital clinical staff and cdc public health professionals perform different roles, and may have different views on infectious disease control and prevention. given that the ability of the public health system (i.e. cdcs) to deal with emerging and re-emerging infectious diseases in the context of climate change has been extensively studied (tong et al., a; tong et al., ) , a similar investigation of the clinical health system where diseases are diagnosed and treated, is warranted. this study will contribute to a better understanding of infectious disease diagnosis, treatment, prevention and control in the face of climate change in china, and may also benefit disease control in other countries . the study employs a cross-sectional questionnaire survey among clinical professionals to explore china's capacity of hospitals to deal with infectious diseases in the context of climate change. further, the study explores participants' views on capacity building in the hospital sector to curb potential emerging and re-emerging infectious diseases due to climate change in china. a questionnaire was administered to clinical professionals in november . the questionnaire instrument design was informed by previous studies and relevant literature on climate-sensitive diseases (national development and reform commission of the prc, ; semenza et al., ; tong et al., ; wei et al., ) . the questions asked about clinical professionals' thoughts on climate change, disease occurrence, the capacity of hospitals to deal with infectious diseases, and strategies to build the capacity of the hospital sector and curb the health impacts of climate change related to infectious diseases. an open-ended question was included to explore in greater depth, participants' understandings of disease control, diagnosis, treatment and management in the context of climate change. the questionnaire is included in appendix a. the study site of anhui province was selected because it has a high incidence of infectious diseases, especially malaria and hfrs (bi et al., ; jiao et al., ; the people's government of anhui province, ). anhui province is located in east china (see fig. ), and has a warm-temperate, semi-humid monsoonal climate with an average annual temperature between and °c, and annual precipitation between and mm (the people's government of anhui province, ). participants were clinical professionals from three major hospitals in anhui. a total of clinical professionals were surveyed in two general hospitals in the capital city of anhui. a further were surveyed in an infectious diseases hospital in a prefectural-level city. the clinical professionals included both doctors and nurses whose roles pertained to infectious disease diagnosis, treatment, and management. investigators administered the questionnaire in the three hospitals. to maximize the response rate, the principal researchers selected six key senior contacts from hospitals to assist with the distribution of questionnaires to potential participants. the process of participation was voluntary, and no incentives were offered. there are clinical professionals relating to infectious diseases in the three major hospitals, and for ease of administration, the entire population was used as sample. in total, questionnaires were distributed, and after omitting incomplete questionnaires, were analyzed, with a response rate of %. all returned questionnaires were entered using epidata . software (lauritsen, ) to create a database. statistical analyses were performed with stata . (statacorp, ). participants' demographic characteristics were descriptively analyzed. binary logistic regression was used to explore the association between binary responses and demographic variables. ordinal logistic regression was used to explore the association between ordinal responses and demographic variables. the demographic variables were age, gender, professional level, length of employment, education, and occupation. the binary responses were "yes", and "no/unsure". the ordinal responses were "very concerned", "concerned", "slightly concerned", and "not concerned"; or "extremely important", "very important", "important", "less important" and "not important". data were analyzed with a two-sided test and p-values less than . were considered statistically significant. table shows the demographics of the participants. in this study, % of the clinical professionals who participated were doctors, and . % were nurses. while ages ranged from to years, . % of professionals were aged below years, with the mean age being . years. of the participants, . % were female. junior level professionals accounted for . %, intermediate level professionals . % and senior level . % of the total participants. over % of the participants had worked in hospitals more than five years. more than % held a university degree or higher qualifications, especially the doctors who were mostly highly educated with a bachelor of medicine degree or above (see supplementary table s in appendix b). as shown in table , more than % of the professionals were either very concerned or concerned about climate change. there was a statistically significant association between age group, professional level and the concern about climate change (see supplementary table s in appendix b). those who were over years (or = . , % ci: . - . , p = . ) and the senior staff (or = . , % ci: . - . , p = . ) were more likely to be concerned about climate change. furthermore, % of the professionals agreed with the statement that the weather was becoming warmer, especially nurses and the professionals who had been employed more than ten years (see supplementary table s in appendix b) . nearly all ( . %) believed climate change would have an adverse influence on population health. furthermore, . % and . % of professionals agreed that predicted increasing temperatures and changes in precipitation patterns would affect infectious disease transmission. specifically, . %, . %, and . % of professionals were either extremely or very likely to believe that there was an association between climate change and malaria, dengue, and hfrs, respectively. table shows that about % of participants thought that there had been an increasing number of patients with malaria, dengue, and hfrs, and roughly % and % of professionals thought that climate change and population migration, respectively, were contributing factors. more than % of participants believed that malaria and hfrs hospital-reporting protocols were in place, and . % believed so for dengue. in terms of the diagnostic capability of hospital laboratories, . % of professionals responded that their hospitals were 'always' or 'mostly' able to rapidly provide diagnostic tests for malaria, . % for dengue, and . % for hfrs, respectively. some % of participants rated diagnostic and treatment capacity as excellent/good for malaria and hfrs, while % thought this was the case for dengue. moreover, if an unusual cluster of cases was noticed, most professionals would take actions such as discussing with colleagues and laboratory technicians, informing the public health officer, and consulting with the cdc. in addition, there were no significant differences in these perceptions of infectious diseases between doctors and nurses. clinical professionals' perceptions of the current capacity of their hospitals to deal with infectious diseases are shown in table . specifically, . % of participants agreed that they had sufficient staff to deal with disease outbreaks. also, more than % believed that their staff were well informed about current infectious disease trends, and that the quality of reported data from their hospitals to the cdc was excellent. however, . % of professionals thought that logistical support in hospitals (for example administrative and maintenance services) needed to be strengthened. overall, . % either agreed strongly or somewhat that hospitals were well prepared for the threat of a serious emerging disease. there were no significant differences between perceptions of doctors and nurses regarding the current capacity of the hospital. additionally, . % agreed that more research on the health impacts of climate change was needed. to build capacity to meet the challenge of emerging and re-emerging infectious diseases due to climate change, . % of participants thought that prevention and control measures were extremely important strategies, and . % believed more collaboration with their local cdc was extremely important (table ) . furthermore, multivariate ordinal logistic regression analysis showed that compared to those under years, professionals aged over years were more likely to indicate that prevention and control measures are extremely important (or = . , % ci: . - . , p = . ). senior level staff were more likely to believe that more collaboration with the cdc was extremely important (or = . , % ci: . - . , p = . ); while doctors were less likely to believe so (see supplementary table s in appendix b). about % believed more funding was required for rural health care and that improving the accessibility of the online infectious disease reporting system for rural hospitals was also extremely important. more than % of those surveyed believed that the health impacts of emerging and re-emerging infectious diseases due to climate change could be addressed with strategies such as: better response mechanisms; strengthening the climate change poses a significant threat to global population health (intergovernmental panel on climate change, ). clinical professionals are at the frontline of health care provision to the population and are likely to witness firsthand the health impacts of a changing climate (blashki et al., ) . they, therefore, provide a unique perspective on the health impacts of climate change and an indepth understanding of local community health (blashki et al., ) . to the best of our knowledge, this study is the first of its kind to gauge clinical professionals' perceptions of the capacity of china's hospitals to manage infectious diseases under climate change and to provide insight for policymakers, practitioners and medical educators regarding climate change adaptation in the health sector. concern about climate change was acknowledged by most professionals, especially among senior staff and the group aged over years. the majority of professionals, particularly nurses and those with longer terms of employment indicated that the weather was getting warmer. nearly all respondents indicated climate change would adversely affect population health, and most indicated it would facilitate infectious disease transmission. less than % of participants thought climate change was unlikely to be associated with malaria incidence compared to % for dengue and hfrs. the results were consistent with our previous studies among cdc staff who indicated climate change was more likely to have an influence on malaria than hfrs, whilst the perception of an association between climate change and dengue was not as strong as in a previous study conducted in guangdong (tong et al., ) . this is likely due to guangdong having the highest incidence of dengue in china, whilst in anhui there are fewer dengue cases reported (lai et al., ) . moreover, compared with previous studies among cdc public health professionals in other provinces (tong et al., a) , the hospital clinical professionals in this study were more likely to indicate that climate change would have an influence on hfrs. this could be due to the relatively high number of hfrs cases in anhui province, and hence firsthand knowledge of the disease and its determinants. additionally, another study conducted among health experts in europe also indicated their perceptions of the negative impact of climate change on vector-borne, food-borne, water-borne and rodent-borne diseases (semenza et al., ) . despite . % of participants agreeing strongly or somewhat that hospitals were currently well prepared for the threat of a serious emerging disease, climate change may present new challenges for health sectors such as increased incidence in certain climate-sensitive diseases, or diseases either not previously seen in the area, or not seen for some time. such an influx in cases could stretch the coping capabilities of the present system. this may explain why the public health and clinical health sectors share concerns about potential emerging and re-emerging climate-sensitive diseases. the majority of professionals indicated that climate change and population migration were considered as the most significant factors associated with the increase of these infectious diseases. this is in line with other studies that indicated the impact of climate change and migration would affect infectious disease transmission (gao et al., ; mcmichael, ; sang et al., ) . higher temperatures and changing precipitation patterns can contribute to the increasing m.x. tong et al. social science & medicine ( ) - population of vectors/rodents and more frequent contact with humans. simultaneously, population movement can facilitate disease transmission from one region to another. in china, currently it is estimated that approximately million people have migrated from poor rural areas to gain work in the cities (national health and family planning commission of the prc, ). this "floating population" (highly mobile population) of internal migrants chiefly work in insecure lowwage jobs. moreover, they often lack health insurance and defer seeking timely medical treatment, which could have a negative influence on infectious disease control (qin et al., ) . future infectious disease control and prevention should be cognizant of climate change impacts and population movements on the transmission of these diseases. in addition, building a more comprehensive national health insurance system covering the internal migrant population's health expenses in any part of china may provide benefits for population health (qin et al., ) . most professionals claimed that there was a hospital protocol in place for the reporting of notifiable diseases and believed that the capacity for disease diagnosis and treatment was either excellent or good, especially for malaria and hfrs. as anhui historically has a high incidence of malaria and hfrs (chen and qiu, ; gao et al., ) , professionals are more likely to have experience and confidence in dealing with these diseases, whereas historically there has been a low incidence of dengue in the province. the majority of professionals believed that their hospital laboratories were able to provide rapid diagnostic test results for malaria, dengue and hfrs. additionally, most professionals purported they would take comprehensive actions if they detected an unusual cluster of cases. this indicates there is a strong likelihood that outbreaks of emerging or re-emerging diseases would be detected early by the clinical professionals or the cdc. this detection of unusual trends could aid in curbing transmission if preventive measures are activated early. regarding the capacity of hospitals to deal with disease risks, most participants believed they were well prepared for the threat of a serious emerging disease. of particular note, participants indicated there was a need to strengthen the hospital's logistical support. this is in line with another study conducted by xu and chu in focusing on logistics capacity in hospitals, which advocated that a reliable logistical support system is one of the most important components in modern health care systems and should be given higher priority to sustain and promote better health care services in the long-term (xu and chu, ) . currently, the main obstacles to improving hospitals' logistical support in china are the lack of high-quality staff and regulatory frameworks for logistics management within hospitals (lin, ) . promoting specialized training for logistics staff and implementing management and quality control guidelines would be important. moreover, the problems in rural areas were highlighted, especially regarding the funding for rural health care support and accessibility to an online reporting system. the severe acute respiratory syndrome (sars) outbreak demonstrated that rural health care was the weakest component of china's disease control and prevention system (knobler et al., ) . more resource allocation to rural areas would be a vital step in advancing china's capacity building in response to emerging infectious diseases due to climate change. in this study, older and senior staff were more likely to indicate that primary prevention measures and interdisciplinary collaboration were important, compared with the younger or non-senior staff. such differences could be due to the rich working experience of the groups, who may have a better understanding of the infectious disease control and prevention strategies. most participants also suggested a need for improved environmental health, more health education programs and financial support to improve vector/rodent-borne disease control, diagnosis, treatment and management in the context of climate change. some studies have made future predictions about the effect of climate variation on dengue and malaria (caminade et al., ; ebi and nealon, ) , although there are many confounding factors to consider. although some participants in this study indicated that changes have already been noted, it would be useful in future studies to gain an insight into stakeholders' perceptions of when global warming would reach a point where there is a marked effect on the burden of climate-sensitive diseases. some limitations of this study deserve mention. firstly, this study was conducted in one province. the results may not be generalizable to clinical professionals in other provinces of china. secondly, as dengue is an emerging and uncommon disease in anhui, participants in this region may be less likely to have hands-on experience in dealing with dengue cases, and feel less confident about dengue diagnosis and treatment. lastly, the study was conducted among three major hospitals in cities, and results may not be generalizable to rural areas, townshiplevel hospitals or village-level clinics. nevertheless, these findings may provide information for policymakers and clinical professionals pursuing initiatives to strengthen the capacity for hospitals to cope with a potential increase over time, in cases of climate-sensitive infectious diseases. this study revealed that most clinical professionals thought climate change would have an impact on infectious diseases, and believed climate change and population migration were significant factors associated with infectious disease transmission. health professionals in our study thought that the overall capacity of the hospital healthcare system to deal with infectious diseases was excellent. however, logistical support should be strengthened in hospitals and more climate change related research is needed. issues that could be addressed include prevention and control measures, collaboration with the cdc, inhouse staff training and improved healthcare systems in rural areas. the multiple direct and indirect impacts of climate change will continue to threaten the health of the chinese population. these findings may help health policymakers develop organizational adaptation policies to address the adverse impact of climate change on health. the authors declare no conflicts of interest. el nino-southern oscillation and vector-borne diseases in anhui, china. vector borne zoonotic dis general practitioners' responses to global climate change -lessons from clinical experience and the clinical method impact of climate change on global malaria distribution epidemiologic surveillance on the hemorrhagic fever with renal syndrome in china china's national climate change programme. people's republic of china: national development and reform commission dengue in a changing climate change in rainfall drives malaria re transmission of haemorrhagic fever with renal syndrome in china and the role of climate factors: a review bringing into full play the role of general hospitals in the prevention of contagious diseases communicable disease control in china: from mao to now epidemiologic characteristics of haemorrhagic fever with renal syndrome in mainland china from climate change : the physical science basis climate change : 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health professionals' perceptions of hemorrhagic fever with renal syndrome and climate change in china. global planet perceptions of malaria control and prevention in an era of climate change: a crosssectional survey among cdc staff in china infectious diseases, urbanization and climate change: challenges in future china perceptions of capacity for infectious disease control and prevention to meet the challenges of dengue fever in the face of climate change: a survey among cdc staff in guangdong province the impact of climate change on infectious disease transmission: perceptions of cdc health professionals in shanxi province strengthen control of vectorborne diseases to lessen the impact of climate change in western pacific region with focus on cambodia, mongolia and papua new guinea: final project report. who regional office for the western pacific impact of climate change on human infectious diseases: empirical evidence and human adaptation association between dengue fever incidence and meteorological factors in animal reservoir, natural and socioeconomic variations and the transmission of hemorrhagic fever with renal syndrome in adhere to scientific development to improve the overall logistics capacity in hospital historical patterns of malaria transmission in china climate variability and hemorrhagic fever with renal syndrome transmission in northeastern china spatiotemporal transmission dynamics of hemorrhagic fever with renal syndrome in china meteorological variables and malaria in a chinese temperate city: a twenty-year time-series data analysis malaria situation in the people's republic of china in . chin geographical, meteorological and vectorial factors related to malaria re-emergence in huang-huai river of central china this work has been funded by the department of foreign affairs and trade through the australian development research awards scheme under an award titled 'how best to curb the public health impact of emerging and re-emerging infectious diseases due to climate change in china' [project id: ] and the national basic research program of china ( program) [grant no. cb ]. the views expressed in the publication are those of the authors and not necessarily those of the department of foreign affairs and trade or the australian government. the commonwealth of australia accepts no responsibility for any loss, damage or injury resulting from reliance on any of the information or views contained in this publication. we thank the anhui medical university, anhui provincial hospital, the second hospital of anhui medical university and fuyang no. people's hospital getting involved in this study for their assistance in the distribution and return of questionnaires. all survey participants are greatly appreciated for their valuable contributions. supplementary data related to this article can be found at http://dx. doi.org/ . /j.socscimed. . . .m.x. tong et al. social science & medicine ( ) - key: cord- -p jyyt authors: taylor, steven title: anxiety disorders, climate change, and the challenges ahead: introduction to the special issue date: - - journal: j anxiety disord doi: . /j.janxdis. . sha: doc_id: cord_uid: p jyyt climate change involves ( ) increases in the prevalence of extreme weather events (e.g., wildfires, floods, hurricanes), ( ) more gradual climatic changes (e.g., rising sea levels, desertification), and ( ) increased risks of pandemics and other widespread disease outbreaks. anxiety evoked by the threat of climate change can be either adaptive or maladaptive. adaptive anxiety can motivate climate activism, such as efforts to reduce one’s carbon footprint. maladaptive anxiety can take the form of anxious passivity, where the person feels anxious but incapable of addressing the problem of climate change, and may take the form of an anxiety disorder triggered or exacerbated by climatic stressors. such stressors may involve exposure to extreme weather events or may involve exposure to other stressors such as forced migration due to rising sea levels or desertification. three types of interventions are needed to address the various types of climate-related anxiety: ( ) programs that motivate people to overcome anxious passivity and thereby take action to mitigate the effects of climate change, ( ) treatment programs that address anxiety associated with exposure to climatic stressors, and ( ) programs that build resilience at an individual and community level, to help people better cope with the challenges ahead. at no point in human history have we faced such an array of both familiar and unfamiliar risks, interacting in a hyperconnected, rapidly changing world. new risks and correlations are emerging. decades-old projections about climate change have come true much sooner than expected. with that come changes in the intensity and frequency of hazards. risk really is systemic, and requires concerted and urgent effort to reduce it in integrated and innovative ways. (united nations office for disaster risk reduction, , p. iii) the anthropocene, which is our current geological epoch, is a period during which human activity is the dominant influence on climate and the environment. the anthropocene is an epoch that will be filled with climate-related existential threats. the goal of the special issue for the stressors such as rising sea levels can also lead to the loss or damage to places of cultural importance such as places of worship. people who are strongly attached to their possessions, homes, or areas of residence may find it especially difficult to evacuate during extreme weather events. in the case of wildfires, this can result in injury or death when people refuse to evacuate (conaway, ) . climate change-related forced migration will become a growing problem in the coming years, as coastal towns are flooded and other regions become uninhabitable due to heat and desertification (robinson, dilkina, & moreno-cruz, ) . clayton ( ) observed that stressors can be directly experienced or encountered vicariously through news or social media. research by gibson et al. ( ) in this special issue illustrates the distinction between direct and indirect impacts. in studying the effects of climate change on the pacific island nation of tuvalu, gibson et al. observed that the residents were anxious about both the direct, local environmental impacts of climate change on their island nation, and also by news reports about global climate change and its future threats. clayton ( ) further observed that climate-related anxiety may be an appropriate reaction to a realistic threat, or it may be excessive and disproportionate. the distinction is important. appropriate levels of anxiety can serve as a motivator, providing an impetus for people to take action to address climate threats, such as by finding ways of reducing their carbon footprint. unfortunately, however, as noted by hrabok et al. ( ) in this special issue, some people experiencing appropriate levels of anxiety may lapse into helplessness and despair if they believe that their personal or collective acts are insufficient to deal with the challenge of climate change. excessive anxiety-that is, anxiety that is highly disproportionate given the level of threatcan be severe and debilitating, meriting clinical attention. hrabok et al. ( ) , in this special issue, described in detail the psychological impacts of extreme environmental events, which include posttraumatic stress disorder (ptsd), anxiety disorders, mood disorders, substance-used disorders, and other clinical problems. high levels of anxiety and other disorders are likely to occur for victims of forced migration (jannesari, hatch, & oram, ) . the psychological impact of climate change is likely to be complex and varied, as people try to cope with a mix of acute and chronic stressors. anxiety is one of a range of responses to climate change, in addition to depression, grief, and anger. denial of the threat of climate change is another psychological reaction to this existential threat. little is known about whether clinically significant climate-related anxiety is always part of some dsm- disorder or where it could form some other kind of syndrome. research into covid- -related anxiety revealed evidence of a covid stress syndrome, not readily characterized by dsm- , consisting of worries about personal health, worry about the socioeconomic impact of the pandemic, xenophobia, traumatic stress symptoms (e.g., nightmares and intrusive thoughts about covid- ), and covid- -related compulsive checking and reassurance-seeking . something similar could be true of climate-related anxiety; it may be part of a broader syndrome of worry, checking, and intrusive thoughts, rather than neatly fitting into an existing dsm- diagnostic category. further research is needed to investigate this issue. several of the contributors to this special issue describe how climate-related stressors have a greater impact on some demographic groups than others. clayton ( ) observed that j o u r n a l p r e -p r o o f younger people, compared to older adults, tend to report greater climate-related anxiety, most likely because younger people, unlike older adults, will be most likely to live through the climatic adversities in the decades to come. climate-related anxiety also tends to be greater in people who deeply care for the environment (clayton, ) . levels of climate anxiety are likely to increase over time as increasingly more people are directly impacted. people in poverty, indigenous peoples, children, and people living in precarious circumstances (e.g., in areas prone to droughts or wildfires, or regions vulnerable to rising sea levels) are among the at-risk groups for experiencing severe effects of climate change (clayton, ; hrabok et al., ; gibson et al., ) . living in communities where there is a lack of social support or a lack of other stress buffers is also likely to be a risk factor for anxiety-related pathology when climate-related stressors such as floods occur. people with pre-existing mental health problems, such as pre-existing anxiety disorders, may be strongly affected by the stressors of climate change. although this remains to be fully investigated, research suggests that the stressors associated with pandemics (which may be climate-related), can worsen emotional problems for people with pre-existing anxiety or mood disorders . climate change entails many uncertainties, such as uncertainties about whether one's home will be destroyed in a wildfire, or uncertainties about one's safety and livelihood in cases of forced migration. accordingly, people who have a great deal of difficulty tolerating uncertainty will find climate change to be particularly stressful. the intolerance of uncertainty is a personality trait that is a vulnerability factor for a variety of types of anxiety-related pathology (rosser, ) . other personality traits that might provide to be vulnerability factors for climate-related anxiety are likely to be many of the same traits that are vulnerability factors for pandemic-related anxiety. these include negative emotionality (neuroticism), the tendency to overestimate threat, and anxiety sensitivity (fear of arousalrelated sensations) (taylor, ) . people with high scores on these traits may be at particular risk for developing anxiety disorders as a result of climate-related stressors. the various types of vulnerabilities to climate-related anxiety can be cumulative in their effects, and so it is essential to develop and refine interventions to protect at-risk groups. future pandemics are inevitable (taylor, ) . while there is no evidence that the covid- pandemic was climate-related (wyns, ) , climate change and pandemics are linked in many ways. climate change facilitates the spreading of many kinds of infectious disease; for example, outbreaks of tropical diseases in temperate climates. this can occur, for example, when regions become warmer and wetter, thereby providing suitable habitats for disease vectors such as mosquitos and birds. it has been predicted that the outbreaks of numerous kinds of disease may become more prevalent as a result of climate change, including influenza, dengue, cholera, and malaria (chowdhury, nur, hassan, von seidlein, & dunachie, ; curseu, popa, sirbu, & stoian, ; hertig, ; watts et al., ) . some outbreaks have the potential of becoming the next pandemic. extreme weather events such as wildfires and hurricanes typically require people to evacuate their homes and relocate to shelters. during a pandemic, this makes it difficult to implement social distancing, meaning that some people may refuse to evacuate (thereby increasing the risk of trauma or death), while other people may choose to relocate to shelters (thereby increasing the risk of infection). this was a concern during the recent wildfires and j o u r n a l p r e -p r o o f hurricanes in the united states, which occurred during the covid- pandemic (salas, shultz, & solomon, ; shultz et al., ) . extreme heat poses additional challenges to covid- mitigation efforts. for example, wearing a face mask is uncomfortable in high heat and humidity and may exacerbate risks for heat-related illnesses; conversely, not wearing a mask increases the likelihood of spreading covid- (salas et al., ) . extreme weather events such as wildfires can increase air pollution. this can exacerbate respiratory diseases. indeed, during covid- , exposure to air pollution appears to have increased mortality in patients infected with sarscov (wu, nethery, sabath, braun, & dominici, ) . in summary, the co-occurrence of pandemics or other disease outbreaks during climaterelated stressors, such as extreme weather events, will seriously compound the stressors that people may experience, thereby increasing the risk for anxiety disorders and related clinical conditions such as ptsd. coping with combined extreme weather events (e.g., wildfires, hurricanes) and pandemics will involve ( ) improved communication about dual need to evacuate and to engage in socially distancing, ( ) improved evacuation shelters that make possible both social distancing and hygiene precautions, and ( ) learning from each storm to refine operations . timely action, forethought, and trust in science are key to dealing with climate emergencies (manzanedo & manning, ) . this means mitigating realistic anxiety by scientifically-based climate activism rather than denying, dismissing, or discounting the threat of climate change. one of the challenges is to find ways of countering anxious passivity and defeatism, in which people are distressed about climate change but feel helpless about their ability to do anything about it, which can lead them to deny, dismiss, or trivialize the climate problem as a means of coping with climate anxiety. several of the articles in this special issue touch on this important topic. for example, mah et al. ( ) offer recommendations about how to structure communication to the public in order to promote adaptive coping with climate change and to minimize the odds that people will dismiss fear-evoking messages about the threat of climate change. this involves tailoring the message to both the type of climate stressors and the type of recipients of the messages (e.g., the recipient's coping style). in an indirect way, the covid- pandemic may provide people with incentives to take action against climate change. during this pandemic, in which large numbers of people were in self-isolating "lockdown", there were notable reductions in air pollution, at least temporarily (tollefson, ) . these effects were rapid and dramatic, and provide a clear example of how we can have a positive impact on the environment by reducing our carbon footprint. this provides an example of how we can learn from the covid- pandemic in order to cope with the challenges of climate change. communities should be reminded of these effects in order to motivate people of taking action to reduce their carbon emissions. this may be a potent antidote to anxious passivity and defeatist attitudes. chen et al. ( ) , in this special issue, remind us that most people are resilient in response to climate disasters and that enduring levels of distress have been observed only in a minority of people. nevertheless, even if - % of people developed severe anxiety or other disorders, as estimated from research on various kinds of natural disasters (e.g., galatzer-levy, huang, & bonanno, ) , that could overwhelm the healthcare system. understanding the process of adaptation, as reviewed by chen et al., may eventually lead to important new insights about how to strengthen resilience at both an individual and population level. mah and colleagues ( ) delineated the different types of resilience that are needed to cope with adverse climatic events, including resilience at the levels of the individual and community levels. mah et al. propose that resilience arises from a combination of adaptive emotion-focused and problem-focused coping. at an individual level, adaptive coping with climate change stress involves helping people to manage their emotional responses, deal with practical immediate problems, and prepare to mitigate future hazards. various types of programs have been developed to improve a person's hardiness or resilience to stress. for climate-related stressors, these may be important in reducing the risk of anxiety disorders and related clinical conditions such as ptsd. such programs can be made available to people who don't have psychological disorders but may be at risk for such disorders, such as having environmental, occupational, or psychological risk factors. regarding occupational risk factors, there are encouraging but preliminary studies that stress-management and related programs that can improve the resilience among firstresponders (wild, el-salahi, & esposti, ) . there are encouraging findings that the risk of developing anxiety disorders can be reduced by targeting psychological risk factors such as the person's level of anxiety sensitivity (e.g., knapp et al., ) . however, the efficacy of such programs in reducing the risk of anxiety disorders has yet to be firmly established. several other anxiety prevention programs have been developed, for either children or adults, in various formats including face-to-face and internet-based interventions. these cognitive-behavioral programs might be useful in building resilience in people who are at risk of exposure to climate-related environmental stressors. unfortunately, however, the results so far have been disappointing. such prevention studies have been evaluated in several recent metaanalyses and found to have weak or small effects in reducing the risk of anxiety or mood disorders (caldwell et al., ; deady et al., ; moreno-peral et al., ) . clearly, much work needs to be done to improve anxiety prevention programs, although from a public health perspective even small effects can be of some value in improving the lives of people. community resilience refers to the ability of communities to cope with external stressors, and is influenced by social capital (e.g., social networks), infrastructure, and vulnerabilities (mah et al., ) . strong community resilience involves cooperation, altruism, strong leadership, solid social networks, and the necessary resources. building community resiliency may entail partnerships between health authorities and schools and community organizations to reduce mental health stigma and facilitate the accessibility and delivery of mental health services (newnham, titov, & mcevoy, ) . the covid- pandemic, along with previous pandemics, has shown that communities can rapidly come together to support one another (barzilay et al., ; taylor, ) , although the persistence of such altruism may wane over time. to cope with climate change, we need to find ways to develop enduring altruism and other forms of social support within communities, in order to buffer the effects of the stressors associated with climate change. as noted by hrabok et al. ( ) , multiple strategies will be necessary for communities to prepare for and cope with climate-related stressors, including enhanced access to medical and psychological services, inter-agency cooperation, and ensuring that practical resources are available in the event of disasters (e.g., shelters, food, clean drinking water). the covid- pandemic should serve as a wake-up call for governments, communities, and individuals to ensure that healthcare systems are better prepared to meet the challenges of the future, many of which will be climate-related. pandemics, just like climate change disasters, are mass events requiring large-scale psychological interventions. we will need to re-think the way in which mental health services, such as the treatment of anxiety and traumatic stress disorders, are delivered in the future. the luxury of a one-to-one consultation may become a thing of the past as the need for large-scale interventions become necessary. covid- has forced practitioners to move to online formats, and researchers and service providers have begun developing and implementing online platforms for providing mental health advice and services on a large scale. those platforms should not be dismantled once the covid- pandemic has passed, because they will be vital for addressing climate-related mental health problems in the years ahead. challenges in implementing such platforms for climate-related catastrophes need to be carefully considered. access to personal computers may not be feasible during climate disasters, but hopefully, people will have access to their cellular phones, which may prove to be an important way of connecting to mental health services and resources. regardless of the availability of digital resources, screen-and-treat approaches, implemented on a mass scale, will be important for identifying and triaging people with clinically significant psychological problems, such as climate-related anxiety disorders or other clinical conditions such as ptsd, so that they can receive empirically supported interventions. disclosure: the author declares no conflicts of interest. j o u r n a l p r e -p r o o f do pre-existing anxiety-related and mood disorders differentially impact covid- stress responses and coping resilience, covid- -related stress, anxiety and depression during the pandemic in a large population enriched for healthcare providers school-based interventions to prevent anxiety and depression in children and young people: a systematic review and network meta-analysis anxiety and resilience in the face of natural disasters associated with climate change pandemics, pathogenicity and changing molecular epidemiology of cholera in the era of global warming climate anxiety: psychological responses to climate change waiting for fire potential impact of climate change on pandemic influenza risk ehealth interventions for the prevention of depression and anxiety in the general population: a systematic review and meta-analysis 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responders: a systematic review exposure to air pollution and covid- mortality in the united states: a nationwide cross-sectional study. medrxiv climate change and infectious diseases weather effects on social movements: evidence from key: cord- -upyhirb authors: miller, melissa farmer; li, zhongyu; habedank, melissa title: a randomized controlled trial testing the effectiveness of coping with cancer in the kitchen, a nutrition education program for cancer survivors date: - - journal: nutrients doi: . /nu sha: doc_id: cord_uid: upyhirb following a diet rich in whole grains, vegetables, fruit, and beans may reduce cancer incidence and mortality. the aim of this study was to investigate the effect of coping with cancer in the kitchen (cck), an week in-person program offering education, culinary demonstrations and food tasting, and psychosocial group support, compared to receiving cck printed materials by mail on knowledge, confidence, and skills in implementing a plant-based diet. a total of adult cancer survivors were randomly assigned to intervention (n = ) and control groups (n = ) with assessments at baseline, , and weeks via self-administered survey. the response rate was % at weeks and % at weeks. the majority of our study participants were female breast cancer survivors ( %) who had overweight or obesity ( %). compared with the control, there were significant (p < . ) increases in intervention participants’ knowledge about a plant-based diet at weeks and , reductions in perceived barriers to eating more fruits and vegetables at week , and enhanced confidence and skills in preparing a plant-based diet at week . there was a significant reduction in processed meat intake but changes in other food groups and psychosocial measures were modest. participation in cck in person increased knowledge, skills, and confidence and reduced barriers to adopting a plant-based diet. positive trends in intake of plant-based foods and quality of life warrant further investigation in larger-scale studies and diverse populations. the overall aging of the united states population and changing prevalence of risk factors, including obesity, have increased the incidence of many types of cancer while advances in the early detection and treatment of cancer have led to reduced cancer mortality. these factors have combined to dramatically increase the number of cancer survivors [ , ] . nearly million people in the us were living with a history of a cancer diagnosis as of january . this number is projected to grow to more than million by and to more than million by [ , ] . the importance of diet for cancer survivors is indicated by the accumulating evidence that a healthier diet after a cancer diagnosis can lead to improved treatment response, recovery, side-effect management, and disease outcomes [ ] [ ] [ ] [ ] [ ] . the world cancer research fund/american institute for cancer research's (aicr's) third expert report, diet, nutrition, physical activity and cancer: a global perspective, states ten cancer prevention recommendations [ ] . six of these recommendations focus on aspects of diet, including following a dietary pattern rich in whole grains, vegetables, fruit and beans, and limiting consumption of red meat and processed food, to reduce cancer incidence and mortality. a substantial body of research has demonstrated the benefits of adherence to the aicr's cancer prevention recommendations, including a lower risk of cancer incidence, recurrence, and death [ ] [ ] [ ] [ ] [ ] . despite the growing evidence supporting positive changes in diet to prevent cancer-related morbidity and mortality, most cancer survivors' adherence to the aicr's dietary recommendations is low. a recent systematic review and meta-analysis concluded that only % of cancer survivors met the recommendations for fruit and vegetable (f&v) intake and only % and % for fiber and red meat intake, respectively, while % of cancer survivors met the recommendations for smoking and % for alcohol intake [ ] , which suggests potential opportunities for interventions to improve the dietary patterns and decisions among cancer survivors. diet/nutrition was recognized as a common concern in an observational survey research study of cancer patients and survivors in the community across the us [ ] and among members of a community-based cancer care organization [ ] . further, cancer survivors frequently experience low quality of life partly attributed to nutritional problems and thus report high demand for nutritional support [ , ] . in fact, diagnoses of cancer often motivate individuals to make lifestyle changes [ ] [ ] [ ] . nonetheless, cancer survivors may find it difficult to act on their intentions and can struggle to achieve their goals when they are not given necessary tools such as specific action plans and evidence-based information for making and sustaining behavioral changes [ , , ] . coping with cancer in the kitchen (cck) was initiated to help fill this gap between lifestyle recommendations and the sustained adoption of improved lifestyle behaviors among post-diagnosis and post-treatment cancer survivors. cck is an week in-person program offering multidisciplinary support for cancer survivors that includes nutrition education, culinary demonstrations and food tasting. it also offers facilitated group discussions with structured goal setting to address psychosocial health, and it introduces simple and effective techniques for coping with cancer-related stress. the first phase of research was conducted in to test the feasibility and acceptability and to determine the preliminary effect sizes of the cck program. these promising results were used to inform refinements in both the cck curriculum and the training [ ] . in , the next phase of research was conducted as a randomized controlled trial to rigorously evaluate the efficacy of the in-person cck program versus receiving printed cck materials. the objectives of this paper are to describe the randomized controlled trial, report the results, and examine whether the in-person cck program (the intervention group) increased knowledge, skills, and confidence in adopting a plant-based diet as well as made positive changes in dietary intake and quality of life compared to the delivery of the cck program through printed materials (the control group). cck was developed according to evidence-based concepts, including aicr's cancer prevention recommendations, aicr's foods that fight cancer™, and aicr's new american plate ® . cck is a response to the needs of cancer survivors, registered dietitians, and other health professionals who lamented the lack of an evidence-based, standardized curriculum specifically for cancer survivors. a core principle guiding the design and development of cck was to ensure that it benefitted communities in which it would be implemented. to achieve this goal, the design and development of cck's evidence-based curriculum involved multiple partners across its first and second phases of research, including aicr, living plate (far hills, nj, usa), cancer support community (csc; washington, dc, usa), csc of central new jersey (bedminster, nj, usa), registered dietitians and mental health practitioners. the cck intervention was primarily guided by the social cognitive theory (i.e., knowledge and skills development, self-efficacy, and observational learning mediate behavior change) [ ] and the transtheoretical model (i.e., stages of change) [ , ] . its psychosocial components were guided by csc's affiliate model, which empowers individuals impacted by cancer to improve their health and well-being through active participation in community-based programs and active engagement with their health care team [ , ] . furthermore, it is indicated that enhanced learning occurs in a small group defined by a shared cancer experience with professional and peer support to motivate behavior change [ , ] . often the cost of food can be a barrier to trying new foods and purchasing perishable food items, both of which are important to adopting a plant-based diet. with this in mind, the cck program's recipes were designed to include relatively basic, whole foods that can be found at standard grocery stores; the serving sizes are small to limit the quantity of ingredients needed and the potential for spoilage; and no name brands are explicitly recommended. as well, a variety of recipes at various price points are provided so that facilitators can choose to demonstrate the recipes that are most appropriate for their participants and communities, and the curriculum includes time for the facilitators and participants to discuss possible recipe variations and ingredient substitutions. finally, each week, the facilitators probe for perceived barriers to adopting a plant-based diet related to each module topic (e.g., veggies, snacks, whole grains, and breakfasts) and addresses financial concerns. the first cck pilot study assessed the feasibility, acceptability, and pre-post-impact of the program by a single-arm intervention among adult cancer survivors in [ ] . participants reported increased confidence preparing a variety of plant-based foods (p = . ), perceived control over cancer (p = . ), perception of dietary quality (p = . ), and weekly behavioral capability, including food and nutrition knowledge (p < . ). there was a non-significant (ns) trend towards increased f&v and whole grain intake with moderate effect sizes ( . - . ) for intake of beans and legumes, vegetables, and cooked whole grains like brown rice and quinoa. this single-arm pilot study achieved enrollment of % of the accrual target; program attendance at each session ranged from % to %. participant satisfaction was positive with % of participants very satisfied ( or on -point likert scale) with the cooking demonstrations, % very satisfied with the facilitated group discussions, and % very satisfied with the nutrition education. we conducted a two-arm, randomized controlled trial; cancer survivors were randomized to receive the -week cck in-person multidisciplinary program immediately (intervention arm) or to receive cck printed materials (control arm). data was collected through self-administered patient surveys completed at baseline, post-intervention, and follow-up. a maximum total sample size of ( in each group) with % loss to follow-up and type i error of % provides > % power to detect a difference (effect size) of at least . standard deviations between intervention and control groups (two-sample means test). this study was conducted according to the guidelines laid down in the declaration of helsinki, and all procedures involving research study participants were approved by ethical and independent review services (e&i) institutional review board (lee's summit, mo, usa; approval code: - ). written informed consent was obtained from all subjects/patients during enrollment. the trial was retrospectively registered with clinicaltrials.gov (identifier: nct ). cancer survivors were recruited from community members served by csc-los angeles (la, ca, usa) and fanwood-scotch plains ymca (scotch plains, nj, usa) from april to june , largely by social media, emailed letters of invitation to affiliate members, community-based presentations at cancer support groups, and fliers. the research sites in los angeles, ca, and scotch plains, nj, were selected to represent a diversity of cancer survivors-from the west and east coasts of the us, respectively; from urban and small township/suburban regions, respectively; and from sites that provide programming specific to cancer or with a general community-based focus, respectively. eligibility criteria included: ( ) years of age or older; ( ) ever been diagnosed with cancer; ( ) able to attend at least seven of the eight total sessions with mandatory first and last sessions; ( ) willingness to be randomized and adhere to study protocol; ( ) completed active cancer treatment (not including hormonal or other similar agents, e.g., tamoxifen). if potential candidates had not completed active cancer treatment, research staff determined eligibility if side effects of the current treatment had not affected sense of taste causing difficulty eating healthy foods, like f&v or whole grains, and had not caused a level of fatigue that would impede ability to attend an -week program, shop for healthy foods, and prepare recipes. consenting participants were randomly assigned to of treatment groups: cck intervention or printed materials control. a unique computer-generated list for each of the two research sites randomly sequenced intervention and control assignments. allocation to the intervention or control groups was concealed until all participants had been enrolled at a site. once the final participant was enrolled, research staff broke the treatment code and assigned participants in order of enrollment to either the intervention group or the control group by adding them to the randomized list of intervention and control assignments. the local research staff notified participants in the intervention group of the start date for the cck program. participants in the control group were notified of their group assignment and mailed a baseline survey with a postage paid return envelope approximately one week before the start of the in-person cck program with instructions to complete the survey within two weeks. cck was facilitated by a multidisciplinary team. registered dietitians were trained to educate participants about aicr's cancer prevention recommendations and two evidence-based programs: aicr's new american plate ® and aicr's foods that fight cancer™. licensed social workers were trained to facilitate group support and discussions that strategized how participants could overcome psychosocial barriers to nutrition behavior change in the context of cancer survivorship, equip them with strategies to help with cancer-related stress, and encourage goal setting. in addition, the registered dietitian or a culinary assistant demonstrated convenient, easy, and tasty ways to prepare and cook plant-based foods and offered tastings. prior to facilitating cck, facilitators completed a required two-part live virtual training about how to implement the cck program with intervention fidelity, and they met weekly by phone with researchers using a semi-structured moderator guide to address any questions or problems that arose during program implementation. an evaluation of the training program indicated a high level of knowledge about cck research procedures and preparedness to deliver program content. components of the cck intervention are summarized in table . cck participants attended eight, in-person, -min classes convened weekly at their community-based organizational facility. the schedule at each site included a -week midterm break for a national holiday and staff travel, so the complete program extended to weeks. weekly themes included beans and whole grains, one-pot meals, breakfast and snacks, comfort foods, veggies, and building a foods that fight cancer kitchen. to encourage attendance, some participants were sent email reminders before some classes and some absentees were contacted by phone. licensed social workers provided support through a structured and empowering group learning environment to address the complex, important (and, unfortunately, often rarely openly discussed) psychosocial barriers to nutrition behavior change in the context of cancer prevention, treatment, and survivorship. ( ) cooking demonstration culinary experts demonstrated convenient, easy, and tasty ways to prepare and cook ftfc and invited recipe tasting. the last in-person meeting of the program included a time to review the overall experience and engage in discussion. it was intended to explore milestones achieved, recognize precipitous moments of comprehension, connect to feelings related to the program ending, identify ongoing obstacles and/or challenges, identify changes and successes along the way, share ideas and hopes for continued success, and discuss take-aways from the group experience. each week participants completed a one-page worksheet that prompted them to identify one to three specific, measurable, actionable, relevant and time-bound goals that were revisited at the next session. participants in the control group received seven comprehensive summaries from coping with cancer in the kitchen weekly module content and recipe cards (two from each of the weeks of culinary demonstrations). these were mailed to participants in one package upon completion of the baseline survey. knowledge about a plant-based diet participants rated their agreement ( = strongly disagree; = strongly agree) with six custom items developed by the research team, e.g., "i understand the benefits of consuming whole grains versus processed grains". a composite score was calculated as the average of the ratings (range - ; cronbach's alpha = . ). participants indicated "how sure are you that you could prepare the foods listed below in a tasty way?" ( = very unsure; = very sure). the -item scale included whole grains; beans, seeds and legumes; green leafy vegetables; and mixed foods, e.g., healthy one-pot meals. a composite score was calculated as the average of the items (range - ; cronbach's alpha = . ). participants rated their agreement ( = strongly disagree; = strongly agree) with five custom items developed by the research team, e.g., "i am confident that i can create a kitchen environment that makes it easier to store, prepare, and consume fruits, vegetables, whole grains, and beans."; the average of the five ratings was calculated to create a skills composite score (range - ; cronbach's alpha = . ). we adapted items from an existing barriers instrument [ , ] to measure perceived barriers to eating more fruits and vegetables (f&v) (average score of items; cronbach's alpha = . ) and whole grains (average score of items; cronbach's alpha = . ). participants were asked the general question, "listed below are some common reasons why people don't eat more servings of vegetables and fruits each day. indicate whether or not this is a reason for you by marking how much you agree or disagree." ( = strongly disagree; = strongly agree). in addition, using the same list of possible reasons (excluding spoil too quickly), participants indicated whether it was a common reason they did not eat more servings of whole grains. example reasons included take too much time to prepare; my family doesn't like them; hard to find a variety of good ones. the functional assessment of cancer therapy-general (fact-g) questionnaire is a general quality of life instrument that can be used to assess top-rated symptoms and concerns in cancer patients [ ] . the fact-g is a brief -item adaptation [ ] . internal consistency and reliability in the present study was good (cronbach's alpha = . ). the phq- is a brief -item validated screening scale for measuring core symptoms and signs of depression and anxiety [ ] . participants were asked to rate their level of fatigue on the average in the last week [ = not at all fatigued; = fatigued as i could be]. this item comes from the fatigue symptom inventory that assesses the frequency and severity of fatigue and its perceived interference [ ] . participants completed the soc which measures emotional support, or the perceived availability of someone to provide empathy or advice in times of need [ ] . higher scores represent more emotional support. scores are converted to standardized t scores (mean = , standard deviation = ); normative reference groups are the us general population. participants were asked, "to what extent do you feel you have control over the course of your cancer (that is, whether your cancer will come back, get worse, or you will develop a different type of cancer)?" ( = no control at all; = complete control). the control group received printed cck educational materials including written summaries of weekly nutrition content and recipes that emphasized the weekly nutrition themes (see table ). (only written summaries were provided because the eighth session was a reflection and review session with no new nutrition information introduced.) research staff mailed the materials to control participants upon completion of the baseline survey. the control group was not contacted again except for follow-up surveys at and weeks. upon completion of the -week survey, control participants were emailed a $ gift card. all data was collected through self-administered participant survey at baseline (pre-test, week), post-intervention ( week), and at follow-up ( week) . for participants in the cck intervention group, the baseline and post-intervention surveys were completed in person at the beginning of the first and last cck classes, respectively. the baseline and post-intervention surveys (with a postage-paid return envelope) were mailed to the control participants at their home within approximately one week of in-person cck program commencement and conclusion. the -week online follow-up survey was completed by participants in both the intervention and control groups. a link to the online survey was emailed weeks from the last session, or weeks from baseline, with a window to complete the follow-up survey of to weeks. participants were contacted by email or phone as a reminder to complete the questionnaires, as needed. the primary outcomes included knowledge about a plant-based diet (average score of items; cronbach's alpha = . ), confidence preparing a variety of plant-based foods (average score of items; cronbach's alpha = . ), and skills to practice a plant-based diet (average score of items; cronbach's alpha = . ) (see table ). secondary outcomes were measured using validated instruments for dietary intake (national cancer institute dietary screener questionnaire) [ ] ; general quality of life (fact-g ) [ ] ; psychological distress (phq- ) [ ] ; fatigue (single item from the fatigue symptom inventory [ ] ); and emotional support (soc ) [ ] . we modified an existing scale [ , ] to measure perceived barriers to eating more f&v (average score of items; cronbach's alpha = . ) and whole grains (average score of items; cronbach's alpha = . ). 'perceived barriers' was originally conceptualized as a moderating variable (those with fewer perceived barriers might experience a greater benefit from the cck intervention) rather than an outcome variable and, for that reason, was not measured at weeks follow-up. however, the findings indicate a reduction in barriers in the cck intervention group, so we included 'perceived barriers' as an outcome. additional survey items included sociodemographic characteristics, disease characteristics, and health status. descriptive statistics were calculated overall and by study group. means and standard deviations are presented for continuous variables and frequencies and percentages are presented for categorical variables. we assessed the comparability between study groups using two-sample t-tests for continuous variables and fisher's exact test for categorical variables and pre-post differences within study groups using paired t-tests. we used multiple regression analysis to estimate the difference between the cck intervention and control groups at weeks (post-intervention) and at weeks (follow-up) adjusting for baseline (pre-test) levels of the dependent variable and research site (stratification variable). we considered a p-value < . statistically significant. effect-size calculations were also used as a standard for determining a meaningful treatment effect using cohen's criteria for small, medium, and large effect sizes of . , . , and . , respectively [ ] . standardizing the observed changes by the standard deviation (sd) allows for the comparison of the effect size magnitude across outcomes and can provide a meaningful reference for the future evaluation of the program in its implementation and dissemination. we calculated the es statistic for the effect size, a form of cohen's effect size index, as the mean of the changes in outcome scores for each study group at baseline and post-intervention ( week) divided by the baseline sd [ ] . thus, the consolidated standards of reporting trials (consort) flowchart for the trial is shown in figure . a total of adult cancer survivors were randomly assigned to intervention (n = ) and control groups (n = ). the majority ( %) of study participants learned about cck from csc-los angeles and ymca staff; % from their oncologist; and % from other care providers. there was only drop-out in the intervention arm ( %) who declined participation after randomization but before the cck program commenced, indicating they could no longer commit to the duration of the program. the retention rate was % at weeks and % at weeks. there were no statistically significant differences between those who completed the online -week follow-up survey and those who did not with respect to sociodemographic variables, disease characteristics, and baseline levels of primary and secondary dependent variables. one participant in the intervention arm was excluded from the analysis due to an ineligibility discovered after completion of the program. attendance rates ranged from % for the first session to % for sessions and . characteristics for the total sample and by treatment group are shown in table . study participants were, on average, years of age and primarily female breast cancer survivors with a college degree. the sample was % non-hispanic white and % hispanic or non-white race. approximately half of the participants were married or living as married; % resided in suburban regions and % in urban areas. most study subjects had overweight or obesity ( %), and less than half ( %) indicated they ate enough plant-based foods like fruits, vegetables, whole grains and beans in the past month ("most of the time" or "all of the time"). the cck intervention group and printed materials control group were similar with respect to sociodemographic and disease characteristics. despite the randomization of participants, there were notable imbalances, though not statistically significant mean differences, between study groups in baseline levels of primary and secondary outcome measures (table ) . participants in the control group, on average, entered the study with higher confidence preparing a variety of plant-based foods and skills to practice a plant-based diet, and they also reported better quality of life, lower psychological distress, and less fatigue. thus, we adjusted for baseline levels in regression analyses. knowledge about a plant-based diet significantly increased in the intervention arm (in-person cck program) compared to the control arm (printed materials); this increase was sustained at weeks post-intervention (figure a) . confidence in preparing plant-based foods significantly increased at weeks ( figure b ) as did level of skills to practice a plant-based diet (figure c) . perceived barriers to eating f&v decreased in the cck intervention group and increased in the control group, and the adjusted difference between intervention and control groups was statistically significant (− . ; % confidence interval (ci) − . , − . ; see table ). in addition, there was a larger decrease in perceived barriers to consuming whole grains in the intervention group compared to the control group (− . v − . ), but the treatment effect did not reach statistical significance (− . ; % ci − . , . ). participants began the study consuming, on average, . and . cup equivalents ( . and . servings) for f&v per day in the intervention and control groups, respectively. intake of whole grains was approximately . and . servings per day, respectively. the between-arm differences in intake of f&v or whole grains consumption were not statistically significant. further, intake increased in both groups over time with adjusted differences between groups post-intervention of . cup equivalents ( % ci − . , . ), or . servings, per day for f&v among participants in the intervention group compared with those in the control group, and . ounce equivalents ( % ci − . , . ), or . servings, in whole grains. the cck intervention group also had significantly lower daily servings of processed meat in comparison to the control group at and weeks. in the intervention group, intake of processed meat was . times per day at baseline, which is equivalent to approximately one ( . × = . ) time per week, and it decreased to approximately one ( . × = . ) time in the past month. no statistically significant differences between cck intervention and control groups were observed in self-reported assessments of quality of life. nonetheless, the baseline to -week change trended in a positive direction for general quality of life (+ . v + . ; fact-g ), psychological distress (− . v + . ; phq- ), and fatigue (− . v − . ; -point likert). similarly, the level of social support and perceived control over the course of cancer were relatively stable in both arms, and the results suggested a trend for increase at weeks in the cck intervention group. the magnitude of effect sizes for the changes between baseline and weeks (post-intervention) in the cck intervention group, as measured by the es statistic, are graphically presented in figure . the effect sizes varied across categories of outcomes with large or nearly large effect sizes for outcomes measuring knowledge, confidence and skills. barriers to consuming f&v and whole grains showed medium reductions. the effect sizes were in the range of small to medium for total f&v, whole grain and processed meat intake but were greater (with medium to large changes) for specific components of those dietary factors including beans and legumes, whole grain bread, and cooked whole grains (like quinoa). we detected small changes in quality of life measures. the decrease in fatigue was nonetheless large. we also considered using cohen's d as a measure of effect size, which is the -week difference between the cck intervention and control groups divided by the pooled standard deviation, which is common when comparing two independent groups. however, baseline differences between the cck intervention and control groups were large relative to the variability, or standard deviation, of the factor of interest, and, therefore, may have underestimated the true treatment effect. this trial investigated the effectiveness of cck, a multidisciplinary behavioral intervention incorporating both nutrition education and psychosocial support, in modulating several motivational, action, and environmental mediators for implementing a healthy plant-based diet and for improving quality of life among cancer survivors. previously published interventions have shown that motivation, goal setting, action planning, social support, and instruction regarding how to perform desired behaviors are key elements in successfully promoting behavioral changes whereas self-monitoring is often less effective in doing so [ ] . cck reflects those concepts in its curriculum, and the results from this randomized controlled trial favored in-person delivery of cck over receipt of cck printed material only. in-person delivery of the cck program resulted in significant increases in knowledge, cooking confidence, and skills in adopting a plant-based diet over and weeks compared to the control group that received written cck materials. participants who attended cck in person also reported a greater reduction in perceived barriers to the consumption of f&v and whole grains compared to the control group. as the literature indicates, people with higher perceived barriers tend to have poorer diets and are less likely to engage in behavioral changes, even when they are aware of the benefits of lifestyle changes [ , , ] . lack of access to accurate nutrition information, disbelief in diets and their relationship to cancer outcomes, low reinforcement from friends and family, and unfamiliarity with certain plant-based foods are commonly cited reasons for people not taking actions [ , ] . in particular, limited knowledge and skills in selecting and cooking healthy foods often demotivate cancer survivors from making dietary or lifestyle changes [ , ] . cck addresses those concerns and obstacles by providing evidence-based nutrition education tailored to cancer survivors and delivered by registered dietitians. moreover, cck sessions included facilitator-led group discussions to enquire and consider approaches to reduce barriers to preparing and consuming a plant-based diet specifically for cancer survivors, and they contained weekly thematic cooking demonstrations using evidence-based aicr's dietary recommendations and inviting participants for recipe tastings. facilitated group discussion, access to trained facilitators, and experiential culinary support may contribute substantially to the observed difference between the intervention and control arms. participants in the cck intervention group were able to observe thematic, plant-based foods and recipes being prepared, ask questions, receive verbal information in real time about the health benefits of the ingredients in cck recipes, interact with group members during the recipe demonstrations, and were encouraged to taste new foods. fredericks et al. state that nutritional education with experiential features provides further drivers for behavioral change including collaboration, peer support, and palate development [ ] . though telephone and web/app-based interventions can be more accessible to a wider audience, especially in remote areas, they rely heavily on self-monitoring and often face challenges in retaining participants [ ] . conversely, cck's in-person classes achieved high attendance (≥ %), which is comparable to other effective nutrition education programs targeting cancer survivors, such as cocinar para tu salud, a -week nutrition education program, and the home vegetable gardening interventions [ ] [ ] [ ] . the study showed the benefit of in-person implementation over provision of printed materials only. however, given the current covid- pandemic, future research could investigate virtual implementation of cck using an online platform when in-person gatherings are prohibited, not possible or not preferred. the cck program is available to survivors of all types of cancer. its broad relevance increases the efficiency of delivery, the adaptability to local communities, and the scalability regionally and nationally. the demonstrated enhancements in knowledge, skills, and confidence in practicing a plant-based diet at the end of the program, which continued their upward trend even at weeks follow-up, implied, though not directly measured, the cck effect on improving self-efficacy, which likely led to a higher level of patient empowerment [ , ] . self-efficacy is a critical indicator of patient empowerment and a key construct of the social cognitive theory and transtheoretical model used to guide the design and implementation of the cck program for health behavior change [ , , ] . low self-efficacy (i.e., low confidence in one's ability to execute a course of action) is an important barrier impeding behavioral change among cancer survivors [ , ] . further, practicing and experiencing are among the most important sources of self-efficacy [ ] . we indeed observed that cck's positive effect on self-efficacy was larger in the th week follow-up survey than immediately post-intervention ( weeks) as participants had had more time to practice a plant-based diet by the th week. higher self-efficacy has been indicated in studies to associate with higher probability of achieving and maintaining healthy behavioral goals and overall higher quality of life [ ] [ ] [ ] . further, empowerment also positively correlates with healthier behaviors and better decisions as well as health and clinical outcomes including improved disease management behaviors, use of health services, and health status [ , ] . the effects of cck on total f&v and total whole grain intake were not statistically significant, but the observed net gain of . cup equivalents, or . servings, in daily f&v intake was similar to other studies of nutrition interventions designed to increase adult f&v intake [ , ] . a systematic review of the literature documented increases of . to . servings of f&v, and when targeting smaller focused communities, increases of . to . were observed [ ] . the cck participants had high baseline dietary intake of f&v at nearly cups per day (exceeding the minimum intake recommended by aicr guidelines). increasing people's intake of nutrients or foods when the baseline intake is already sufficient is expected to be challenging [ ] and might have contributed to our study not observing significant post-interventional dietary changes. as hypothesized, we demonstrated a significant reduction in the consumption of processed meats in the cck intervention group and observed medium to large effect sizes in specific components of total whole grain intake (e.g., cooked grains and bread). these changes, coupled with significant increases in mediators of behavior change (knowledge, confidence and skills) suggests that with longer follow-up, participants are likely to continue making important changes in adopting a plant-based diet. additional investigation in diverse populations and communities whose adherence to the recommended dietary guidelines is low is warranted. there was modest impact on measurements of quality of life (qol) in the current study, and effect sizes were generally small. though not statistically significant, findings were within the range of effect sizes reported in prior research of interventions for cancer patients [ , ] . the data suggested positive trends in qol, reduced fatigue and lower psychological distress at weeks, though these trends did not reach statistical significance and were not sustained by the th week. our limited sample size could be one of the major reasons for not finding statistical significance despite the observed positive trends. likewise, a randomized controlled trial conducted by uster and colleagues reported similar non-significant improvements in qol among palliative cancer patients after nutrition and physical exercise interventions [ ] . living with cancer is undoubtedly stressful and associated with reduced qol. previous research demonstrated as many as % of cancer patients have clinically significant psychiatric comorbidities [ ] [ ] [ ] and one in two reported significant distress [ ] . chronic stress is linked to several biobehavioral mechanisms related to the development of depressive symptoms and poorer cancer prognosis that may discourage people from making positive changes in their lifestyle; these factors may contribute to a vicious cycle of persistent emotional distress and accelerated physical deterioration [ ] . furthermore, it has been indicated that up to % of cancer survivors have never received psychosocial support due to limited access to such programs, suggesting the existence of unaddressed needs in this population [ ] . we chose to measure health-related qol using the fact-g due to its brevity, validity, and reliability for use in cancer patients [ ] . the yanez ( ) study reported a mean score for the fact-g of . among cancer patients and . in a general population sample [ ] . in the current study, we reported an average baseline score of . among participants in the cck intervention group with a one-point increase at weeks post-intervention. the cck intervention has the potential to close the gap between cancer survivors and the general population in health-related quality of life and mental health directly through learning coping strategies to reduce stress and indirectly through improving diet quality [ ] . furthermore, in a naturalistic study, giese-davis et al. showed that csc therapist-led support groups provided an experience in which the development of a new attitude was valued [ ] . this trial was small, and a substantial proportion of participants did not complete the online follow-up survey at weeks. the low rate of response to completing the -week survey may in part be attributed to the online platform, which was a departure from the pen-and-paper format of the previous surveys. retention rates may have been higher if we had disseminated the questionnaire in the same format as the previous and additionally provided an incentive upon completion of the survey, either financial or educational by disseminating preliminary study findings to interested participants. limitations in our dietary measurement methods may also have prevented us from fully uncovering the true effects of cck on dietary intake. the use of -h recall, food records, and objective biomarkers may be more sensitive to changes and warranted in future evaluations of cck, which would allow us to quantify more nuanced changes in dietary intakes, such as replacing processed grains with whole grains rather than an overall increase in whole grains. furthermore, follow-up was short. we recognize that behavioral change and changes in quality of life may take longer than - weeks. we hypothesized that participation in the cck intervention would indirectly influence health-related behaviors through self-efficacy mediators. with more time to practice acquired skills, intake of plant-based foods may continue to increase in the cck intervention arm with longer follow-up. another limitation of the study was that clinical outcomes were only measured through patient-report. we did not include body mass index or percent body fat as outcomes since the cck program was not specifically designed to be a weight loss program. while adopting a healthier diet can result in weight loss or changes to body composition, the goals of cancer survivors can be varied with some aiming to increase body weight and others to lose or maintain weight. as the program is more widely disseminated and administered in academic and clinical settings, there may be increased feasibility and interest in measuring pro-and anti-inflammatory biomarkers (e.g., c-reactive protein, interleukins , , , and , and tumor necrosis factor-α) and plasma concentration of antioxidants. other limitations included self-selected samples of participants who are predominantly female, white, fairly educated, and a substantial proportion of individuals with breast cancer. these limitations impact the study results' generalizability to a more diverse population. however, while this sample is not representative of all cancer patients and survivors across the us [ ] , it is representative of those who tend to seek nutrition education as well as social and emotional support in their community. future work is required to understand the impact that cck has on participants' long-term behavior changes, and to evaluate its applicability and cultural sensitivity among other diverse samples and settings. participation in the in-person cck intervention led to improvements in nutrition and food-related knowledge and skills as well as confidence in adopting a plant-based diet among cancer survivors. as an evidence-based, experiential nutrition education program that embeds psychosocial health elements, cck can serve as the standard for a high-quality community-based survivorship program. further studies and larger-scale implementation of cck in more diverse populations are needed to further understand its effect on health-related behaviors and wellbeing. promoting lifestyle behavioral change through programs like cck has potential and value to improve cancer survivors' nutritional status and quality of life. silver tsunami": prevalence trajectories and comorbidity burden among older cancer survivors in the united states obesity and diabetes: the increased risk of cancer and cancer-related mortality colorectal cancer survivors only marginally change their overall lifestyle in the first years following diagnosis changes in prostate gene expression in men undergoing an intensive nutrition and lifestyle intervention telomerase 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bromplon nahonal hean and lung hosprlal, sydney slree:. london sw np percutaneous fine needle biopsy s an established diagnostic technique for lung lesion^ afirm diagnosisofbenign versus malignant is often achieved but histological interpretation of small fragments or groups of cells is difficult manual cutting (twcut) needles provide asuperior histological specimen but are associated with a high complication rate and have been mainly used for pleural lesions this s the first prospective study to assess the feasibility of obtaining histological samplesfrom lung lesionswing apoweredcuning needle (biopty gun) we have biopsied patients using biopty gun there were no major complications histological diagnoses were obtained in patients ( malignant, benign) the malignant lesions identified included nan-small cell carcinomas. adenocarcmamas. squamous cell carcinomas, bronchiolaalveolar cell carcinomas, -cell lymphomas, small cell carcinoma. atypical camnoid and metastatic breast carcmoma. the benign es ons included sarcoidosis. clyptogenic organising pneumonia. wegener's, resolving pneumonias with chronic inflammation the rad~ologistls assessmentaftheamauntoftissueobtainedconelated withgood histology. thethreefalsenegativespecimens obtained the radtdogist noted the inadequacy of the samples ~n two cases and the third was a geographic miss percutaneous biopsy using the biopty gun is a simple and effective means of obtaining good quality histological material from lung parenchyma with a high degree of diagnostic accuracy for bath benign and malignant lesions inorganic particulate matter in "normal" lung: a study using light microscopy (lm), scanning electron microscopy (sem), and energy dispersive x-ray analysis (edxa) london, onlano. l partmeni of palhology soulh sireel london onlano n a g canada h e possible association between inhaled inorganic matter and some cases of usual inter tltial flbrosis (uipi has been of longstanding interest to pathologists and cllnoians. unfortunately. most analytical techniques are impracticable ~n the context of a pathology sewice laboratory in an anempt to find a practicable s utm to this problem and to establish a baseline for inorganic particle load in a "normal" population, a study was undertaken using techniqueswhichwauld beavailable ~nmostlargepathologylaboratories ninecaseswereselectedfram the case(centra andperipheralparliansaf lower. middleand upperlobesjandexamined with thesemand edxaand compared with the lm appearance to determine the panicle type and distrlbutm m a "normal" population. a wide range of inorganic matter was ldentlfied canespanding to siiicb. aluminium and magnesium s cate , rutile and alumina "like" particles varying from < p to p in size in addition. trace elements including zinc. cadmium and increase in number of particles wasalso noted in areas of fibrosis which were present in two cases (old mflammatory disease) preseumably related to problems in particle clearance the findings of this pilot study suggest that although the sem and edxa will likely prove useful tools in the evaluation of lung biopsy soeclmens. the findinq of lnorqanic material in cases of uip must be interpreted with caution autopsy senicb whlch had no known expcsure to lnorganlc dust sectlons were taken from the nght lung each pulmonary adenomatosis has been described as adl~tinctive pathologlcal change seen in the lungsolexperimental area of replacement of normal alveolar lining cells by a taller more glandular type of eplthelwm. usually wlthwt significant cytological atypia. we describe cases i" whlch a smtlar change was seen as an mdental fcndlng ~n resection specimensfor primary pulmonary adenocaranoma. the lesions (usually multlpleand each mm orless m diameter) were identified in lung parenchymaat a distance from the tumour and consisted of thickened alveolar walls lined by prominent, distinctly atypical cells morphologically slmllar to type i pneumacytes and cytologically different to the associated turnour reactive changes " lung involved by obstrmtive pneumonitis were not included !n thts sews all of the associated tumwra were peripheral adenocarcinamas and all showed a pattern of alveolar wall spread at the tumour periphery clinically of the patients were female and all were smokers or ex-smokers the slgnlflcance of this lesion in the histogenesis of primary pulmonary ademcarcinoma is. as yet, unclear animals often i" assoclatlon wlth exposure to inhaled camnogens morphologically the lesion is a clrcumscrlbed departmen ofli~slopafhology sf richard's hosprlal. chrcheslei wesf sussex. po se a series of consecutive personally conducted autopsies in patlenfsdying suddenly outside hospnal and where the death was reported to h m coroner presented cot deaths were excluded. in cases the death had been reported because theattendingdoctarwas unwillingtoissueadeathcertlficate. in theother cases. deathwasnot duetonaturalcausesandfallowedsuicidearan accident oftheformetgroup, therewere t unsuspected malor findings cn patients ( . %). that s to say findings which were either the cause of death or would have led to admission to hospital far assessment and possible treatment they had been discovered in life the largest group were cardiovascular ( ) but there were cases of unsuspected malignant disease there were only five malor unsuspected findings in the group of unnatural deaths years). these results highlight the loss of teaching material in the coroner's system, at a time when hospnal postmortem rates are in universal decline. this matenal would be of value to medical students and to pathologists in traming. and largely comes from cases of lmle or no medico-legal significance. %) and these were in older sublects (mean age . a total of postwnortem specimens refened to the royal victoria ho~pdal electron microscopy unit dunng the years - havebeen reviewed thkscompnsed . vo of thetotalnumber of sewre-relatedcaaesreferred to theunit.oulof ocasesreferred. wereexaminedandrecorded indetail theremainderdidnotundergoelectron microscopic examination for various reasons, such as a concius~ve diagnosis being reached by light microscopy alone. and semi-thin sections showing severe tissue mtoiysis. the most common tssues referred for examination were lung, kidney. liver, brain and head the range of e m. studies carried out included transmission electron microscopy item). scanning electron microscopy (semi and x-ray microanalysis on sem. theelectron micrographs wererevlewedwlth respect totlssuepresewatlanand th wa correlatedwlth the timeintervalbetweendeathand autopsy. electron microscopy was considered, an review. to have been diagnostically useful in % of cases an which it was deployed c s herringtan, a k graham, k cooper, j mcgee it was shown prev usiy that the discrimination of human papillamavirus (hpv) types and by nlsh in archival biopsy matenal requires dlfferent conditions from those predicted by conventional solufion kinetic analysis the parameter tm' (tissue tm) was defined in order to describe these differences in this study. these pnnciples were extended to the discrimination of hpv . and in casesol cln the results of nlsh analysis werecompared with both immunohistochemistry for viral capsid protein and pcr typing these data demonstrate that crosshybndisatm of high nsk viral types occurs ~n clinical lesions under conventional hybridisation and stnngency washmgcondit ns thiscross hybridisation isnot due to thepresenceofviralcapsidproteinand sm relikelyto be areflectionoftheendpoint usedinn sh.i e thepresenceofvisiblesignal.practcally.multiplenlshsignalsduet closelyrelatedprobesinarchival materialarenot indicativeofmultiplehpvinfectionunlesstheyarepresent either ~n morphologically discrete areas of the biopsy or their presence has been confirmed by another molecular technique. motegenerally, thepresenceof asignal in nlsh usingaparticularprobedoes not implythattheidentity of the target nucleic acid is that of the probe medical students in the autopsy room shows that % thought faces should be covered during autopsy, and % thought that genitals should be covered. about half the group were indifferent to both proposals % thought the patient's identrty should be concealed from observers three students thought there should be no conversation at all. % thought that conversation should be limned to procedures and findings and % thought it should be limited to professional maners;conversely. % thought thereshwldbenolimitson thetopicsdiscwaed nearlyso% thoughtstudents should beencouragedtoassistbutnotpressuredintadoingsa. . % thoughtthatallshould becompelledtoassist, but % thought that students should onlv obsewe. and not be allowed to ass s.t. % of students thought that they should oni!iobsene autopsies on patients they had clerked. whereas % thought they should onlfbe on autopsies on patients they had not clerked. fmally. % thought that relations should give spmfic perm on before students observed autopsies. whereas . % thought not a quantitative study of the effects of fibroblast growth factor on wound strength and cellularity fibroblast growih factor (fgf) has potent angiogenic and fibrogenlc effects and is lmpllcated in the formatlon of granulation tissueand healing few attempts have been made toquantity theseeffectsmnvo. we havestudled arat skinwound modelusing red cellghostsasan fgfvehicle tensiometv ofthewoundsshowed amaxlmaleffectof the fgf after seven days when the wound strength was % above that m controls (p < ) this effect had disappeared by fourteen days. computerized image analys~ using a joyce lo& mini magiscan measured total nuclear content of areas i" the wounds. permitting a topographic analysis of cellulanty versus distance from the wound centre cellularlty effects showed adifferent time course from wound strength. a % increase at fouldays anda % decreaseat sevendays. relative tocontrols(bothp < . ) attwelvedaysthecellularityeffectwasstill sqnlflcant at a vo decrease but by twenw-one days it had dwappeared the results suggest that fgf causes an early transient increase in cellulardy and more rapid increase in wound strength: most of these cells are macrophages and fibroblasts suggesting a connection between thesetwo obsenat ns. the adhesion molecule$ integrin (cd ). integrin (cdl ). and intercellular adhesion molecule- (icam- , cd ) are essential to the intimate co-operation of antigen presenting cells (apcs), t cells and keratinocytes cyclosponn. which is an effective treatment for psonas~s, may cause immunosuppression by altering antigen presentation we have pertormed a quantitative immunohistochemical assessment of the effect of low dose ~yclosporin on the expression of p -integnn. p -integnn and icam-f m the epidermis in chronic plaque psonas s. staininglevels werecompared withclinicalresponseasassessed bythepsoriasisareaseverihllndex(pas score) pt-lntegnnand cam expressionon keratioocyteswerenotaltered bytherapy buttherewasasignificantdecrease inthemeanlevelsof p positivelargedendriticcells(apc )within theepidermis.bz-integnn wasnot expressed by keratinacytes there was a strong correlation between p expression and pas score after three months on cyclosporln and one month off therapy these results lndlcate that p stalnlng on large dendrltlc epidermal cells previous studies enumerating silver stained n~c l~l a r organiser regions in problematic cutaneous melanocytic lesions have yielded inconsistent. but generally favourable, resulis. it seems probable that such inconsistencies. arise largely from differences ~n fixation. staining and counting strategies. our group, having devised improved methods of agnor staining and counting. is now able to re-examine the potential role agnors in borderline lesions pilot work demonstrated potentially significant differences in agnor dispersal between benign and malignant lesions and i" this study bath agnor numbers and dispersal patiems have been evaluated a range of melanocytic lesions including banal naev~. dysplastic naevi. typical melanomas, spiu naevi, atypical spih le~ions and minimal deviation melanomas were collected virtualty all of the unusual lesions had been diagnosed by specialists in dermatopathology. using a combined assessment of agnor numbers and dispersal, it proved possible to discriminate borderlip lesions from banal naevl and typical melanomas. benign borderline lesions -such as spit naevi ~ possess numerous nor$ but display tight clustenng in contrast to malignant melanomas wherenordispersa isaprominentfeature discriminatinganetypeaf borderlinelesionfromanathercauld not be achieved however, in practice this distinction is probably less important than assigning a aoirary specimen to a benign or malignant group a larger prospective trial of agnors in melanocytic lesions currently in progress with a mean total exposure of pack-years. alihough p was expressed more commonly in adenocarcinoma ( rof lo)andsquamouscarc~noma( ~~of )than~nsmallcelltumours(l %of o),thiscouldbeaccountedfor by the smoking history. since patients with non-small cell carcinoma smoked more (a mean af pack-yeas) than those with small cell lesions (mean of pack-years). there was no relationship between p expression and sunlyal. s. a. sun. j. r. ~a s n e y depaitmenl ofpathology university of liverpol, p box , l!vemooi. l bx activation of thec-myconcqene with overexpression of itsancopratein product ocurs in bronchlal malignancles ofalltypes, but has beenmost extensivelystudied insmall celicarcmoma, wherensaverexpression~n culturedllnes has beenassociatedwithdevelopmentoffast-growingvanants whlchlosemuchofthelrendocrlnephenohlpeand aner their morphology. it has been suggested that these vanant lines might be the equlvalent of the large cell bronchial endocrine carcinomas sometimes seen i " yiyo, but this is not proven. we have used th myc - e monoclonal antibody and the avidin-biotin technique to study the panem of expression of the p oncoprotein product of the c-myc gene in turnour deposits of twelve subjects coming to necropsy with disseminated small cell carcinoma in an attempt to relate l o morphological vanablity and metastatic site. anhough expression bore no relationship to morphological vanation. it ohen differed markedly horn site to slte. whereas parts of the primary t m w r strongly overexpressed the protein ~n all but one wbfect, there was considerable vanability between secondary deposits. oossiblv mdicatina arelation~hi~ between c-mycex~resslon and propensltvfor metastasls to certain iocations. tumour growth rate is a key parameter of neoplastic aggression. and is determined by the balance of cell gain and loss.apoptos~sisama]ormadeoftumourcellloss, but linleis knownof itsregulation. dieremesin turnourgrowth conferred by hpvtypes and werestudied in aratfibroblast modelsystem. immortaiisedcells weretransfected with hpv and expression vectors. either alone or with activated c-ha-rasl. monoclonal cell lines were established. and their vector dna content was confirmed by pcr tumour cell ddfered in their growth properties m wvo and m vilro. hpv containing turnours were larger and showed less apoptosis than those containing hpv , although bothshowed moreapoptasls than thenodulestormed bytheparentfibroblastsalone. in all turnours the presence of ras greatly reduced apoplosis and increased the growth rate. very similar propetlies wereobswvedin culture, and apoptoticratesshowed astranglnveneconelatlonwlthratesofnet cellgrowth. hpvs appeared iostim"latetumourcei apoptasis, butthiswas suppressed byras. melowerapaploslsassoclatedwilh hpv compared with hpv may partly explain the more aggressive phenotype of cervical c a n m containing hpv . it hasbecomeapparent thatanumberof maleculesmaybeexpressed byrestingorqu~escentcellsandarelostwith transition into the cell cycle. in addition to being of biological interest, such molecules may prove useful as operatcanal mahers of quiescent cell populations in histological material and allow the further characterisation of cellular subpopulations. one such molecule is statin. a kd protein previously reported to be expressed only by cells ~n go. we have shown bylaserconfocalfluarescence microscopythat thestatin antigen is associated with the nuclear envelope with a dstnbution m l ar to that of nuclear lamins. using a monoclonal antibody ( - ) that recognises slatin we have defined the tissue distribution of statin immunoreactivlty in a range of continuously renewing, conditionaily renewing and non-renewing tissues. the distribution of immunoreactivlty s essentially as would be expected of a maher of quiescent ceils. in contrast. in established pancreatic carcinoma and other epithelial ceil lines. we have found $latin immunoreactivity ~n cycling cells using biotinylated k slatin double labelling techniques. in conciusion. statin lmmunoreactivitv in normal tissues correlates with auiescence but this realtionship is lost, at least in vifro. loss of cell-cell and cell-substratum adhesion are imponantfactors during turnourprogression. tumour promoten are compounds which although not carcinogenic themsews increase the frequency of turnour development in animals previously exposed to carcinogens. we have used the turnour promoter tpa on cultured human renal epithelia ~ells mimic nwplastlc transformation. following tpa treatment we haveexamined thedistribution of vlnculin, b integrin and actin within thetreatedcells byfluorescencemicroscopy.treatment of therenal epithelium by tpacauses arounding up of the cells and a loss of adhesion toeither laminin or fibronecttn substrata fiuorescent microscopic examination reveals a progressive loss of reactivity for vinculin and b inlegrin within local conlacts. these changes are accompanied by a redistribution of the actin microfilaments from orientated bundles of stress fibres to a circumferential arrangement these changesofareduction in focal contact componentsand disarganised actin cytoskeietan mimic the changes we have previously described in renal carcinoma. glutathione s-transferases are a diverse group of enzymes with an important role !n the metabolism faelgn compounds including some carcinogens and cancer chemotherapeutic agents. increased expression of gst pi is seen in manyanimal and humancancersandisassociated with resistancetocalboplatinandcisplatin in humanlung cancet cell iines. gsts are involved m steroid hormone transport and metabolism and have a role in the complex metabolic relationships between settoli ceils and germ cells. we have studied gst isoenzymne expression by an immunohistochemical method in stage i teratomata. stage i tetatomata, stage i seminoma, cases of intratubulargerm cell neaplasia( tgcn) and a groupof cryptorchid and normal testes. the stage i teratomata had beentreated with cisplatin based therapyand bothprimmaryandpost-therapymetaftatictumourtissuewerestudied. gstalphaexpression correlated with morphological evidence of epithelial differentiation in teratomata.therewas no difference in gst expression between stage i and stage i pnmary testicular iumours nor between primary testicularand post-therapy metastatic tumours. gstexpression did not correlate with survwal. gst pi was strongly expressed in the neoplastic germ cells of itgcn but was weak or negative in normal germ cells. this may be significant in view the potential for later contralateral turnour development tn patients treated by cisplatin based therapy. in summary, gstexpression in testicular germ cell turnours reflectedlheirdifferentiationandappsared to be unrelated l o therapy and subsequent survival. a case illustrating the usefulness of electron-microscopic examination of fine needle aspirates s described fine needleaspiration wasperlormedon asubcutanwusnoduleinthechsstwall ofaneldedyman whowassuspmted tobesuffering from bronchogeniccaronomaanclinicaland radiologicalfindings.mesmears werenotdiagnan~c. but ~n view of the history af asbestos exposure, the needle washings were submined for eiectron-microscopic examination, which showed mesathelial differentiation and a diagnosis of metastatic malignant mesotheiioma was suggested. an autopsy perlormed eight months later confirmed the diagnosis of malignant mesotheboma. t. dorman, a ti ismail, b cunan, m. leader pathology department hoyal college of surgeons m ireland, dublln many histologi~allymalignantfeaturessuch as hypercellularltyand high mitoticcounts, but clinically followa benign course. demoid tumours and fibromafoses can be densely cellular but usually quite bland histologically and are associated wdh infiltrative marginsand repeated local recurrences. in addtion irradiated tissue often contains many cellswithcytologicalfeaturesol malignancy thisstudyexaminesthepioidyofsuch lesionsbyboth olthecunently availabletechniques using dlsaggregated formalin fixed paraffin embedded tissue sixteen cases nf, dfsts. dts. fibromatoses( nci~ding palmar, plantar. retrcpentonealand soh tissue) and miscellaneouscases including leiomyoblastoma, inflammatory pseudosarcoma and plexiform histlocytama. one malignant fibrous histimytoma and t paragangliama were included as possible poslive controls. cases containing numerous irradiation fibroblasts were alm analysed all 'pseudosarcwnas' were euploid by both image analysis and flow cytometry the malignant fibrous histiocytoma was aneuploid by image analysis and flow cytometry and the paragangliomawasaneuplaid by imageanalysis and tetraploid by flow cytometv themndwonsof thisstudv are that pseudosarcomas are euploid and aneuploidy would appear to be confined to malignant tumours of mesenchymal ongm. other sites we collected cases to study their morphology and antigenic profile after staining for the epithelial markefficam . flow molecularweight keratins)andlp (high molecular wefghtkeratins), thegeneralmelanoma and neural markers s w. neurone specific enoias (nse). protein gene product . (pgp) and the melanoma s w i f l c marker hmb , the neuroepithelia markers leu and glial fibnllary acidic protein (gfap), and the intermediate ftlaments vimentln vim) and nsurofilament (nr the following turnour types emerged: i pure spindle celltype(n = ), ,purepolyganalcelltumaun(n = )comp sedofpleomorphiccells(n = ).undwmcellswith an alveolar in = ) or sheet arrangement (n = ). and mixed pleomorphic spindle and polygonal cell tumouffi (n = ). two tumouffi proved to be lymphomas an intraepithelial melanocytic component could be established in cases within adjacent respiratory or squamous melaplastic epithelium. in cases n e w trunks a~mclated with tumour contained increased numbers of atypical schwann cells. consistently expressed were s w ( and colon ovanan tumour antigen (cota). csais a heat stablemucin associated antigen present m normal colonic epithelial cells and is expressed m greater quantitites tn colon c adenmarcinomas cota is a heat stable antigen present n colanicneoplasiaand mumowwananturnours but not ~nnormalcalonicepithelium psmionsfrom to primary adenocarcinomas ( ovanan, colo-rectal, gastric. breast. oesophageal. prostatlc. pancreattc, endometnal and t gallb adder)and two adenmarcinomas metastatic tothe liverwere incubated with anti csa and anti cota antibodies using the p.a.p technique with positive and negative controls. while anti csa positivitywasseenin d ~ loni~aden carcin ma~( wsakand lo~tr~"g).~twasalsaseen n af ovanan ( weekand strong). of gastric ( weakand strong), of breast( weak and strong). of oesophageal ( weak and strong). of prostatic (all weak). of pancreatic ( weak and strong). of endometnal (all weak) and the gallbladder ademcarcinoma. both liver metastatic adenocarcinomas were negative in additim while anti cota staining was positive m of colonic adenocarcinomas ( weak and strong) and of ovarian adenocarcinomas.~twasalsapositive~n af gastnc( weakandzstrong). al lobreast( weakand stlmg), all esophageal(allweak) of pr ~tati~(allweak), pa"creatic(low akand t strong),all endometnal(all weak) and the case of gallbladder endocarcinoma (strons) both liver metastatic adenocarcinomas were negative. m e c o~c~u s~o~ of this study s that anti csa and anti cota are not adequately specific in the identification of a ~olonlc or ovarian origin af an adenocarcinoma and cannot reliably be applied to the identification of a metastatic adenocarcinoma of unknown primary site the early intfa-epithelial changes of adenocarcinoma of the nose and paranasal sinuses were sought in the histological sections from high grade adenocarcinomas and cyclindric cell carcinomas from this region none of which had had previous radiotherapy treatment. of the cases came from wwd-workers. i r m a polishw and from a plasterer attention was directed to the non-neoplastic epithellm of the surface and of the sera-muclnous glands. in casesofadenocarcinomaand t of cylindriccell carcinoma surfacechaogeswere detected these took the form of hyperplasm of goblet cells of the rsspiratq epithelwm accompanied by dysplastic changes of other epithelial cdls: m the latter these became enlarged and irregular m shape with large hyperchromatic nude and prominent nucleoli. lnthsmajorityof iesionsthesechangeswereisolatedin theepithelium. changesof thesetypes were never seen in sefomucinous gland epithelium. in addition similar changes were not noted m the coverlng respiratoryepithelium in casesofnasal polyps evenwhen severely inflamed thesemsmchangesso described haematoporphynn derivative sensitised tumwr tissue. s underwing evaluaton " widesptead and m i s t a n t papillary tumoursand ,n casesof rvldespreadseveredysplasra/carnnoma,nufu". arewewof spallents ( , m, ages years. pre-and post therapy) has revealed no change ~n histological grade and stage ~n patients, progression to invasion in t case and a redunion to mild urothellal atypla alone in case. local tissue changes following treatment were limnedto oedema and an acute lntlammatoryreactlon. no metaplastlc. stromalfibroblast or nene changes were seen variations ~n bladdw size. both increased and dlmtnlshed, wwe encountered the histological grading of tcc following pot shows little improvement. but studies are in progress to improve lhght delivery in the bladder, and hence improve treatment outcome. a armour. a. s. jack bpartment of pathology, umverslty of i d s . leeds. lsz jt follicular lymphoma is a disease characterised by widespread lymph node involvement usually at the time of presentation proper tie^ of lymphocyte homing and circulation appear to be mediated by a variety of cell adhesion molecules. the a m of this study was to compare mrmal nodal lymphocytes with the neoplastic populatlm m folllcularlymphoma. hall casesalloftheneaplasticfolliclesexpressed lcaml but thisyanedfromonlyafewcellsto % of cells within a follicle this dosltlvlty included dendmic retic~l~rn cells. germinal centre cells and neoplastic lymphocytes. leu (lymphocyte homing receptor) showed an inverse pattern of expression in all but cases the phenotype (icaml + l a is a feature of germinal centres and scanwed paracontcal blasts in reaclive nodes anhough there is uniformity of expression of lcaml in germinal centres which is not apparent in any folllcular lyumphoma case. this study showed a loss of lcamt and increased leu expression by neoplastic lymphocytes within follicles this may relate to the propensity of this disease to spread widely throughout the lymphatic system activation leads to an alteredfunction and aconcomitant aneration in the chemistry ofthe cell surface, which ~s a site nchincalbohydrate. rat lymph nodelymphacytes.eitherunactivatedoraclivatedfot days namixedlymphocyte reanlon. were treated with biotinylated iectins from a large panel. chosen to probe surface glycans lmlns were revealed with awdln-phycoerythnn and cell populations were analysed in a fluorescence activated cell-sorter double-stalnlng with fitc-monoclonal antlbodles defined the functional lymphocyte subsets. uea- and lta boundtonoce sof anytype. unactivatedb-cells boundall theremaininglectins.savempa. toagreaterextentthan did unactlvatedt-cellsand b-cell actlvatlon produced no change i" glycan sxpresslon. unactlvated and actlvatedtcells all expressed a . -linked sialyl residues, but o- . -linked sialyl expression was hetrogeneous, did not conespond toanysubsets,and wa~unchangedonanlvatian.alargegroupof lectinsshowedlow wno binding to unactlvatedt-cells, but boundonactivatianexactly in parallel to l- receptorexpressionctacantigen) thes~ngle structure gawgalnacal, galpl, glcnac-) galigalnacat , gal~ , glcnac-) r could account for their binding the presence of neoplastic (light chain restricted) b cell follicles in low grade b c d gastrolntestlnal (gi) mmphomaof although malt s not present in normal human gastric mucosa, lymphold t,ssue ,* acqulr& ,n response lo colonisationof themucosa by helrcob~cleipyioii. we have investigated the possibility that thisacquiied lymphoid t ssue is malt type whlch may povovlde the background i" which lymphoma can arl~e w e examined gast,,c b~ p ~e fr m casesafh~l~~~bacrerassaciatedgastntis.and biopsyandresectionspecimensfram casesaf t"mours has contr'buted to a peklstence of ihe more "iew arefofl'cle (fee) gastrlc cell lymphomaof malt in cases of hebcobaciergastritis prominent lymphoid follicles were idenfilled in aftheee b cell clusters were identified within the gastric epithelium. reminiscent oltheteatures seen the dome epithelium of sm intestinal peyer'z patch. thls b cell-eplthelh assoclatlon was not assaclated with the eplthelm cell changes or the glandular destructm seen ~n lymphoeplthellal i~s i o~s malt lymphomas. in / cases of gastric maltlymphoma helicobaclercould be identified oltheremaining cases. weiegastrecfamy specimens n which specimen washing may have contributed to the negativefindings. we suggest that gastrlc malt s acqulred in response to local immunological stimulation as a result of mucosal colonisation by hel~cobaclerpylon, and that the development of malt lymphoma is a subsequent event mucosa associated hmphold tlssue (malti has been explained on the bas of speclftc colonization at reactlve b lymphomasbe included in lhecatqoryof malt lymphoma but the frequent presenceotafoll~culapanem in these foltic es by ihe neaplastlc cantrocyte-like(ccl) it has been low grade cellthyro d we have ihe and 'nvesflgated the and genotype of' of primary low gradebcelllymphomasofthethyro'd alsodemonstratedfeatures ofmalt ymphama'ncludtng cclce and l y m p h~' t h~' a l l~' o n s . m e appearances and immunoh'sto'og~ofthefo'l'c'es werethoseofto'l'cularcalon'rat'on described 'ngimaltmmphomarather thanfccfoll'cular'~mphoma thepredomlnant iwg ~ane'naffoll~cularcolon~rat~~nconformed tothatdeslgnated cclcells show'ng astnk'ngly h'gh prol'feratton late no evidence of the '( ' ' transiocat'an was found in any on dna extracted from fresh (n = ) or paraffln embedded (n = ) t sue mese ilndlngs argue against a fcc lineage lor primary thyroid lymphomas and support their ncius on " the maltcategory we have used a panel of antibode$ to demonstrate stages of granulocyte maturation by immunoh~st chemistry i" decalcified. wax-embedded bane marrow trephine biopsies antibodies reactive with muramidase. u- -ant~tryps,n. neutraphil elaslase and cd react with early granulocyte precursors. cd and calgranulin identity later granulocytes. ihdiylduai antibodies dlfier rn the populations cells ldentlfled theantlbcdtes also react with cells of mwlocyte llneageand provide information concerning theorganlsatlon afmonopolesls about whlch liffle is known in normal marrow granulopoiesis is zonal wlth maturation occurring radially around trabeculae and blood vessels this pattern is exaggerate in reactive hyperplasia and chronic granulocytic leukaemia (cgl) there is marked disruption of this zonal organisation in myeloproliferative and myelodysplastic states, with considerable overlap patterns between these conditions in chronic mvelomonacvtic leukaemia (cmml) there is comdlete absence of zonal arrangement of granulopaiesis possibly due to monocytic proliferation obscuring the underlying marrow spaces and that by analogy with cgl cmml represents an exaggeration of this normal panern granulopoletlc panern we hypotheslse that monopoles normally occurs " a randomly dispersed fashion within as previously shown by us in animals untreated with cyclosparin a. the cell birth rate fell from an initial prelmmunisation valueof cellsit cellsihour to cells celldhouran day followed by a rise cellsit cellslhour on day however, in cyclosporin a treated animals the cell birth rate ( ce s/looo cells/hour) was signiticantlydepressed belowthecontrol pre-immunisation levelandremained suppressedupfaday fallowed by an abrupt rise these results are consisten with the hypothesis that t lymphocytes or their pmducts not only drwe the morphological appearancescompnsed lymphoepithelia lesions (in onecase). numerous lymphoid follicles and a diffuse infiltrate of monocytoid cells (centrocyte like cells) there was striking plasma cell differentiation and colonisation of lymphad folllcks by monacytoid cell?. and neoplastic plasma cells. lmmunahistochemistry convincingly demonstrated heavy and light chain restriction ~n all the biopsies from both the cases the bladder s developmentally related to the hind gut and this manifests " the variety of metaplastic epithelium seen at this site circulating cells. encauntenng the endothelial surface. make contacts an environment rich n glycan. a large panel of biotinylated lectins was used to probe far variations in the glycans expressed on endathelia of artenes. veins, arterioles, venules. capillanes, high endothelia vessels and lymphatics m a range of normal and pathological human tissues formalin-fixed, paraffln embedded specimens from the files of manchester royal infirmary and manchester royal eye hospital were used lectins were revealed with an avidin-peraxidasesystem no differences werefound between arterial. arteriolar, venular, veinous or lymphatic endothelia all expressed abundant complex-type n-linked glycans. of several subtypes. capillaries were highly variable and showed heterogeneity ~n their expression of ) outer chain sequences fn n-linked glycansand ) mucin-type sequences. both between different. normal organs and within an organ. implying that the surrounding tissue probably had a regulatory effect where endothelium was reactive, additional aneratiom wete seen and actively growmg endothelium in granulation tissue expressed hqhmannose siwctwes. high endolhelial ybss~is showed a much lower density and narrower range of glycan expression thandidad~acenlnormalcapillanes,despite theirknownvery highrateofglycansynthesisand secretion the iymphocyteiln-homing receplor(s) would be a component of this restricted porlfolio of glycan monocyte margination in atherosclerosis associated with immunological injury n. j. combs, p j gallagher. p. s bass clinical and experimental evidence indicates that immunological injury is associated with accelerated atherosclerosis. allograft recipients may develop accelerated atheroscleross and anlmalsgwen serum sickness and a high fat diet develop more extensive atherosclerosis than controls fed the diet alone we have tested the hypothesis that atherosclerosis ~n these animals is associated with increased adhesion of monocytes to the aortic endothelium. chronicswum sickness was induced in genetically hyperlipidaemic rabbits with nativeanionic bovine serum albumin (nbsa) or highly cationised protein (cbsa) rabbits given nbsa showed a spectrum of glomerular endocapillary prollferativechange thasegiven cbsa developed early membranous glomerulopathy in controls the numberofmanocytesadherent to theendothelium ranged from y isq mm and m animals given senm sickness - isq mm (nbsa)or ~ isqmm(cbsa) as therewerenosignificant intergroupdifferences theseiesultsdo not support the hypothesis that immunological injury increases monocyte adhesion to the aortic endothelium departmen! oipatholcgy, souihampfon universily hospdals soufhamplon. so xy amyloidcan beidentified nupta % of elderty hearts,especiallyin themyocardium oftheatrialappendages in a small prapanionofth%lecasesamylaid~salsapresent inthecardiacvalve~ butisolatedvalvulardepos,tsarerare. a year old male presented with bilateral leg weakness and an intradural turnour. he died days after spinal surgeryanddeposltsofaglloblaslomamunlforme wereldentlfledm both thecerebrumand thecord therewasno hlstory of cardiac disease but all four valves showed translucent verrucous thrkenlngs. these had a uniform eosnophilc hi~lologicalappearance. stainedwithcongo red and had ultrastnucturalfeatures of amyloid amyloid p proteinwas identified immunohistochemically ~n thesedeposits but negative multswere obtained withantibodies t o m . aland a proteins. mwewas no evidenceof cerebrovascular ormyocardial amyloidosis the involvementof all four cardiac valves. the stnking absence of amyloid i" other organs and the a~~o~l a t i o n with a widespread glioblastoma are unusuai but unexplained features of this case the ima s used as a bypass of narrowed coronary arteries it is said to be less prone than vein gratk to develop subsequent occlusivedisease. thisstudy of pairs of imasfrom subjects of vario~s ageswas to see if the histological structure ofthevessel might explain immunityto graft disease sixteen imas were fixed in distension byformalln at cm of water pressure and secllon~ taken at the level of each nb (first to stxth). all arteries undelwent similar changes along thelr length. wlth no slgniflcant difference between left and rlght lntimal changes were minor conssting of lbro-elastic muscular thickening in all age groups and in those who had died from vascular disease theinternalelastic lamellawaswelldefinedat all levels. buttheexternallamellawasclearlydefinedonlyatthelevel ofthefmhandslxthnbs(i.e. mthedetalartwyj. medral thrckne~~decreasedalongthelengthofthearter~esandinall cases changes from an elastic to a muscular structure, generally at the level of the fourth or fiflh rib the ratio of medial thickness to numbw of lmdlae in~reases along the artery. notably at its point of change muscle fibre orientation changes from inter-mixed cir~ular and longitudinal in the elastic part to predominantly circumferential in the muscular pan. the pronounced stnctural differences of the ima compared to the similar sired epicardia coronary arteries. which are muscular. may be of relevance in explaining their markedly different incidence of atheroma. naomi carter, s variend fatty change of the heart is a pwrly defined pathological entity which in the adult hean can be caused by severe hypoxia nutritional disorders. poisoning by selected drugs and catecholaminerelease it is most commonly seen ~n association with coronary artery disease little data exists with regard to the paediatric hean in pediatric deaths coming to post mortem over a year period. there were cases of myocardial fatty change of varying seventy and distribution detected ~n red -stained sections infection and congenital disordenwereimplicated n deaths infectionand cangenitaldisordenwereimplicated n deaths. fallcasesoffarmchangeand % of all deaths seven ofthe e~ases had acombined ~nfecti~u~andcangennalaetiology othercausesofdeath included turnour, traumaandcomplicationsofbinh % of casesofsudden infant dealhsyndrome(s ds) hada fatty heart only one case of the total cases showed deflnlte hlstaloglcal evldence of ischaemic myocardial damage. insome instances, thedegreeaffattychangemayberelatedtathedurationand seventyoftheundertyng condition someofthesechildren may havean occunnutritional orenlymedisarderwhich ~sexpressedatacellular level ~n the form o! fatty change and that contibutes to their early death. we havecampared thereparting oftemporalarterybiopsiesbetween - ( cases)and ( cases). the overall incidence of positive biopsies was . % and . % in each period the number of patients with clear clinical evidence of cranial arteritis was % but in - , % and in , % of these had positive temporal artery biopsies when the histology was reviewed approximately % of biopsies in each period had been erroneou~ly reported as healed or atypical artentis. in contrast. a true histological diagnosis of artetitis was missed in~nlya~inglepatient approximately % ofall patientswithaclinicaldiagnosisof giant cellarteritisdevelopedan additional symptom or pathological change associated with steroid treatment frequent final clinical diagnoses in patients with negative temporal artery biopsies were transient ischaemic attacks, cerebrovascular accidents. unexplained headache or migraine. polyarteritis or polymyalgia hwmatica these resuns confirm that one third of patients with deflnite clinical evidence of cranial arteritis will have negative biopsies pathologists continue to misinterpret normal arterial ageing changes as evidence of healed or atypical arteritis depanmenf hslopafhology and depanmen! resp raioiy medicne. sl banholomew s hospifal london ecla /be adhesion ~fepithelium to extracellularmatrices is mediated partly byafamily of heterodimeric molecules known as megrins we haveexamined theexpressionofthealpha- toalpha- integnn subunits in~unured human branchia epithelial cells. and in bronchial biops es from normal subjects and atopic asthamtics. we have also studied the expression of intercellular adhesion molecule-t (icam- , cd , rhinovirus receptor) br~nchialepithelia cells from surgical specimens were grown as explant cultures on glass covenlips. bronchial biopsies were taken from right upper and middle lobe carinas lmmunastaining was performed on acetone fixed cells and frozen tissue sections using alkaline phosphatase and immunoperoxidase techniques all biopsies showed strong positive staining of epithelium for alphe?, alpha~ and alpha- integnns. stainingforalpha- was weak or negativeand epithelium was negative foralpha- and alpha- except in twoasthmaticswhere itwas weakhi alpha- posittve. in contrast. cultured bronchial epithelial cellswere positive for all these mtegrinsexcept alpha- . epithelium was positive for cam- in asthmatics but negative all other biopsies. cultured cells were ~trongly positive for this molecule it concluded that expression of some adhesion molecules bv bronchial emhelium may vaw ~n relation to the cellular environment and that ths may be imponant tn disease l%panmen! of pafhology univeisrty of birmingham and departmenrs o! 'immunology and his!opalho!ogy eas! blood eosinophils are in a relatively inactive state with migration into tissues eosinophils became more activated these activated cells are hypodense compared to most blood eosnoph s low affinity receptors for both ige (fcerii. cd and igg (fcgriii. cd ) have been documented on activated. hypodense eosinophils this study assessed the expression of these proteins on ttssue eosmophk derived from nasal wlyps the blood and nasal polyps of seven patients undergoing nasal polypectomy were studied nornodense and hypodense eosinophils were isolated from venous blood by centrifugation an a discontinuous percoll gradient percoll wassimilarly used to obtainaneosinophilrichpreparationfromcellsearactedoutafthenasalpolyps cytospin preparationswetem?.de of these samples frozen sections of each polyp were also prepared lmmunostaining using an alkaline phosphataseianti-alkalln phosphatase detection system demonstrated that neither blood nor nasal polyp eosinophilsexpressed detectablecd orcdt l t i~p~~~i b l e t h a t e~~i n~p h l l s o f na~alp~lyp~aresimilarto blood eosinophils and are in a relatively inactive state lavage and biopsy studies. the relationships between mucosal inflammation. bronchospasm and bronchial hyperreactivity are unclear since bronchial smwth muscle has an essential rolein the pathophydogyof asthma. we haveexamined theextentto which it ~sin~ol~ed~nallergicinflammation bi p~ie~in~l~ding~moothmu clefr m asthmatics (age range who did not use steroids and controls (age range ) were embedded i" araldite and stainedformast cells ~monocional ant bodyaat)and eosinophils(monoclona antibody eg ). mast cell numben in the lamina propria and smooth muscle were similar for both asthmatic and control sub e m (mean values. astham lamina propna mm . smooth muscle timm , controls /mm and blmm'respectively) eosinophil numbers in the lamina propria were increased -fold in the asthmatics (p = ) but there was no significant increase in the number of eosinaphils rn the bronchial smooth muscle eosinophil numbers in the asthmatics correlated positively with fev, we conclude that the role of muco~al inflammation in the pathophysiology of asthma has yet to be determined we present cases ofextra-pulmonary pneumocystosis diagnosed on routinesurgical specimens (two biopsiesof liver. and one each of gastric mucosa. small intestine and a pen-anal mass). in each case. the histological features were similar to those seen in the lung. and as in other material from cases of aids, munlple pathology was often found extra-pulmonaly pneumocystasis s now being reported from a widerangeof clinical specialties. one reason forthismcreasemaybe that impraved patient suiyiy~i with"topicay inhalatiooalpentamidinetherapyallowsvisceral foci of infection to become clinically apparent this hypothesis is supported by the finding that ofour cases were taking nebulised pentamidme inthesehypersensltivitystatestothatobselved mbronchiectasis (a chronic suppurative lung condition not thought to in~olve a hypersensitivity aetiology) and in smokers and non-smokers with no evidence of active pulmonary inflammation ourresuns havesh wnthattheb:tlymphocyteratioisnodifferent ineaaandsarcoidfromthat seen ~n bronchiectasis and in normal lungs. we believe that thns is further evidence to suggest that bal is an unrepiesentativetechnlquetorlhestudyof interstitial lung diseasesand that morecansiderationshould begivento the possibility of humoral immune components in the pathogenesis of ea determine its effects upon the number of pulmonary neuroendocrine cells and their peptides. in one experiment. the concentration of noradrenaline in the lung was estimated by chromatography, and that of the peptides bombesm. neurotensin and caknonin gene-related peptide (cgrp) by radiaimmunoassay. there was significantly ies noradrenaline and bornbesin in the lungs of test rats than in controls but the levels of neurotensin and cgrp were unchanged. in a second experiment, pulmonary neuroendocrlne cells in histological sections were labelled with antiserato bombesin,calc,t~tonin,cgrp andproteingenepmduct [pgp s ) a~d =~" " t~d . t h~, e w =~ nochange in the number of labelled neuroendocrine cells expressed per unit area of lung or per unit length of airway between test and control rats for calcitonln. cgrp or pgp . . bornbesin-containing cells could not be identified in either group. an increase ~n pulmonary neuroendocrine cells could not be identified ~n either group. an increase in pulmonary neuroendacrine cells immunoreactive for bomtesin and calcitonin occu~s in the early stages of plexogenicpulmonaryartertopathy in man. theabsence ofsucha change in monocrotallnedulmonanhydertenslon in the rat suggests that this is a poor model for the human disease. this preliminary study was carried out to assess the quantitative expression of neuroendocrine and mast cells in adun humanlungsofcasesofasbestos-relateddisease.tencaseseachofasbestasis, pleuralplaques,carcinoma and mesothelioma were studied m comparison with ten normals wnh no history exposure to asbestos the lung sections were stained lor neuroendocrine markers neurone specific enolase (nso and chromagranln. and, chloroacstate esterase and toluidine blue for mast cells. there was a notable variation in the number of neuroendocrine and mast cells between the control and asbestos-related disease group the variation was also seen between the various asbestos-related diseases. though not statistically sbgnificant. the trend of the vanatton indicated that the individual diseases follow a particular pattern a n lhe expresston of these two cell popuipioiis. we have studied fibrous turnours of the pleura using morphology. immunohistwhemislry and eleclronmaroscopy. the findings were compared and contrasted with reactive pleural fibrosis and desmoplastlc mesothelioma. the fibroustumwn hadarangeof histologicalappearancesand % weremalignant ~nnature.theimmunophemtype was uniform and consistent with positive staining for vimentin and alpha m w t h muscle actin. this was ~n sharp contrast to findings ~n reactive pleural fibrosis and desmoplastic mesothelioma. uttrastructural appearances of the fibrous tumours of the pleura were supportive of a myofibroblastic ongin. we propose that fibrous tumours of the pleura arise from the submesothelial myofibroblast. the malignant fibrous turnours have a distinct immunohistochemical profile and electron microscopic features to differentiate l from themalignant mesenchymai mesothelioma. a study was undenaken to evaluate the use of immunaperoxidase stains on paraffin embedded tissue lo define the cell type in routine lung cancer preparations. and in particular to identdy a subgroup of turnours showing neuroendocrinedifferentiation. forty lour consecutivethoracotamycaseswere selected. following apilot study of cases. to assess digestion times and potentiaily useful antibodies, the remaining cases were processed using a battery of monoclonal antibodies: cytokeratr iaelfae , e ). neuron specitic enolase (nse), chromogranin. and beta microglobulm. in addition to the carcinoid turnours and oat cell carcinoma in the study large cell carcinomas and adenmarcinomas demonstrated positive neuroendocrine markers. uiirastnrcturally, dense core granules could be demonstrated in only / of the large cell carcinomas and in / adenocarcmomas. the discrepancy between the lmmunoperoxldase staining and electron microscopic features likely reflects the heterogeneityafthese turnours. in thisstudy noneof the turnours co-expressed neuroendocrine markersand beta microglabulm. however, the staining panern was inconsistent m the remaining cases high molecular weight cytokeratin ( pe ) was stronglyposltlve m all casesot squamouscell carcinoma and negativein everyihing else. in summary. monoclonal nse and chromogranin appeared to provide sufficient information to identity neuroendocrinedifterentiatian in thecasesexamined m this study. high molecularweight cytokeratinwasfound to bea usefuldiscriminaforforsquamouscelicarcinoma. beta microglobulin was negativemall the turnoursshowing neuroendocrine differentiation. butthe inconsistency of staining m non neuroendocrinetumaurs. made it less helpful for routine laboratory use although endocrine differentiation is the essence of small cell carcinoma of the bronchus, its occurrence m other morphological ("on-small cell) types of bronchial umour (large cell, squamous and adenmarcdnoma) is welldescribed however, its prevalence m such tumours is uncertam, estimates differing from study to rudy and accordingto how it is sought. we have examined, byimmunalabellmg, expression offiveendocrine markerproteins (neuron-specific enolase (nso, protein gene product [pgp) . , the bb isoenzyme of creatine kinase [ck-bb), synaptophysinands-t protein)m bronchoscopictissue biopsiesof "on-smallcellcarcinomaandasse~ns varlabiltty within and between tumour deposits in subjects coming to necropsy with disseminated disease exactly half of the tissue biopsy specimens immunolabelled for one or more marken; one for four, four for three, twenty lor two and five for one. possibly indicating an element of endocrine dtflerentiation inapparent from their morphology. expression was even more prevalent amongst the extensively-sampled turnours at necropsy, but since theinlraductionofthenewgeneralpractnianercontractmapril ,lhere hasbeenasignlicant increasein thenurnbeisof skin biopsiesreceivedmhistopathologydepa~ments in ourdepartment there has beenalhreefold increase in numbers of general practiioner skin biopsies. the aims of this study were to crnlcally appraise these biopsiesand comparethem tosimilarlysized skin biapsiesreceivedfrom hospital in-patientsviageneraland plastic surgeons for the six months prior to and aner st april . data collected included numbers received, ranae of pathological diagnoses, quality information supplied on the request card, accuracy clinical diagnoses. adequacy of excision, age. sex and sites of lesions the resuns showed a similar range of pathological diagnoses. the quality of clinical information supplied was comparable in the two groups as was the age and sex of patients. general practitioner biopsies were less common from the face. clinical recognition of lesions was somewhat less accurate amongst general practitioners than amongst hospital surgeons inadequate excision was more common m general practnioner cases. . % of general practitioner lesions were found unexpectedly to be premalignant or malignant (eight cases) and all ofthesewere inadequatelyexclsed. important implicationsemergingfromthisstudy are dlscussed an audit of skin biopsy specimens from general practitioners in grampian region: changes in requesting practice and specimen type assessmen ofresaction marginsofsurgicalspecimens~s becming more important m manyfieldsofpathology. we wwe interested in developing a means of assessing surgical margins dermatopatholqy in both conventionally removed skin ellipses and m skin ellipses removed during m o~s chemasurgery technique many of the conventionally used markers, such as indian ink, alcian blue and tipp-ex correction fluid are difficult to use i" that they are messytoapply. slow todry and show insufficient contrast wiih oneanother both lnthegrass specimen and microscopically weused"superman'paint,apaintusedforresinandplastermodels,wh,chcomesin awlderange of colours. the paint was easyto apply. did not run and dried qwckiy becauseof the variety of colours avaciablewe were able to apply contrasting c~l o u n to the vertical and horizontal axes of b x c~s i o~ af skm elhpses removed by mohschemosurgery.thepaint proyidedagaodmarkergrasslyand wasnot affected byfreezinqthetissue model paint provides another marker lor surgical bxcij~ii margins and s particularly useful for moh's chemasurgery where horizontal and vertical axes aremarked inorderto assess theadequacyoftheexcision.the model paint may also be usetul in other branches of surgical pathology where resection margins are important. in psoriasis there is altered bpidermal dmwentiattan and increased epidermal turnover both which involve changesin intercellularadhesion aquantnative imm~noh~st chernicaicompa~~sonof the expression of the integrin staining of integrin p subunlts - and e subunlts were disclosed usmg an andin-blatln peraxidase techn!que. large epidermal dendritic cells (antigen presenting cells) expressed p- the psoriatic skm.showed increased - a- . - . and p- expression. buta- and~- showednosignificantdifferencefromnormal thelncreasedlntegrin expression by keratinocytes seems to be a reflection of awered epidermal differentiation rather than increased keratinoc ?turnover themcrease in ~- pasitivedendriticceilscauidbeareflectianofalteredanfigen handkngin psonatic gin. interleukm- (il- ). used m the treatment of patients with metastabc disease fallmg to respond to conventional treatment, can induce regression oftumwrbuik in certain patients however, the systemlc administration ofil- is associated wlth a number of toxic effects, including dermatological compl#catlons. these have been poorly documented we have prospectivehl studied the dermatological reactions patlents treated wiih il- for metastats cdorectal ~arconoma. pre-and past-treatment biopsies were obtwied where possible, arid sect~ons stained with h e. giemsaand pas: hesh tissues weresubjected to immunophenotyplng.there were femaleand malepatients.with treatmentrour~sranginginnumbwfrom - onlyonepatlenthadapastmedlcal hlstoryof any skm complaint (eczema]. four patlenk suffered a dlffilse eryihematous reaction with mild desquamatm and dryness the other patient developed genwalised erythroderma and an additlonal photosensitivity type reaction. histology, anerthe initialcourse. revealed patchyspmgios s. exocylosis and basal layer epidermaldamagewnh a mild perivascular chronic inflammatory cell mfinrate. with suhsequent treatment there wasthlckenmg ofthe epiderrnls, plgrnent incontinence. dermal oedema and more marked chronic perivascular cell tnfiltrate. immunohistcchemistry revealed markedchanges inthe expression of cdt , hladr, cam- and cd m the derm . these changes were greatly heightsnedwith subsequent liealmsnts. with addnional changes tn other t cetl markers. clearly il- enhances the parenchymal expression of antibody-dependent and antigen-mdependent accessory molecules which are important in focusing the immune response. claire m. thornton. maureen y. waish weexaminedalfprimarycutaneaus malignant melanomas seen rn thmdepartmmtover a fweyear permd from to . the total number of tumoum was the number of cases of malignant melanoma both invasive and m m u , increased from cases ~n to cases in . superfmal spreading melanoma was the most common type of melanoma, accounting for % of the total cases this was followed by the nodular melanoma whch accountedfor %. wlthlentigamallgnamelanomaandacral lent~g~nousmelanomabe~ngtheleast commontypes. therewasanincreasmg numberoftumourspresentlngwlth abreslow'sdepthoflessthan t mm. thefigurermng from % of cases in lo % of cases m most cases were stdl of ciar*e kud ivanhough mcreasing numbersaftumour presented at clarke levels i and i wlth a corresponding reduction ~n cases presenting at clarke level v more lesions presented with a flat cross sectlonal profile ~n the later years the figures lncreaslng from % in % in i w o he numbor of i c s~s showlng surface ulceratan at predentatlon d-reased from % in t to % in themltotlcact~vlty. thedegreeofp~gmentalion. theintensiiyofthe~nnammatorycell infiltrate. the predominant cell type and the mcldence of vascular lnvaslon showed no change over the study penod classification of benign vascular tumours is notoriousiy difliwlt and ciinicopathological correlation is often imprecise. this almost certainly reflects the tendency of pathologists to lump together different lesions under the broad heading 'haemangioma', sometimes with capillarylcavernous subtyping. twelve cases of a distinctive subset of cavernous haemangiomas, to be known as sinusoidai haemangioma, are presented. these presented in adws ( female. male. mean age years. range & ). five arose in the upper limb and five an the trunk (of which two developed in mammary subcutis). all were solitary and presented as a bluish cutanmus swelling up to . cm in diameter of variable duration. one case was associated with ipsilateral gynaecomastia. average foiiow-up of . years has revealed no tendency far local recurrence or metastasis. histologically these were subcutaneous/deep dermal lesions with a lobular, sieve-like appearance and focally ill-defined margins. they were composed of dilated. thin-walled interwmmunicating vascular channels with a pseudopapillary architecture. thrombi were common and two cases showed central infarction. vascular spaces were lined by monolayered endothelium which was often plump and hyperchromatic but n d mitotic. distinction from wnventional cavernous haemangioma and angiosarcoma (particularly in breast lesions) is discussed. current methods forthe identification of herpes simplex virus (hsvj may fail to identity the presence ofthe virus in biopsy or autopsy material. we have investigated autopsy cases and neurosurgical biopsy cases of clinically suspected herpes simplex encephalitis by a nested polymerase chain reaction (pcr). dna was extracted from routineiyprocessedand paraffin embedded material byproteinase k incubation, phenol chloroform extractionand ethanol precipitation. nested pcr was performed using known oligonmlaotide primers designed from the hsvtype giywprotein d gene from an area with the lowest homology with hsv type . two of autopsy c a w and of neurosurgical biopsy cases ware pcr positive for hsv. the third neurosurgical biopsy case was not confirmed by pcr as being due to hsv. such primers lo hsv allow the rapid retraspeaive diagnosis of herpes simplex encephaliis and should be of value to neurapathologists. tissueembedded at lowtemperaturein lowicryl-k m resin hasbeenshownto besuitableforimmunogoldlabellng of cellular antigens, and to be capable of withstanding the processing required for hybridization of nucleic acid probas.theaimofthisstudywastoestablish basic wnditionssuitabieforhybridirationof digoxigenin labelled dna probes to lowicryl embedded material, both at the light and electron microscope levels. cultured haemopoietii cells wereembedded afler brief aldehyde fixation. digaxigenin labelled whole human dna or plasmid per (negative control) probeswereappliedtothinandsem-thinsectionsafterproteolytictilgestion and/or denaturation by heat or alkali. hybrids were detected in semi-thin sections by standard wlourimetric methods. and in thin sections by immunogold techniques. elaborate blocking procedures and prolonged washes were found to be unnecessary. specificnu~l~earsignaiwasseenatbdhthelightandemisv~iwiththewhalednaprobe,revealingdetailsof nuclear dna distribution not evident in paraffin sections or cytospun preparations. non-specific binding and background were minimal. signal was greatly reduced if the denaturing step was mined, and was slightly increased by protwlytic digestion, though at the expense of cytoplasmic morphological integrity. while the sensitivity of this system is limited by the fact that hybridization occurs only at the suriace of the section, it is a rapid and specific means of nudaic acid detection, and offers the dossibilitv of accurate iocalization of intracellular human and viral nucleic acid sequences at a fundamental level. of prostatic carcinoma, mouse liver, kidney and gut were used. acp was demonstrated using an kc-dye cwpling method. the acp was unaffected by the addition of mm tartrate. although the acp was known to be 'tartrate sensitive'. the addition of mm tartrate or mm sodium fluoride weakened but did not eliminate the reaction. mouse tissues, prostatic carcinoma and leiomyosarcoma tissues were processed using variausfixatives and embedding procedures. these were tested for acp and trap. the acp in the muilinucleated giant calk of the leiomyosarcoma survived standard formalin fixation and paraffin wax processing and was tartrate resistant. as expected mouse liver, kidney and gut acp and prostatic acp did nd survive most fixatives and embedding procedures. however acp could be demonstrated in those tissues processed bythe 'amex' method. i.e. fixed in acetoneat - o' c. processed throughmethyi benzoateand xyianeto paraffin wax. theacp.thus imalised in paraffin blocks was eliminated by the addition of mm tartrate to the incubating medium. if needed the amex pracedure prrsenes some tartrate sensitive acp in paraffin blocks. acp that survives standard fixation and embedding procedures is iikeiy to be tarfrate resistant. showed acp only in osteoclasts. various elements ot tissue processing procedures were examined to find the wnditionsnecessarytoachievemaximum acp localisation. tissue blockswere pr-ssbdintowaxusingstandard embeddingprocedures. threefixativeswereused. % neutral bufferedfmalin, formalcalcium and ethanol allat + 'cfor hours.twodecalcifyingfluids wereemployed, %ethylenediamine-tetraaceticacid(edta)ph . at + %, zo' c and 'c.andformicacidisodium citrateat+ %forw hats. formalinfixed edtatreatedtissuaat + % produced maximum acp activity. acp was shown in osteociasts, some ostwcytes, chandrocytes. cement lines., tide mark, periosteaicellsand large macrophage likecells inbona matraw. formalinfixed tissues decalcified in edtaatzo'cand 'c werenegativeforacpaswerealltissuesfixedinethana . lnformalcalciumfixedtissueand formic acidlsodium citrate decalcified tissue the acp reaction was weaker. with some elements, e.g. chondrocvtic acp missing. all the acp preserved through paraffin processing was tartrate resistant. it is well recognised that morphology and optical resoiution are vastly improved with resin as opposed to paraffin embedding of tissue. however, difficulties in producing consistent immunopetoxidase reactions on resin sections have caused many departments to abandon thetechnique. although antigenicity is preserved the low optical density of diaminobenzidine (dae) means that the reaction product is barely visible in thin resin sections. the aim of this study was to develop a method whereby antibodies wmmonly used on paraffin sections wuld be successfully applied to pm resin sections. tissues fixed for - hrs in % formol saline were partially dehydrated and infinratedwithlr whiteresinat °cfollowed bypolymerisationat cusingacatalyticmethod. pmsectionswere reacted with polyclonai and monocionai antibodms using a standard indirect immunoparoxidase technique. visualisationwas with a silveramplification systemfordae (amenham) appiiedasthetinalstage. excellent resuns have been obtained with a rangeof antibodies including s , von wiilibrand factor. immunoglobulins, uchll and l (dakoltd.). usingthistechniqueitisnowpossibletocombinehighresoiutioniightmicroscopywithaprecise immunocytochemicai reaction. the advantages are obvious and are particularly relevant in the field of lymph node pathology. however, at and gythe number in muscle dropped significantly . and hoursfoilowing treatment, but hadrecoverad to w n t m levels byfivedays. in the lamina propria, agnor numbersincreasedinitiailyaflerthe . and gy treatments but returned to control values by five days. with gy the agnor numbers showed a significant fall hours after irradiation: this decline continued up to five days. it is evident that &nor numbers within the small intestine are affected following irradiation. the variation in counts is dependent on dose, cell type and time since irradiation. karen m. britten, w. r. roche oepartment of pathology, southampton unwewty general hosp~lai, southampton, so xy in response to ever-increasing demands for immunophendyping in inflammatory disorders, we have developed anernatwefixation and embeddingtechniquesforsmall biopsy specimens. bronchial biopsieswerefixed in buttered formaiin and processed lor embedding in araidite or were fixed in acetone containing protease inhibitors and embedded in the water-soiubk resin glycol methacrylate (gma). gma allowed for the investigation of a full phenotypic profile akin to that which may be wfwmed in frozen section while yielding far superior morphology and greater numbers of d a n s from small biopsies. the phenotypic markers included those for t-ceiis (cd . cd , cd ,cd andlfal),macrophages(cdllc, cd ),mastcelis~b b andaat)andeosinophils(mbp, egl and eg ). wehaveaisodemonstratednautrophiieiastase,cytokinesandtheceiiadhesionmolecuiesicaml .elamand vcam. similar high qualty sections were obtained with araidne but the repertoire of antibodies was restricted to thoseantibodieswhichcannarmailybeappiiedin paraffin. wesuggestthatforsmaii biopsies whichrequiredetailed immunohistochemistry, such as in the areas of transplantation and mucosa immunology, fixation in acetone at - % with the inclusion of protease inhibrors and processing into glvcolmethacwiate with careful temderature control gives optimum results. these included % formaiin at vanous temperatures, microwave treatments. bouin's fluid and a wmmercialiy avaiiabieproduct "rapid fix".thetissueswereroutinelyprocessed. embedded in paranin wax andstained wilhthe haematoxylinandeosin method. an eval"ation~fthefi~alion methodswasaisocarriedoutforimmunocytachemicai stains. from microscopic examination of the results t was evident that for the haematoxyiin an+eosm stains. % formaiinat o'cwa~ wnsistentiytheoplimum methodof choice. farimmunocytochamistryali methodsrasuited in a poor performance using a standard trypsinisetion time. however, an acceptable result was achieved from % formalinat °canda microwave fixative method. byreducingthetrypsinisation time. this. however, required strict wntroi. thus it is possible to fix tissue within one hour. by a method which is cost effective and which can be used within limitations for immunacytochemistry. we set out to study the extent and time dependency of storage related artefacts in cytospin fluid (shandon). we assessedtheeffectof stwage in cytospin fluidon the nuciearmarphoiqlyof breast fnac. fnaspecimsns were coiiected on day and slides were made from each specimen on day . day and day . these were fixed with a spray fixative. between each sampk preparation, the cytospin colleaion fluid was kept at %. the slides were fauigen stained and nuclear morphological parameters (area, perimeter, form ar. farm pe. and convexity-concavity) weremeasured using aseescansoiitaire plus imageanalysis system. there wasasignlticantchange in ail the parametem betwwn day and day , and between day and day (p < . ). the measurements were repeatableon differentoccasionswithoutsignificant difference in theresuns. these results indicate thatstorage in cytospin fluid significantly aners nuclear morphology. the nuclei become progressively larger and more irregular in shape. il is therefore important to standardize the storage conditions of fna specimens. if accurate objective comparisons are required. the identification of macromolecular components of hydrated patholqlicai tissues revealed by low temperature scanningeiectron microscopy (ltsem) isan emergingfieldof enquiry. in orderthatthetechnicaidetaiisforltsem labeilingmaybeestablishedanexperimentaiprotocoi has beendrawninwhichiabeiparticiesofhighatomicnumber are visualized. the experiment comprises a mcdei system in which bovine serum albumen as a known amigen is dissolved in phosphatebutteredsalineand adsorbedontonitroceiiuiosemembtane. theantigen onthemembrane is subsequently reacted mth rabbt anti-cow antibody and then elther protein a-goid or goat anti-rabbn-gold. aner treatmentwithadeveiopertoaddaiayerofsiivertothegoidparticies,thepreparationsareanachedtostubs,rapidiy frozen in nitrogen slush at - ooc, coated with aluminium and observed on the ltsem stage at approximately - °c. backscaneredeiectronimagingathighacceleratingvoltages( kv)isusbdtodetectsilver-enhancedgoid partic es.these areciearlyvisuaiized. thefrozen hydrated preparationsarestabieunderthe described wndtions of ltsem operation. in contrast dry, wnventionai. sem preparations are beam sensitive; their initially observed delicate uitrastncture quickly dearades an exeosure to even the moderate i kw electron beams emdioved in secondary electron imaging. a new approach to systematic storage of pathological specimens for low temperature scanning electron microscopy as greater numbers of pathological specimens are stored for wnvenient future imaging by iow temperature scanning electron microscopy (ltsem). it is increasingly important to maximise expensive cryostare capacity. a cassenesystem hasthereforebeendeveiopedwhichincreasesthe hoidingcapacityof cryostoresbyafactord . over conventional bee storage. each c-he consists of an aluminium disc . mm in diameter wilh cylindrical wells mmindiametereveniyspacedand . mmapart. specimens. mountedan jeolstubs,areretainedinthe wells by set screws. five cassettes fit into each x mm glass storage jar, gwmg a capacny for a canister cryastore(w th @rs percanister)of oostub-mounted specimens an addtionai benefit ofthecassettesystem is that each specimen is afforded a mechanically and thermally protected environment. specimen collection for ltsem may take place in a distant operating theatre or laboratory. lmmobiiiration of specimens whilst they are in transit excludes any possibility of their being damaged during normal handling and improves the prospects for survivalof accidents.afurther benefitisthataparticuiar mountedspecimencannow bemorespeedily identlledand removed for ltsem from rs well. many pathological condtions are charactensed by the presence of cellular degeneration accompanied by cytoskeietai abnormalities. in many such diswders it is not clear whether cytoskeietai abnormalities are pftmary process or are part of a secondary response to cellular insun by other agents or mechanisms. we have used a fibroblast ceii culture mcdei to study the effects of physical distension on the cellular cyioskeielon. inert beads of and pm were introduced intoceils byallowingendocytosisor bymicroinjection.thecytoskeietai response tothese beads was studied using immunofiuorescence microscopy. beads introduced by endocytosis migrated to the prinuciear region and over hours became enmeshed in intermediate filament and mlcrotubuiar aggregates. actln microfilament organiselionwas not alfected. incontrast. microinjectionof beadsproduced an immediatecollapseof morofilaments,wiulvimenbnandtubuiin distribution being presened.the immediateresponseto microlnlectlon is similar to the collapse of actin filaments s w n upon thermal stress. this experimental model has shown !hat aggregates of cytoskeietai proteins may be produced within cells as a secondary responseto intraceiiuiar debris, and that microinjection may induce cytoskeietal abnormalnies similar to those seen in thermal stress. thedetection of numerical chromosome aberrations m interphasetumourceiis bynonisotopic insitu hybridisation has been previously described but the application d t h s technique to paraffin embedded material has been wmpiicatsd bythe requirementfottissuesectioning with the production of partial nuciei. inthisstudy, the analysis - pm thickparaffin sectionsolconventionally processed caski ceilsusing both human papliiomavlrus(hpv type l e a d achromosome specificalphoid probe wascompared with resunsomained usingintactceils.the use of sectioned material did not give signal distributions comparable to those obtained using whale cells. this is consistent wnh a mathematical model derived for the relationship between section thickness. nuclear size and nuclear retention in paranln sections. a method was therefore developed for the extraction and analysis of nuclei fromthick( pm)paraltinsectionsandappiiedtotheanaiysisof squamouscelicarcinomasofthecervix(n = ). the number of copies of chromosome varied from to and this variation was apparent both between lesions and between tumour ceiis within the same ieaon. this is comparable to resuns obtained wth cervical carcinoma derived csll lines. there was. however, no clear relationship between the presence of hpv sequences and chromosome number. these preliminary results suggest that the postulated loss of host suppression of hpv gene function by deletion of genes on chromosome does not occur through gross chromosomal abnormalities. membranous giomeruionephritls (mgn). an immune-mediated disease, is a frequent caused renal morbidity in man. cationic bovine serum albumin (cbsa), given to nzw rabbits in a chronic serum sickness-type protocol. is knowntoinduceglomerularchangessimilartothe humandisease. we~ssedtheenectofashortcourseofthe immunosuppressivedrug cyaon thedevelopmentof early stage.cbsa-induced mgn. fourteen male nzwrabbts received an iv immunismg dose of mg cbsa and p g e. coliendotoxin. one week later they commenced daily iy inlectionsaf mgcbsafor ~onsec~t~vedays. three rabbtsweresacftficed atthistime. sixofthe remaining rabbits were commenced on a short cou~sb of o m cya. whilst continuing to recewe daily doses of cbsa. the remainingrabbitswsregivencbsaonly.after consecutivedosesofcbsathesell anlmaisweresacnficed.ali rabbns given doses cbsa showed early stage mgn and those given doses of the cationic protein showed a more mature, established disease (thickened glomerular capillary wails wth diffuse. global. granular deposition of igg/c and subepithelial electmn dense deposits). four of the cya-treated cbsa rabbns showed a marked reduction in giomerular capillary wail c deposition. three of these rabbns had considerably less severe disease ultrastructuraiiy. these results suggest that cya may aker the course of cbsa-lnduced mgn. 'oroartment of pathology university of edmburgh m&~aischooi: late renal aliogran loss s due to arterial intimai proliferation and iumenai narrowing. there are few studies ofthe phenotypes of the intimai cells. we analysed these lesions by light microscopy and immunmyiochemistry using antisera against t-lymphocytes. b-iymphocytes. macmphages, smooth muscle cells, class ii hla or molecules and the proideration antigen pclo vessels were studied from gran nephrectomies resected between and months post-transplantation. we identified an arterial endolheliaiilis, r-rted in cardiac aliografts, but not emphaszed in renal graftrejection. panernsolarterial pathoiogywwerewgnisd: ( ) endotheiialitis in nnteriobuiar arteries without intimal proliferation, ( ) endothelialiis in larger arteries accompanied by intimal prdtferat!on of smooth muscle. ( ) '"inactive" lesions with thickened intima (i foam cells) but no endotheliaiitis, and ( ) 'natural" atherosclerosis of larger arteries. the endothellaidis tended to m u r in shmw sumving grafts. the pedominant cell was the macrophage. with fewer t-lymphocytes. pclo was expressed in mononuclear ceiis, smwth muscle cells and endothalid ceiis. pmicuiariy. but not exclusively in younger grafls. we proposb these ledonsevolve, variably. from an early endotheiiaiitis to late chronic vascular rejmlon or gralt athwmierosis. the predomlnance of the macrophage at ail stages, suggests it plays a significant roia in the evolution of these lesions. the rat is used to study the response of the renin-angiotensm system m diseases such as hypertension there are structural differences in the jga butthere are few comparisons of its response to stimulalion between species we used renin antisera and an lmmunoperoxidase technique to stain renin-containing cells (rcc) ~n rat (n = i t ) and human kldneys(l nephrectomy and autopsycases). westmulated therenin-angintensin system experimentally by clipping one renal artery ( rats) and inducing sodium depletion ( rats) and studied the analogous human diseases-renal artery stenosts ( cases) andaddison'sdissase( untreated and treated eases). wecounted the rcc and plotted their distribution on scatter diagrams. there were some dinerences in distribution between the species but in boththerewasagradient in distributionof rcc which predominated m the superficial renal cortex. in sodium depleted rats. recruitment of rcc in the iuxtamedullary jgas abolished this gradient. while in some of the animals with renal artery clip hypeitension the normal gradient was reversed, with most rcc in the deep cortex. in bdhu~nreatedaddison' diseaseand~nrenalanerystanosistherewasanoveralincrease(xs)m rcc butthenormal gradientoftheirdistnbution withintherenalcortexwasmainlained.theseresults haveimpl,cationsfortheroleolthe intrarenal renin-anglotensln system in the control of renal haemodynamcs. we have developed a model of adult human prostatic epdhelium that allows arch$tectuml and cytologioai features to be maintained. epithelial organolds produced by enzymic digestion of human benlgh prostatic hyperplasla tissue weresuspendedm type collagengelandsubcutaneouslyxenogranedintointact malenudemice.thexenogranis progressively invaded by mouse stromal cells thesesurround theepithelial organoids and supportthe reformation of epithelial structures with a lumen, lined by a mixed epithelial layer. as the lumen forms tall columnar epithelial cells begin to be seen, these progressively express the prostate specific epithelial markers. psa and psap further the xenograflsexpress appropriatecyiokeratin markers in boththeluminaland basaleplthellal cells. gelsplaced within a . pm millipore chamber, which do not undergo stromal invaston. lose all epithelial organisation with disorganised sheetsand ballsofcellsbeingfound.these cellsdonot express thesecretory markers. in the absence lanandr genic~tim~i~sep,th~l~~l structures wsth a lumen are formed but there are no tall columnar secretory cells and noexpression ofthesecretory markers. thismodel hasfurther been investigated todetermine theresponseof human prostatic cells growing in vivo to the antiandrogen flutamide and to a -aza-steroid e-reductase inhibitor these observations indicate the essential role of both stromal cells and androgens m dictating functional dlfferentlatnon this model will allow the dissection of the regulatory processes involved in prostatic differentiation. hepatocyte growth factor (hgf) is the most potent known mitogen for adult rat hepatocytes in pimary cuiture and isthoughtto haveanimportant rolelnlivergrowihandrepalr linle~sknownaboutthemechanismofactionof hgf on hepatocytes. since the adenylate cyclase system has been implicated in hepalocyie growth control. we examined the role of adenylate cyclase and cyclic amp wmp) in hgf-stimulated dna synthesis. human recombinant hgf (hrhgf. numl) had no elfect on baa or stimulated adenylate cyclase activity in membranes prepared from freshly isolated rat hepatocytes. similarly, hrhgf had noeffect on intracellular camplevels in cultured hepatocytes. furthermore, agents which increase camp inhibited hrhgf-stimulated dna synthesis m primary hepatocyte cui ures glucagon had an i . c. , of lo-'; fotskolin ( . ibmx ( "m). -broma camp ( pm) and dibutyryl camp ( pm) completely inhiblfed hrhgf-stimulated dna synthesvs. from thts. we conciude that adenylate cyclase and camp do not have a role m hgf-stimulated dna synthesis m pnmarycultures oladult rat hepatocytes. the receptor for hgf has recently been identified in tissues other than the liver, as c-met a protooncogenewith intrinsictyrosinekinaseactivitv. whether or notc-metactsasthsrecedtorfor hgf ~n heoatocvtes s currently under investigation a novel in wvomodel of intestinal differentiation is descnbed. fourteen-day, undifferentiatedfetal rat small intestine. stripped of the major part of s mesenchyme. then suspended in a type i collagen gel and renografted ~n a nude mouse. undergoessmall mntestbnal morphogenesis and cytod$ffeienbat$on. all tourmapr epithelial imeages. namely paneth. goblet. columnar and endocrine. are present. double labelling m sit" hybndlzatlon, employing biotinylated and digoxigenin labelled dna probes to whole rat dna and whole mouse dna, reveals an unusual ]uxtaposition of species specific stroma. the outer longitudinal smooth muscle layer, and the major part of the lamina propria. p isthemost commdnlyalteredgene~n humanturnours. mutati~nleadstothep~oducfionolanabnormalprotein which can be detected by immunohistology. such abnormalales are seen in a wide variety of turnours, including colon cancer. abnormal p protein levelshavebeen detewed in & % of sporadiccolorectaltumours. in order to determine i p mutations occur in carcinomas arising from dyspiasla. we have investigated the prevalence of such mutations m colorectal carcinomas from patients wr)h long-standing ulceratwe colitis %). lmmunocytochemicalstalnlng waspwformedon freshand paralflnembeddeduccarclnomasand sporadlc carcinomacontrols matched forsite, stageand grade six areas ofdysplasla, (fourassoclatedwlth uccancers)and sporadic adenomas were also stained. p protein was detected by immunohistochem~stry m the fresh uc cancers and / ( %) ofthe paraffin embedded uc cancers usmg the antibodies cmi, pab , pab and pab . slmllarresuitswere obtained in thesporadic carcinomasw,th fresh cancers positlvefor p and / ( %) of the paraffin embedded cancers. two areas of dysplasla which were associated with p postlve uc cancer~also showed positive p staining, alongwith an adenoma. our results indicatethatunlike k-ras mutations, p proteinabnormalities occurat a similarfrequencyln sporadic colorectal carcmomas, carcinomasar sing !n uc as well as being present in uc dysptasasla. this work suggests that p mutations play a mle m the dysplastacarcinoma sequence. threepanernsofstainingareloundin humancolonusingatechniquetodemonstrateo-acetylationofsialomucins (mpasj. % afindivdualsshowunifwmmpas-positivity. theremaindwareeitherent~~ympas-negativewmpasnegativewith occasional positive crypts. we suggestthatthisrepresentspalymorphismof an autosomal gene ( sa) controlling -acetylation of sballc acid. isolated crypt-restricted mpas-posltivky in otherwise negative indivtduais representing somatic mutation of the ma* gene in crypt stem cells of osa'/osa-individuals. to test this we have studied colons from patients with rectal carcinoma, half of whom had received cgy radiation days preoperataely. radialiond~dnotaffecttheprevalenceofthethreepnenotypesbutincreasedthefrequencyofmpasposltlvecryptsinanegativebackground(t ~ ~~~ . ~ to*, p < . ), largelyduetocryptsshowingseclorial mpas-positivlty ( . x lo* ys . x to , p < o.c€ql). consistent with incomplete crypt colonisation by a recently mutated phenotype. the prevalenceofthisosa*/osa~phenot~e(radiated %, non-radiated %) isveqcloseto the predicted heterorygosily rate ( . %. hardy-welnberg law). thew resuns suggest that human colonic crypts are monoclonal with a longer stem cell cycle than the mouse and that mpas staining provides a method for measuring human stem cell mutattonal load. pouchrris m leo-anal resarvoirs is a frequent complication of restorative practocolectamy which is associated wtih constderable morbidity. long term complications of pouchitis are unknown, however patient follow-up wlth sigmoidoscopic surveillanceis mandatory to assessdysplamc or neoplastic changes in resldual rectal mucosa. we examined epithelial cell proliferative activdy in the ileal mucosa of pouch biopsies using the antibody pclo which detects nuclear expression of the kd nuclear protein proliferating cell nuclear antigen (pcna). ten patienls with functtoning ,lea-anal pouch reservoirs of at least one year duration were biopsied and assessed histologically for evldence of pouchitis formalin fixed paraffin embedded biopsies from anterior and posterior pouch wall were examlned with pclo using the pap technique. ten terminal ileum sctions from right hemicokctomy specimens werenormalcontrols. cryptsin pouch biopsiesand normalcantralshadasimilarpciolabelling indexmeanof k and % (mean values - and w ) respectiveiy the sides and tips of villi had a significantly greater pclo labellmg indexinthepouch biopstes( mean. range - ). compared withnormaicontrols(t % mean, range - ) these resulk demmstiate an expanded proliferatwe epnhelial compartment in ileal pouch mucwhich in contrast to normal terminal ileum involves villous surfaces. considering the frequency and long term natllrs pouchitis. thesefindingssupportthe need for continued pathalogical and clinical assessmentof the ileal mucosa in the neo-rectum of these patients. we, cui, i c talbot, j. m. a. northover signiticant alterations in structure. function and gene expression of mltochondna have been reported in cobrectal turnours. but it $ not known rf these abnormalities are due to mitochondria genetic alteration. in this study, total cellular dna was isolated from l lo rectal carcinomas, adenomas and their adlacent histologically normal mucosa. these dna samples were digested separately with different restriction endonucleases. and then analysed by southern blotting using apurified mtdnaprobe. the restriction fragment pattern oftumourmtdnawas comparedta thatofeorrespondingnormalmucosalmtdna.theseresultsshowedthattherearenalargedeletions. insert~ons, or rearrangements $n turnour mtdna. and no single base changes m the delectable regions in spite of some polymorphic vanations. our results suggest that mtdna changes are unlikely to have a malor role in human colorectal tumoungenesis. hence, alterations in colorectal turnour mitochondria must be dependent upon other mechanisms crescentic colitis: the clinicopathological spectrum of a distinctive endoscopic feature in the sigmoid colon n. a. shepherd, s. gore, s. p. wilkmson oepariments ofnsfopafholcgyand gastreoterol~y, gloucestershre royal hospml, great western road, gloucesfw, crescentic coibs describes an endoscopic appearanc of the sigmoid colon charactwised by mucosal swellmg, eqhema and haemorrhagestrictly localised tothecrescentic mucosal folds. in alive yearpericd thisdiagnosiswas made m patrents, representvlg .a% of ail fbeopta endoscopies. there was a male predominance and most plients were middle-aged or elderly. dwerticulosis waspresent m most ( %) but theabnomallf~es wareconfined to the crescentic mucosal folds with sparing of the divenicular onfices. the malorlty of patients presented with a history of bleeding perano. histologically there wasaspectrum of changesvaryingfrommlnorvascularcongestian to florid active inflammatory disease with crypt architectural abnormalities mimicking ulcerative colitis. three patients presenting with crescentc colitis later developed the clinical, endoscopic and histopathological features of distal ulcerative coliitls: two other patients with a history of distal ulcerative colitis were found to have the charaderlstlc changes crescentic colitis only at endoscopy. three cases showed the histological features of mucosai prolapse the findings in thisstudy demonstratethat arelativelyspecificendoscopicfeaturemayexhibit a wide spectrum of pathological changes whilst luminal mucosal inflammation of the sigmoid colon, usually in a sociat on wiih diverticuioss. may mimic the pathology of chronic inflammatory bowel disease. asmall propartion of these cases may represenl a strictly locallsed form of chronic ulcerative colitis. of genome and antigen a n various anatomtcal sees the resuns have imolications lor the mechanism of e n t~ of mv into neurons and for mechanisms of transynaptic viral spread neuiopathology, fnstitute of pathology j chow, j tobias.' k co ston. t j chambers estrogen is generally considered to maintain bone mass through suppression of bone resorption we have previously demonstrated that administration of pharmacologic doses of estrogen increased bone formation in ovary-intact rats to assess theeffectsof physiological concentrationsol estrogen on boneformatian. estrogen was administered to ovariectamised rats fi n which bone resorption was suppressed by ahprbp. animals receiving exogenous p-estradtal (ed ( wglkg. t o wgikg and w pgkg daily for days) showed a dose-dependent increasemtrabecularbonevolumeaft %. . % and . % respectively, compared wnhthoseratstreated with ahprbp alone. the increase in bone volume was due to bane formation in e,-treated animals, in which bone resorption had been almost completely suppressed by ahprbp neither ovanectomy. ahprbp nor e,-treatment had asignificanteffecton thevolumeorrateoflormationofcortical bone thus. theiocreasedbonerasorptionwhichis aconsequence estrogen-dellclency entralns lncreased bone formation, whlch masks a slmunaneous reduction in estrogen-dependent bone formation. it thus appears that estrogen maintains bone volume not only through inhibition of bone resorption. but also through stimulation of bone formation. pgs m bone formation m vmo. which may represent a pathway common to bone anabolism that s observed !n response to many e""lio"mental t m"ll pgf , was wlthoul effect we found that nodule mductton by pgs occutred early the cunures, belore nodules lntegrln expression e n human bone was examkned by immunohistolqlical stalnmg of edta-decalcrfec and undecalcllned cryostat sections of fracture-and tumour-associated callus dbtalned at surgery and neonatal costochondral )unctions obtained at autopsy. cases were stained wlth a panel of well-charactsnsed monwlonai antibodies against p - . el- and uvp integrios using abc peraxidase and indirect immunofluorescence techniques. osteoclasts stained lor pl , p . u and ovp mtegnns. indicating that they express u p nlp- ) and uvp (classm vttranectin receptor) ostmblasts stained lor pl, and u , and osteocytes stained for p l and . indicating that ostmblasts express n pl nlp- ) and m gl nlp- . classical fibronectin receotorl and that vlp- expression s reduced or lost during differentiation to astwcytes j qulnn. n a. athanasau nufield depanment of paihology and bactenotogy, level , john radcoffe hospital, oxford, ox du osteoclasts are known to effect bone resorption in inflammation and malignancy but whether other cells of the mononuclear phagocyte system. particularly macrophages and macrophage palykaryons, are similarly capable of pathological bone destruction is uncertain macrophages derived from tumours (human lung camnomas. murine mmw-assmated mammary carcinomas) and inflammatory lesions (munne foreign body granubmas) were cuituredonboneslicesbothin thepresenceandabsenceaf stz slromalcells.theseneoplasticand inflammatory iesions contained a heavy macrophage infiltrate but no giant cells and no calcified tissue. there was superficial roughening ofthebonesurfacebymacrophagesboth inthepresenceandabsenceofst cellsand.aher t days. scattered areas of lacunar resorption n co-cultures of macrophages and st cells. normal pulmonary tissue macrophagesdid not produce resorption lacunaeunder these conditions. the results show that macrophages alone are capable of atype of low-grade boneresorption and that a subpopuwion of turnour inflammation-assaoated macrophages following specific interaction with slromal cells, can differentiate intocellscapable of the special sed function of high-grade lacunar bone resorpt on macrophages may thus directly contribute to the mteolysis associated with metastatic turnours and inflammatory lesms in bone differences in the grade of osteolysis may also account for clinical differences tn the degrees and rate at which pathological bane resorption occurs the george washmgton university medtcal center, washnqton, c , u s a for decades, new technological advances have been hailedor condemnedas representing the extinclion cla~s~cal diagnostic surgical pathology, but so far the reports of the death of our specialty have been grossly exaggerated. indeed, the newtechnologies of the sare seenas aids torathet than replacementsfor careful and intelligent gross and micmscopic examination and interpretation, which havealways remained the 'gold standard" against wnichnewtechniquesaremeasuted thedecade will bemarked by anexplosionof pos ibilitiesfort ueas well a non-tmue diagnosis, balanced by a shrinkage in both specimen sizes (already evident in breast pathology) and healthcare budgets.thus. thesurgical pathalogist will haveto becometoanevengreaterextentthecomplete physician. in order to beable to choose wiselyand economically from thediagnostic"menu"availab e. thesurgical pathologist w d also have a greater need than heretofore to be a competent cytopathologist as well, as many of his or her cases will also have fine needle aspiration material and a nuclear grading will assume greater significance o n tumor pathology. finally, the roles of the surgical pathologist ~n both research and patient care (including direct patteoi contact) will require emphasis m order to attract more medical students into our specialhl. womenorthosewithlowgradedisease.this hasledto theviewthathpv s~nielatedtocer~ c~l cancer asimple pcr protocol was developed using standards containing . - fg of hpv dna in ng of normal human placental ona toestimate levelsaf hpv dnain smearsor biopsies. the quantltativedistribution of hpv dna tn normal and abnormal ~ervlcal epithelium was mapped thmughout loop biopsy and hysterectomy specimens using micro-dissectionand histologyof alternate millimeter slices levels were measured in parallel c~n~c~i smears. preliminaryresuits show a low lwel of hpv dna(equiva ent to one copy per cells) was usual in women with normal smears or low grade abnormalities replication of hpv dna to a level of one copy or more per cell was limited to areas of cin ~ which may be very small and to atypical immature metaplasia. the level was reflected ~n the smear aswitch to a high level of hpv dna s a biological and potential diagnostic maherfor high grade precancer. cerv cal biopsies were stained by the cracker technique to demonstrate agnors. for the purposes of the study, b~opsiesweredividedlntaflvegroups-normal, kallocytosis.cini.cin iiandciniii.usinganiemps/ hastingan a m s photon framestore a customised image analysls system was created. the image s dllated and eroded to close any cavities an outlining procedure locates the agnors and records their mid-pants. from this st of midpants themeandistanceofeach agnor'slhreenearest neighboursiscalculated.thlsprocedurescanledoutfor several lelds of view. this study attempts to relate the shape of the associated "mean distance" histograms to the histologlca diagnoses in flry cases. primary adenocarcinoma of the cervix: a retrospective clinicopathological study of cases r ananoos, kamta nahar.' alison b,grigg.'s. roberts? sergin m. sma the patientfen healthy with a tendency togain weight being heronlycomplaint. gynaecological referral revealeda week gestation abdominal mass and bilateral varicose veins. laparotomy, lee oapharedomy, total abdominal hysterectomy and right salpingo-oophorectomy were carried out the len ovarian turnour was cyst~c, smoath surfaced. unilocular,cantainedturbid brownnon-greasycontentsand was cmln diameter.al cmsquarearea of lhe lining showed papillary tutmg. and the remainder was smooth. hiatology of these papillary areas revealed a papillomatow proliferative squamous neoplasm with keratinization and mild acute inflammatm. wthout evidence of cytologicalatypia.stromal invasionora benigncysticteratoma.theremainderofthecystshowednat swamous. cuboidal, or low-coiumnar type epithelium with an underlying vaguely endometrial type stroma in minute focl, with occasional small secondary cysts thls unusual squamous neoplasm was diagnosed as a proliferative epbdermoid meglomerularperipolarcell isan epithelial cell snuted atthe vascularpole ofthe glomerulus recently, theexistence of the penpolar cell has been doubted the aims of this study were first, to establish whether the peripolar cell is a m q v e cell type ~n the mammalian kidney. and second, to compare their numbers and morphology in different speces we used scanning electron microscopy to study - kidneys from each of mammalian species including man. we removed the glomeruli by microdissection and examined a minimum of vascular poles from each kidney penpolar cells were largest and most numerous m goat and sheep kidneys ( w% and % of glomerul~) meywerescant~estandsmallest humanand bovinekidneys( % and %).whileaspectsotsome per~polarcells resembled podocytesand otherperipolar cells had features reminiscent of parietal epithelialcells. in each ~pe~ie~wewereabletadistinguishperipolarcellsasaspeciflccellhlpe. weconcludefirst. that theglomerular peripolar cell s a unique cell type ~n the mammalian glomerulus, and second. that their morphology and number cystlc renal disease occurs in various forms characterised by dilatation of different pans of the nephron. the morphologoal, clinical and genetic features of these diseases is variable but some animal models have been developed m an attempt to understand the mechanism of cyst formation within the nephron. we describe a polycybtic . .ofthe kidneyinthscea-n mousew~hanx-linkedrecessive~mmunodetic,entsyndrome there is progressive cystlc dilatation affecting all parts of the nephron. the cyst lining is composed of a single l a y m c epithelium with focal nuclear crowding and the formation of micropapillary structures. the cystlc epithelial cells show subnuclear vacuolation focal basement membrane thickening is also a feature. there is no signdicant inflammatory infiltrate present within these ktdneys. election rni~roscoptc examination w e a l s that the subnuclear vacdation s due to loss of the membrane infoldings at the basal pole of the eplthelial cell wlth flud accumulatlon within the extracellular space. the basement membrane thickening is due to expansion of the lamina dense. the finding of a plycystic kidney lesion on these mice offers an opportunity to investigate the relationship beween the immune system and renal cyst formation. eqthropomtin. a circulating glywprotem, is the principal humoral regulator of elythropoiesis. it s produced in the kidney butthe precisecell oforigin iscontroversial. erythropoietin participates in a~ ~ssicaif dbackcontroi system whch attemots to restore oxvaen deiivew lo the tissues. normallv erdhrowiatin is resent in the sbnm at picomolar c mntrations bui revels may h e up to oid durilg ?&erehkpoxic ;tress. the mechanism linking renal oxygen sensing with erythropoietin synthesis is p o~l y understood but there s evidencethat cells of the innsr cortex respond to tissue hypoxia by producing elythropoietln. eryihropoietin geneexpression in the kidney can be detected by northern blot anaiysis within one hour of exposure to hypoxie stimulation. the cellular location of erythropaietin m-nger rnawasdetected inamurine model byinsdu hybridizationemploying radioiabeiled dna probes and autoradiography techniques. tubular cells of the inner renal cortex wbi b identifled as the main site of ervfhropoietin gene transcription. the location oterythropaetm production was confirmed by immunohistochemistry with anti-sera ra sed to pure recombinam dna-denvea human elythropoetin. the specific antibodies bound oniy lo tubular cell cytoplasm, confirming the tubular location of erythropoiet,n-producing cells. severe reversible acute renal failure and iga nephropathy r jackson, k l. c. mclay unrvemty diws~lon of pathology, giasgow ropl infirmary, castle sireel. glasgow, g osf whilst progressive, irreverable renel failure is well known in the evolution of a stgnificant proportion of cases of iga nephropathy, disease-related. reuerstbie, severe deteiioretion in renei function s a less well recognized phenomenon. relatively few cases aredocumented in the literatureand these have invariably been associated with m-iw haematuna. renal failure has been largely attributed to a combination of tubular obstruction by red - cast~andacutetubularn%crosisthaughttabetheresunof adirecttubulo-toxiceffectof released haemoglobin. in a review of cases of i@ nephropathy we have encountered two cases of this type. the clinical. histological. immunohistochemical and ultrastructural data relating to these cases are presented. on the basis of this evidence and an analysisof the relevant literature. isconcludedthat the accepted pathogenetic hypothesisdnot provide an entirelysatisfactaryexpianationof the phenomenon. itisfurlhersuggested that immunologically mediated injury dfrected at the glameruiar vascular pole and possibly the extraglamerular mesangium could compromise thetubular blood supply and might also lead to haemorrhage into the distal tubule at the macuia densa. scanning electron microscopy (semi when combined with electron microprobe analysis (mpa) s an expeditious and accurate method of identitying substances deposited in tissues. we report a case in which these techniques were used to study crystals in a transplanted kidney. the patient presented at age three with vitamin d resistant rickets and fanconi syndrome on the basis of cystlnosis. he gradually went into renal failure and received a kidney transplantfrom hismother. itfunctionedwsiifarafewmonths butthen deterioratedandwas removedone year after transpiantation.afewcrystalswereob~ed by light microscopy ma biopsytakenattwomonths. arepeat biopsy at four months was examined by sem and contained the typical hexagonal cryystais of cystine. the s o i i i sulphur peak obtained with mpa confirmed their elemental compojltion. the hydrophilic nature of the cystlne prevented their identificetion within routine sectionsfor transmission electron microscopy (tem). the hexagonal o~tline of aystaiswas confined to interstiitat macrophages rather than tubular arglomeruiar ceiis. pm frozen sections mounted on carbn pianchets diowed vlsualisation and analysis of the crystal deposits by sem. the use of appropriate processing methods may be crucial m identifying crystal dspasition within pathological specimens. during the year period - . new cases of dense deposit disease (m~~b,a"op,olif~=ti"~ giomerulonephritis type ill were diagnow in nonhern ireland. two further known patients developed recurrent disease in renal transplants. the mean age of onset was years (range eight- years) with five males and eight females affected. renal function was impaired in % patients at presentation which ranged from nephrotic syndrome ( %). nephrilidnephrotc syndrome ( %) and macroscopic haematura with mild protelnuria ( %) lo mild protemuria f microsopic haematuna ( %). serum c was universally low. haw of initial biopsies showed cr-entsusuailyaiatedwith rapidansefrenaifailure. % ofpatientsrequired diaiysisatorwithin twomonths of presentation. a further % d patmnts developed renal failure within three pars of disease onset of the six patientstranaplanted haw havedaveioped rmrrent diseasin their grafts six patients havenot required dialysisat ameanfoilaw upoffour yearr(ranse neloeightyears). mhaugh reialweiyuncommon, dense deposit disease isan important causeof renal failure in children and adolescents. we have confirmed that this disease has a poor outlook in terms of renal function with a tendency towards recurrence in grafts a review of renal biopsies from patients with diabetes mellitus. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . showed with diabetic giomeruionephropathyalone, with other renal diseaseand with both diabetic giomerulonephropathyand other renal disease. eight with the histologiwl appearances of diabetic nephropathy without known diabetes were alsn identified. clinical details were available on ofthe patients. these showed that in type (insuiin dependent) diabetes ( ). most ( ) showed diabetic giomeruionephropathy, showed both diabetic glome~lon~phropathy and another diagoaas, and showed aniy another diagnosis in type (non insuiin dependent) diabetes ( ). showed diabetic glomerulonephropathy with showing another renal diagnosis without diabetic giombrulonephropathy, and with both diabetic glomeruionephropathy and another diagnosis. patients investigated with nodular glomerulosclerosis resembling that seen in diabetes but not known to be diabetic at the time of biopsy, showed abnormal glucose metabolism on investigation and t showed a paraprotein. this review showsthatinpatientswithciinicairenaldiseaseanddiabetesmeliitus,therenaldiseasecannotalways beassumed to be diabetic nephropathy especially ~n type diabetes where a wide range of other renal disease can occur histological findings of diabetic nephropathy may occur in patients before diabetes is diagnosed cllnlwiiy. a j. hawie, r. l. bryan in a biopsyof arenaltransplant. we notedan artmalabnormalitythat wasunrecorded in standard texts. in an arcuate artery. theiewastheappearanceafformation ofanewartery inside theoid, with layersof muscleandelastic fibres that in places resembled an internal elastic lamina. separated from the original internal elastic lamina by loose connective tissue. in a systematic study of consecutive transpiant biopsies and consecut~ve transplant nephrectomies. another six examples of this abnormality were found. these six and the index specimen showed changes cons~stent with chronic vascular rejection. in a systematic study of consecutive nan-transplant renal biopsies showing interstitial nephritis. another example ofthis lesion was found, in a kidney showing chronic interstitial nephntis. thischange isprobablyavmantfom of muscularisation ofthsarteriai intima, isseen in chronlc renal damage, and occurs in transplanted and native kidneys. localised myloidasis ofthe lower genito-unnarytractisararedisease. few studleshaveanempted tocharacterise theamyloidtypeusing immunohistochemicalstains. wereportaseriesofnin~casssinvoivingthebladder( ), lower ureter ( )and penile urethra( ). thesecomprised malesand pfemaies with an age ranged & years. nonsof the patients had evidence of systemic amyioidoss. three patients had a past history of lower genito-urinq tract infection and of these, one had repeated instrumentation. one patient had prostatic carcmma the cmmonast presenting wmpiaint washaematuriaand the mostfavouredsurgicaldiagnosiswasa neoplasm. fowpat#ents had repeat biapsiesfor persistent orrecurtentamyloidosiswith atimeintmal ofupto t yearsfram initial presentation. bght microscopy showed amyloid deposmon throughoutthebiopsies, in lamina propria. muscle. adiposetissueand vessel wails. with a variable giant cell reaction and lymphoplasmacytic infillrate. an abc-immunaperoxidase techniquewas usedwith antibodmsto pcamponent, serum amyioidaprote n prealbummandkappaand lambda light cham in an attempt to classify the amyloid type. this was found to b e d non-pa. non-prealbumm type. a negative or equivocal reaction was seen for kappa and lambda light chains however, such antibodies may not necessarily be immunoreactive with light chains or fragments of light chains m amyloid deposits. features of bile reflux type gastntis: glandular distmion (branching, "carkscrewmg"). nuclear regeneratwn, intramucosal smooth musclefibresandpaucityof inflammation. helicobacterpylanarganiams werenoted bileacid concentration #n gastrc aspirates collected by endoscopy at the time of biopsy was estimated using an optical densitymethod.gi casesshowingglandulardistorti~ hadraisedgasttic bileacidconcentration.m cases with mahednuclearregeneration hadraised bileacidsandof case~withinbamucosalsmoothmuxlefibres had raised bile acids nine of cases with raised bile acids showed a chronic inflammatory response the histological features of bile reflux type gastritis which correlate best with estimation of bile m gastric juice are glandular branching, "corkscrewing" and intramucosal smooth muscle fibres whereas pauclty of chronlc inflammation does not h pylon was identified in of cases with raised bile acid. aims to compare the bacterial flora of normal and inflamed appendices. and to conelate this with various histological features. to establish the incidence of yeninla infection ~n acute appendicihs !n southampton. methods resectedappendicesweresentheshta histopathologyandal cm portionwasremovedandcultured for aerobic and anaerobic organisma. the remainder was fixed ~n % buffered formalin and pocessed for rwtine histology results: appendices showed acute ap+endicitis, and were normal. histology there were statistically significant differences between the two groups for the p s e n c e o f faecollfhs (p < . ). fibrosis (p < . ) and prominent follicles (p < ) -all were more common ~n the normal appendices. microbiology no yer~inia was cultured in enher group. there were statistically significant differences in the number in each group which grew anaerobes and streptococci. anaerobes were more common in the normal group (p < . ). and streptococci morecommon in inflamedappendaes(p< ) conclusions. .thereisanaiiwed bactenalflora in acute appendicitis. yersinia does not contnbute to acute appendicitis in southampton. faecoliths. fibrosis and prominent follicles are significantly more common in normal appendces le antigen (using cat - ) a i d type h antigen using a specific moncbnal antiserum. we f w n d a widespread distribution oftype structures in both benign and malignant epnhelium oftheextrahepatic biliarytract and ampulla of vater. type h antigen expression was seen in benign epnhelium and in non-papillary tumoun and ampullary carinomas. lmmunohistochemical detection of tyw blood qrou antlqens does not adpear to be ofprwnostlc the majority of cases bearclose relationship to human ulcerativecolitisclinically, endoscopcallyarid i" re-nseto treatment one hundred postmortem iiyerswerestudiedfromconontoptamanns with totalseversul~ataecolitis who were pathogen free and had a histological picture resembling human uicerallve cdhs only iivwere normal in there was a mild penportal chronic inflammation. had extens~ve steatosts, had an appearance resembling chronic active hepatitis and ~n the histology resembled that of sclerosng cholangitja. other hepatic pathologies were s e n in smaller numbers. these changes parallel the itver disease seen in association wlfh "icbrativbcolitisinman. webelievetheconon toptamannprovidesthefirst modelof l i~e r d i~~~~e m u l~e r a t i v e~~l n to allow sturty of the pathogenesis of the enralntestinal manifestations of ulcerative colitis. r-rvolr and ilm-anal anastomosis. pre-and pan-surgical specimens were studied and compared using routine histological and histochemicaltechniqm themalontyofcasssexhibitedan lncreasem chronc lnflammatlon wlth n or noacute inflammation.the levelsof chronic innammation were found to bemost severe i" those cases which ~~fferedhompouchiti~. morphomstncalanalysisrevealedan~ncreaseincrypt depthtovillousheightration(cdvh) ~n % of cases. the cdvh in the pouchitis cases was greater than n the other specimens studied the resuns of m u m histochemical analysis did not show any charactenstic changes occumng. in the cases studied. lectm histochemistry demonstrated an increase an supranuclear staining of the pouches with dba. sea, wfa and wa. which is similar to that fwnd in poximal colon. staining with psa, lca. u f and lta revealed charactenstic changes in binding panem th-may mdjcale changes m tucosylation of certain cellular canponents. certain changes in lectin binding were found to be more sjgnificant in the pouchitis group of cases. the changes in the ra~ewoir mucosa are most irkely to be an adaptive response to the new mtra-lummal environment wlth the acqmdfon of cmain mime chaiacteristics. thereare alsosp~ciftc changes which occur to a greatsrdegree m the cases with wuchits, these may occur as a result of or lead to the occunence of puchms. these resuns may be cryostat sctionshomten patientswith ileal pouches lor extensive colitis, ten distal ulcerative ~olitics and five normal mall intestines were assessed immunohistochemically for macrophages (leu m antibody). rfdl gnterdigitating antigen presenting cells). and rfdt (resting macrophages)antigencantainingcellsin lminapropna. result~areexpressbdasape~=~~t=g~~f thetotal. normal small intestine contained a mean ? % and % (ranges - . - , and - ) leu m . rfdl and rfdt positive ~ell~re~pecl~~elywh~chcontrastswiththoseh~ghervaluesinpouchbiapsies- %, %and %(ranges - , - and )respectively. distal ulcerative coilticscontalned increased macrophagenumbers( . and % for the three antibodies) but have a predominance of the rfdi positive population in contrast to pouchitis patients in whomthe rfd positive cells predominate thesefindings demonswate remarkably high macrophagenumbersin pouchitis, howeverthe rfd positive sub-populations predominance suggests an aetiologv other than ulcerative colitis for muchitis. margaret balsltis. yah mahida the wide family of cell adhesion molecules (including the subgroups of the immunoglobulin supergene family. the integnn receptors andtheselectins) are involved in controlling interactions between endothella cells and leucacytes !n inflammatory states. this is partly by their influence on adhesion and migration of leucacytes to and through endothellurn. using monoclonal antibodies, we have compared expr~ss~on of the three cell adhesion molecules icam-t (interceiiular adhesion molecule- ). elam-t lendothelial leucocyte adhesion molecule-t and vcam- (vascular cell adhesion molecule-t) ~n normal colonic mucosa with muwsa from cases of inflammatory bowel disease. wehavefoundexpressianafthese threemoleculesto beincreased~ninflammatarybaweldiseaseandthis change involvesendothelial cells as well asleucocytes we will dixussthbrelationship between adhesion molecule expression and dlsease actlvlty and posslble therapeutic implications granulomatous enterocolitis associated with therapeutic irradiation . . mangham. k m newbold, s dover the universty of bmmgham department of palhology, sci ool of m&mi science the medrcal schwl, edggbaslon. chronic radiation-induced entero~olitis s a weli recognised complication of radiotherapy for mtra-abdominal or pelvbcmalignancy. the pathological features includepwforatlon. fistula formation. segmental necros~s. and stncture formation. the histologicalfeaturesareessentially lxhaamic in nature. consequent upon the characteristic vascular damage. two case of inadtation-induced granulomatous enterocolltis are described ~n which non-caseating epithelcoid granulomas were present ~n the bowel well. to our knowledge. granulomas have not been previously &="bed in radiation coldisor any other formof ixhaemic bowel disease thsgranulomaswere largely confined to the mmosz-associated lymphoid tissue and the draining lymph nodes. naked submucosal granulomas were, however atso present. this distribution is similar to that seen m crohn's disease and tuberculosis. no evidence of these, or any othersystem!c ~r a n " l~r n a t~~~~~n d i t l~n~ was present these cases suppon theview that granulomas in bowel disease are secondary to an tmpaired muwsal bamer to antigens and highlight the non-specifiaty of granulomas m the diaynoscs of inflammatozy bowel diseases between and % of crohn's disease patients have involvement of the upper gastrointestlna tract the identification of granulomata is normally required to make a specific diagnosts but this may be dinicult in small endoscopic biopsies. we present t o cases where there was a strong suggestion of upper gastrolntestinal crohn's disease with a definitive diagnosis established on more distal invoivbment. four of the biopsies contained granulomata. the remaindershowedeitherpatchy inflammation, local ulceration orvillo~s dtjtmtlon only in aneffort to establish whether crohn's disease biopsies contained specific macrophage or lymphoid populations we applied the~onoclonal antibodies. mt (cd ). uchll (cd ro)). muramidass. mac , alpha- antltwsin and kpt (cdg ) using immunohistochemistry. theresuns were compared with casesot "on-specific duodenitis orlelunltis crypt-restricted loss of g pd activity has been used to quantify carcinogen-induced somatic mutation of the x- samples of peripheral nerve (sciatic nerve) of mice from varylng age groups were examlned over the past years. development olage~related penpheral neivefibredegeneraflon was observed among mlce over months age. the spontaneous peripheral nauropathy was characterized by walierlan type degeneratlon. teased nerve flbres from mice of years old showed evidence of swelling of the myelin sheath. fragmentation d myelin into myelin balls or ovoids, with areas of segmental demyellnatlon. light microscopic examination of h e stained sections revealed axonal degeneration, and myelin fragmentation. with vawolation of nerve fibres. on ultrastructural studies. there was evidence of axa-myelin degeneration. axxoplasm showing dark stained bodles suggemng of degenerate mitochondria the myelin sheath a l~o showed disorganization and fragmentation myelin, sometimes with whorl lormatron rithcm the cyroplasm of schwann cetk, wtth evidence of autophagocytosis by schwann ceiis. in some inslancestherawere sgnsofproliferation at schwann cells. nervefibredegeneration affected both non-myelinated and myelmated fibres generally, therewereapparentlygreaternumbers fdegeneratenan-myelinated nerrefibres than myelmated nerve fibres. furthermore the numben of degenerate myelinat& nerve fibres were less than in seontaneous peripheral neuopathy in ageing rats (personal obsenat on) swntaneous peripheral neuropathy of sciat,cnenesmainlyappearedin miceaged manlhsormore, s eenonlvvelyr rely in mlceof months. younger mice did not show any evidence of this change age-related penpheral nelimpathy is not usually accornpan~~d by any clln,cal ,gns neur~. fhology. uhversrty ofiowa, iowa. u s a v~scuiarendothellalcclls(rn)conslitufet~elntertace betweenthe bloodstreamand thetcsske and pertoormsevetal key roles ~n the development immune and inflammatory responses endothellal cell from the braln display slgnlflcantly different momhoiogy from other en. having ilght ]unctlons between them and pauclty at micropinacytolic ves~cles dunng inflammatory conditions of the cns interaction between mlammataly cells and rbb en s an initial important event. it s known that expression of several adhesion molecules on b r m en is upregulated !n intlammatorycondtti~ns and cytokine-induction ofthese molecules has been investigated however an ~mparlanl mechanism of adheson molecule induction and hence modulation of tmflammatq cell/en binding might beviral infect onofbrainen wehaveexamined theabilityofmeaslesvirus(human )andherpes simplex virus to infect cerebral endothelial cells. adhesion of syngenelc splenocytes to both vlrally-infected and mock infected cells was determined using a chromiums assay. by h of infection with measles yins, cytopathic effect was eadent. and splenocyte adherence was increased to a mean of % of the control mock-infected aner h mock-infecled expression of the adhesion molecules meca- and mala-lon virally-infected cells was determined possible mechanisms for enhancement of adhesion will be discussed and subsequent implications of virus inlectiun of cerebral en ~n relation to homing of inflammatory cells into the brain. ultrastructulal dbsenamns were made on the mechanism and route of innanmatory cell diapedesis through cerebral vessel walls in an experimental model of can~ned~stempervlrusencephalamyel~t~s ~n the hamster migrating monocyies and lymphacyiesextended pseudopodiawhich contacted, indented and adhwedtoendothelwm. they lhen invaded the endnthel l cells. becoming enveloped by endothelial cytoplasmic processes which re-establishec the continuity of the vascular lining as the migrating cell passed through although mlgratlng cells were frequently seen close to intei endotreiml junctions. they were ever seen wlthln iprctlons. or between adlacent endothelial cellsand therewas noev!denceof opening of mterendothelial tlghtpnmons aherpasslng through the endothel~al layer. cells squeezed through small pores (migration porest in the t h r sub-endothellal basal lamina me present study confirms and extends previous observations on the operation u;#thr the cns of a trans-endothel~al. paraiunclianal route for d apedesis of mflammatary cells. ireland falbology royal viclona hospital, bcl/asl hi bl lrrlnrld a sewice for the blochemtcal dlagnosts of lysosamd torpge diseases has now been o n operalaon m betlast forslx y n a r~ it currently lids assays available for the measurement lysasamal acid hydrolases in plasma. serum. lkwcocvtes. cultured sk nfibroblasts. amnioticfluld and cultured amniatlccellstordlagnos~s of lysosomalstorage diaurders solar patients have been referred from throughout ireland wtth having a positive diagnosis of these. were diagnosed as being hamozygous for a specific lysosomal enzyme deficiency, were identified as having multiple enzyme acl~ciencies (mucolipidosis type -cell disease) and had heterozygote (carrier) enzyme ibvpis t t v latier. w~i e either parents (obligate hetemrygotes) or siblings of homozygotes and one was a heterozyquti' lor x~lirlhrll recessively inherited fabry's disease. in addttlon. prenatal diagnosis has been performed on motherswithafam,iy hi~toryofi-celldiseaseand/orhurler'ssyndmme.oneofthesepmvedto bepositivefor huller tht' resuiic the biochemical investigations of these cases are presented. high levels of circulating immunoglobulins are common ~n liver dlsbase. in alcohol c hver disease depos s of mmunaglobullna(lga) have been found in the liver and therenal mesanglum, and werepart acaseof a oyearoldfemale with a year history of cardiomyopathy and progressivemuscle weakness anerthe birth of her baby shewas subsequentlyflnedwith apacemaker. clin~cally, therewas sevweweaknessof neck flexors with proximodistal weakness tn both arms and mild weakness of htp flexors. the most stnking weaknesswasin herbreathmg muscles therewas no ptosisorfaaal weakness rellexeswere symmetrical and her plantarswere flexor herckwas normal. aquadriceps musclebiopsy revealed abnormal vanatcon o n fibre diameters affecting bothflbrelypes. occasional pink hyalme ~nclus~ons which stainedforacid phosphataseand wlth pas wwe seen i~i both fibre types electron microscopy showed these i n~l~s i o n~ to cwtsist of aggregates nm diameter filamentaenmeshedwithinacentral coreofdenseamorphousmatenal. inotherareastheamorphausmatsnal layas inegutar patches within !he sarcoplasrn mainly at the level of the "z" tine causing disintegration of the sacomere immunoelection microscopy wing collmdai gold showed that the denseamorphous material reacted slrongiyw<th desmiri antisera and calld therefore represent a phosphorylated form of the pratein. tubular aggrcgatesitas] hayebeendescr,bedinassociat,on witha widevarietyofdisorders wherethey most onen occurin type %fibres recentlytwolam~i~eshavebeenrepored with muscularweaknessandtasasthedom~nant biopsylinding wereporla thtrdfamilyol whatseemsla beadistinctcondition twopatients afalherandda-ohter presented with slowly progressive p.oxima weakness. limitation of eye movements and aciiilles t e o d a r.antractufes creatine kina-was s t times normal by light mcroscopy, basophllic accmulations were ~e e n in both fibre types clecson m!croscopy revealed that these conslsted tightly packed parallel tubular a m p (tas) these varied somewhat m theiiulliast,wct~ral appearance and were therefore clawfled accordingly thisreport lhrows furner bwdexe m support of a otstmct cllntcopathologicai enttry. the c-myconcaprotein (p ) is a dna-bmdmg protem. increased expression of which is assmated wth shartenmg of the gog, phaseof the cell cycle. quantitahon of p m breast cancers may have prognoshc and therapeutc implicatrons. flow cyiometnc quantitatton (fcmo) of p has been shown to correlate wxth certatn parameters m breast carcinoma. however, thsre are factors which we felt could potenttally confound this fcmq m foimalln flxed paraffinembeded(ffpe)tissues. bothnuclear(n)andcyt~plsmic(c)express~oncanoccur. dependlngonthetlssue fixative used. cytoplasmic stripping and exposure of nuclei to the digestwe enzyme, trypsin, occw dwng the preparation of the nuclear suspension from ffpe tissue. this study documents c-myc oncoprotein stainlog i" * tissue senion of p m a r y breast carcinomas: turnour cells, adlacent benign epithelium and stromal cells were assessed. the monccional antibody om- - (crb) was employed using the abc immunocytochemical technique thi~antibodyrecognisesp andacyiaplasm ~ kd protein the resultsoftrla trypsmsatm of tissue sbctionspriortostsiningwerealsonoted. positivbstainingwasseenin y~oftumaun. both nandcstainmg belng present in % benign epithelium, many endotheiial, fibroblast and lymphocyte nuclel showed posltlvlty trypsinisatwn ledto lossof staining ntensity.thusin ffpe breastcawnomas. ~, " g l e p~~~m = t =~f c m q~f om- - anti-c-mycantiobody isconfounded by facts((i) much extratumoural c-mycexpresslon cccurs, ( ) cytoplasmic stripping may iead to oncoprotrin loss and ( ) breast oancercells,iisinducedaver -fald by phorbolssten.amphiregulin bindstotheegfreceptotandcan be stimulatoryorinhibitorydependingan theceil line. ltal~oblndsto~tsown receptor, possiblyerbb- .weevaluated expression of amphiregulin in primary breast cancers using immunocyiochemistry. % of turnours showed staining using amonoclonal antibody toamphireguiin which wasdlffuseorfocal in tumourcells.amphiregulin was not expressed in stroma and much less often in normal breast ep!thellum than m tumours. immunohistochemistry waspositiveincases withthe highest mrnaexprsssion(concordance %)asmeasuredusingacdnaprobeand qualitativenortherblotsanddat blotsanabplatecounter all caseswereassayedforegfraod er. er'egfr-. er* egfr*. er-egfr-. er-egfr'. there was no association of amphtregulln expresston wlth other receptors. these results suggest amphiregulm may be an independant factor regulating growth of a substantla proportion of breast cancers. in h a s t cancer, overexpression of the c-erbb- oncogene is associated with poorer prognosis, and in a prwious study we found it to be associated with lack of response to endocrine therapy bolus doxorubicin with infmons of ifosfamiddmesna appears to be a very active chemotherapy combination for advanced breast cancer in young women. thirty-nine women (median age = yrs) with rapid recurrence of breast cancer (median disease free interval = mth) were treated with doxarubicin and ifosfamlde/mesna. and c-erbb- expression in their pnmary tumours (determined immunohistochsmically) related to treatment response. the tumours of patlents strongly expressed c-erbb- protein and of these patients had objective responses to chemotherapy in patlents with turnourseitherweakly expressingc-erbb- (n = )otshowingnostaining(n = )therewereobject,veresponses,n . these data indicate that c-erbb- expression ~n primary breast carclnoma, while associated with resistance to hormonetherapy. daesnot preclude responsetosystemic chemotherapy. and sugge~taposs~blerolefarc-erbb- status in directing treatment strategies for individual breast cancer patients. pmicularly adpvant therapies ~n early stage disease. transforming growth factor is a muitifuncyional regulatory protein which can either stimulate or inhibit cell proliferation. can inhibit immune responses and can enhance both angiogenesis and the formation of connective tlssue.tgfpmrnahas beendetectedin breastcancercelliines, andtheprotein hasbe~nfoundinthemediafrom several such lines. in view of the prominent stromal reaction seen in many breast cancen. tgfp may have a srgnificant role in tumour development and subsequent behawour. tgfb, protein expression has been examined ~mm~noh~stdchemically using a specific polyclonal antiserum in formalin fixed. paraffin embedded invaswe and in s,fu breast carcinomas occasional staining of normal acinar cells was seen. % of invawe cancers had prominent staining of cells for tgfb. often with associated stromal staining. % showed occasional cellular and stromal staining. and staining of stroma only wasobserved in %. theremainder werenegative staining of ins it^ carcinomas was less with only % having prominent staining and % being negative. there was no conelatkon with type or grade of tumour. or oestrogen receptor status but all carcinomas with prominent reactivity had metastasised to nodes. this along with the differences between lnvasive and m srtu carcinomas suggests that tgfp may be a determining factor for invasion and metastasis. depanment ofpathoiwy, univers,ty ofshemeld, poboxsq , shefiela s ul chromatin changes ~n neoplasticcells are well recagnised quantitation of nuclear chromatin distcibuhon is dcfficult becauseaf thevanetyof parameterswhich couldpossibly bemeasured in thisstmiyamethodof quantttatmg the distribution of dna was developed for a seescan solitaire plus image analysts system. the parameters homogeneity, condensation and clumping were calculated by segmenting the nucleus grey scale intensity into three levels, black, grey and white, byamethod of adaptivethresholding.theseleveis represent the darkwand lighter regions of chromatin distribution. the relative pr~portlons and positions of each of these regions were calculated by viewing the nucleus through a pixel wide computer generated "mesh" the system was used to compare benign and malignant feulgen stained breast fnac specimens. the homogeneily and clumping parameters were significantly different between the two groups (p < ). but complete discrimination was not possible in order to test the valtdity of the system. spicimens were graded subp wely ( to ) and compared with the image analysis results. there was % correlation for homogeneity and % lor clumping. indicating the difference in chromatin distribution s real and not a result of system error the role of nuclear dna analysis. oestrogen receptor and thymidine kinase (tk) activity as prognostic indicators in human breast cancer was assessed. these parameten were investigated in relation to traditional pmgnostlcators. theoveralloblectivewastodeveiopaprognosticindexthat canpredlctforsunlval.tumourmatenalfmm breast cancer patients was anaiysed the nuclear dna content and oestrogen receptor status were determined by static cylophotometry and a multipoint titration assay respectively thymldlne klnase actlvlty was assessed using atp or ctp as a phosphate donor. patients were followed-up for a median of months. the cox proprtlonal hazards model wasused todetermine which parameterscould predict survival.pathologicalgrade, tumours e. totaltkand ctpjatpratiowerefo~ndto beindependent predictonof survival.aprognosticscore wasderivedforeachpatlent and these were assigned to one of three prognostic groups. using these prognostic groups the rlsk of reduced sunwpi could be predicted in . % of patients. inconcluslon, the assesment of pathological grade, turnow s m and thymidine kinase activity provides a reliable means of predicting prognosis year periodarereviewed.the median follow-up was months. on the basiso margln contour, degreeofstmmal overgrowth and atypla, mitotic activity. nuclear pleomorphism and the presence of speclallsed stroma, benlgn and malignant phyllodestumounwereidentified turnoursoccurredin womenof msdlanage years whomost often presented with a palpable, occasionally painful mass with a medlan diameter of cm exclson ( ) or smple mastectomy(l ) werethemorefrequentformsof therapy. localrecurrenceswereexperlenced by gpatlentswhose pnmary tumours although classified as benign contained areas of stromal overgrowth and specnaltsed stroma: all had been primarily treated by a histologically inadequate local e~~i~i~n~r~~b c~t a n e o~~ mastectomy (l).these local recunenceswere successfully controlled by local surgery ( )and mastectomy( ) mf~vemal~gnant tumours treated by mastectomy, none has recurred locally or developed metastases. flve mcompletely emsed turnours whlch showed neither evidence of stromal overgrowth nor speciallsed stroma remaln disease free at years. it s concluded that histologically malignant phyllodes tumoun are unllkely to develop distant dlsease i treated appropriately andthatstromal overgrowth may indicatethe possibilityof recurrence ~n incompietelyexcisedbenign lesions. several recent studies have evaluated the incidence in primary breast carcinoma of "micrornetastases" ~n multiple sections of axillary lymph nodes. howbyb~, the significance of these findings ~n terms of prognosis and sunw remainsunclear. inanattempt toelaborateon this, wereviewedthecasesof patlentsdlagnosedas havlng'bodenegative'' breast carcinomaon routine histologyand in whom therewasafollow-upperladof at least fwe years. the anginal diagnostic h e slides were reviewed in conjunction with eptthellal immunohistochemical pr-aratlons usmgcea,ema. hmfg, andkeratin, andappropriateclinicalfollow-updatawasaccumulated.thestudycasesfell into two categories, those which retained a 'node-negatlve' status after revew, including immunohistachemical appraisal ( %). and those which demonstrated individual or grouped malignant epithelial cells ( %) ~n the peripheral or deeperainuses of the lymph node. of the former group. all pattents but one werealive with noevidence of recurrent breast carcinoma five yean after mastectomy. of the second cohort. posnttve far "micrometastases". % showed nodiseaserecurrence. while % had gross metatatic d i s e a s e o r h a d w c c~m b t o thecarcmoma within hefiveyearpenod.thefindingsdescnbed.aithough basedon low patient numbenln thisprelimmarysludy. indicate the appropriateness of a large multicentre sunw to evaluate completely the prognostic significance of these '"msrometastases". sadhna bhatnagar. j. mcclure. d a. kalbhen the injection of sodium iodoacetate ( . mg) into the avian knee joint on two consecutive days induces changes which macmscopically and radiologically resemble osteoarthrosis. knee-pnts from groups of six hens were studied atregularinterval~upt weeksalterinjection by histological and histochemlcel methods. characteristlcles~ons were identified and assessadon asemi-quantiative basisand indwdual ision profilesacrossthetime course of the expenment were constructed. during the first five weeksthe obvious changes wwe chondrocyte loss and depleted matrix staining. chondrone formation was also noted. fmm six weeks there was also tibravascular proliferation at the chondro-asseousiunction wim modelling activity and new bone fwmation. in addition near the insenion of ligaments there was chondrocyte differentiation and proliferation. the amount of cartilage fibrillation and flsswng increase during the expenment aithough there were so areas of apparent resurfacing. the decreased matrlx staining noted early in the expenmen stabilised and many lacunar spaces are occupied by chondmcytes towards the end. subchondral bone hyperplasia and osteaphyte formation are prominent persisting features. such sequentialcharacterisation may be usefulin a-ssinglhee(fectsofput=~~vetherapeuticcompounds onthe model. rodentachilles tendonsweiesubiected to midpointtenotomy and allowed to recover for various timesin sllu before the operaled tissue was mmved, placed into a millipore dinusion chamber and insened inhaprntoneally into syngeneic hosts. diffusion chambers were removed at weekly intervals such that the tom time post-operation :i.e. time sru plus time wnhin the diffusion chamber) was up to weeks. histological examination demonstrated that ectopiccartilage wasprodwed withinthediffusion chamber byatotal of weeks but only if thefirst weeks were recovery in situ. wnh increasing time. calcified cartilage, mtteoid and pnmary spongmsa bone was obseived evtdence suggests that the camlage forms via direct tendon metaplasia and that the bone may form as a result of dinerentialive changes within hypertrophic chondrocytes. tlbla and fibula appearto be directly exposed to tha surface of the medial femoral condyle and inner ridge of the lateral femoral condyle. the exposed load-bearing area of the femur and tibia contain demonstrable amyloid around chondrocytes and in the matrix below the surface. these areas are also covered with the densest hyalmd fibro-cartilage. amyloid p r e e u~r s from h e synovlum may penehatethe surface ofthe cartilage under pressure or precursm from cartilage matrix or chondrocytes may be converted into amyloid and are napped below the surface. the inpction of sodium todoacetate into avian knee iolnts is followed by the development of an osteoarthrotic pmess to studytheeftectal sodium lodoacetateon chondrocytemetabalism m wire, chickstmal chondrocytes grown i~th~ee-dimensionalcollagen gels wereexpmedtothe compound. sterna from chick embryos(l - days in om) were digested with collagenase releasing the chondrocytes which were then cultured in collagen gels. w i u m iodoacetate was added to -day old cultures at concentrations of . . and wg/rnl. ( , . and .o y lodm)tor hours. theeftectswere assessed lor up to days by: ) staining wnhfluorescein diacetate which pmducesfiuorescenceiniive. metabolisingceilsonly. )thelevelofproteoglycansinthemedium wasestimated by complexing with dimethylmethylene blue and measuring the increase in absorbance at nm. ) histologycultures were fixed in glutaraldehyde, embedded in paraffin, sectioned. and starned wm alcoholic toluidine blue. alcaan blueandpicmsinusred.treatmentwlthiodoacetateproducedadoserelateddecreaseincellnumbersand intheamount of proteoglycmsreleased into themedium (lessthan % at pg/ml).the histolagyshawed thatthe treated cells were smaller and produced less matrix. these results show that sadium iodoacetate reduces cell number and interferes wqth the manufacture of matrix components. instftute of pathology, ro i vcfona hosprtal, belfast. l. irelend theoblectivesof this study were tocorrelatecertain clmcal features of ependymoma. (i e. age, sex, anatomcai slte. turnour size. meningeal dissemination and raised intracranial pressure with certain pathological features. . e dna content, mnotic rate. immunocytochsmistry and ultrastwctural features) with prognoss a total cases was available for clinical pathological assessment univewty of liverpool, p box , liverpooi bx and 'sari andres unrvemty it is not surprising that wmal exposure to high altitude causes increased activity of the hypothalamic-pltultaryadrenocortical axis. but fflidence suggests that continued exposure to hypoxla leads it to perstst long after homeostas sis restored and acclimatization achieved their pltuatary glands were not sgnlflcantly different insizefromthoseof thesea-levelsubpcts, but c~ntainedlargerpopulat~onsofcort~cotrophs~n termsof the total population of their anterior lobes ( . vs . %: p < ) these fmdlngs are in keeping with those of previous studies of the hypothalamic-pituitary-=d~=~~cottical axis in man and animais exposed to prolonged hypoxia at natural high altitude or in the laboratory. they suggest that greater amounts of acth might be necessary to sus ain normal adrenocoltid function under such circumstances and that adrenocortical hyperplasla bone marrow wnsists of a haamopolstic system whch is nn intimate association with aftbrous stramal network.adun mammalian bonemanowstromais knownto have both chondmgenicand ostecgeniccapabilitiesinvlvoand stromal tissue has been shown to tom fibroblastic wlonies capable of giving nse to bone in vitro. however, the mqbility of manow stroma to form cartilage in who has not been addressed when grown in identical conditions to mammalian stroma. mechanically dissociated single cell suspensions of chick embryo bane manow hom > days in ovo and from day hatchlings readily lorm bath fibroblastic and cartilage colonies in vitro. cells within the cartilage colonies are polygonai in morphology, are separated by a relradile exhaceiiuiar matrix and svnthesise catilage specific pmeogiycans and collagens. as shown histochemicelly, biochemically and immunocytochemically. in contrast, adult chicken bone manow does not form cartilage. instead, the cells appear osteoblast-like and synthesise coilagens typical of bone. a. m. fianagan t. j. chambers &wtmment of h~slopatholosy. sl george's hosptal medlcel sch-i, london. sw ore osteaclastshavebeensuccessfullygeneratedinculturesofmurinehaemopoisticcells. itwouldbeuseful ifasmila, model were available to analyse the mechanisms of regulatlm of human mteaclast formaim ir normal and pathological states. although the osteoclast is present in reverai neoplastic conditions. it is not known whether it forms part of other haemowietic malignancies, lor example, polycythaemia rubra "era or chrmic lymphatic leuhemia. we used strategies based on our experience with murine osteoclastogenesis. however, we have been unabletogeneratefunctional human osteoclastscapableoiresorbingbone m vrtro. large numbersofmultinucleate cells developed in these cultures mese cells did not show a typical panem of reactivity with asteociast-specific manoclonal antibodies, nor did they bind ' sct but rath@r possessed an antigenic profile characteristic of macmphage polykaryons. it is pecullarthat human tissue fails to support osteoclast generation stnce cells of the other haemopoietic lineages were consistently generated in our cuitures. in murine cunuras it is known that a particular shomal cell type is requtred for osteoclastagen@sis; it is possible therefore that this cell population is spamin adult humantissue.miswouldreflecttheiownumberofosteoc asts presentinhuman adults comparedto mice elucidation of conditionssuitableforthe generation mthehuman osleoclastm wtrow~ll heldus understand the mechanisms by which it is regulated in health and disease spaceof one year. the other case s b middie-agedwoman who &=being affected bythe disease far sevaral years developed separate sarcomas in each lower limb dunng a nine month pwiod. each tumour was treated bv pamal amputation but she died from widespread metastases within one year of the first amputationthe distribution of proline- -hydroxylase in a range of human tissues shown by a monoclonal antibody, fib s. smlth, p. revell aepariment of mobd anatomy and bone and h m f research unrt, the london hospw medcal cdl€ge, london. e l fib . a commercially available monoclonal antibady to the b subunit at the enzyme proiine- -hydroxyla~ invalved~ntheproductionofcallagen, wasappl'edtaacetonebxedcryostat sect~onsofawiderangeoftissues. in hver, hspatocytesshowedvarying degrsesof labelling with the antibody, along with spindle shapedcells(sscs) in associated connective tissue in both tonsil and lymph node. sscs in the connective tissue were melled with the antibody along with a number of other cells (lymphocytes) within the germinal centres. in addition to this. perivascularcellsand cells intheconnectivetissue iayerimmediatelybelowtheepithelium also labeliedin sections of tonsil. in skin, very few cells in the dermis were positive for pm ne- -hydroxylase. as were also only a small numbetof epidermal cells. afew chondrocyteswere marked bythisantibody in artlcularcartilageand intervertebral disc,along withswneostwcytesin bone.manycellsinthepleuraandtheepimysiumof normal~keletalmusclew~e posotive. there was no staining at all in specimens of kidney. in addition to these 'normar tissues, examples of a seminoma, a breast carcinoma and a thymoma were examined. each of the turnours showed fib labeliiw of ssc~withinthestromalt~ssue.theseresultsshowthat fib b sele~tivelylabelssscs.whlcharapresumedto be f brobiasts. in a wide ranged both normal and pathological tissues. which produce and maintaln the collagen matnx. proime- -hydoxylase is an enzyme involved in the pmduction of collagen and demonshahle in the rer of fibroblasts. it may therefore be cmsldered as a possible matker for fibroblasts in tissue sections. we have applied the mouse moncclonal antlbdy fib , raised against the $ subunn of prol ne- -hydroxylase. to alcohol fixed paramn embeddedsections and acetonefixedcryostat sect~onsolnormal (n = ) and meumatoid (n = ) synovia. synoviocytes labelled very strongly, but the distribution vaned between samples. insome, mostsynoviocytes weremahed, whilein others ~nlythedeepiayerofcells wasmitive. it isnot known whether thts dillerence ~n prdine- -hydroxylase expression is due to the severity and duration of disease. drug treatment or other factors. it is of interest, however, that fib labelling was not wnflned to the type b (fibroblast-llke) synoviocytes and that the more supficlal type a synoviocytes also contained proline- hydroxylase. spindleshapsdeells i" thesublntlmaleonnectiva tissue were labelledma vanablemanner. inaddltlm to these findings for fibroblastic cells. a small cap of fib posnive cells was seen around lymphoidfollicles. sometimes polansed towards the synovial surface. this enzyme expression by lymphocytes is under further invsstlgatlo". vitronectin s an adhesive glycoprotein which shares several functional similarities with fibronectin. it is a major component of extracellular matnx and plays a role in cell-matrix mteradians, monocyte function. and the coagulation and wmplement systems. we have employed a monoclonal antibody to assess the distribution of vironectin in frozen synovial biopsies from patients with rheumatoid arthntis, ostwnhntis. ankylosing spmdyiitis andtraumatic non-inflammato~ control^ ( cases). lmmunoreactive vltronectin was identified in the synovium. similarto the panem of immunoreactiyefibronectin, itwas located in adensefibrillarpanem surrounding cellsof the synovial lining layer. vitronectin was also associated with fibres throughoutthe sub-intimal connective tissues. being most prolific in fibrotic areas. vltronectin was identified in the sub-endothellal layen of blood vessels. but unlike fibronecttn was not associated with basement membranes. similar distributlon patterns wme observed in all biopsies studied thelocalisation ofvitronectitin in synovial tissues suggestsapossibleroie inanachmmtofcellsto the extracellular matrix and that may be important in the pathophysiology of inflammation and repair manolayercultured articularchondrocytesare known to rapidly losethelrexpressionof collagen typeil.the purpose of this study was to compare the expression of various collagen types in twoand three-dimensional cultures articular chandracytes. in addition. the expression of s- w protein and its alpha and beta subunits was studied in monolayer cui ures. bavine articular chondrocytes were isolated by collagenase digestion from ankle joints and cuhured in monolayerandspheroid culturesin dmemwim % fcs lmmunocytochemical studieswere performed using the indirect peroxidase technique an monolayers affer methanollethanol ( vlv) fixation and on frozen sections of spheroids. the reaction was scored semi-quantitatively as negative (-), weakly (+) moderately (++) ontha basisofthenumbersafcasesrefsiredtoustottheidentificalionofjointcry~talsintissuesectians, webelieve that calcium pyraphosphatedihydrate(cppd) deposnion disease isbeing underdiagnosed byhistopathologists. a crltical light microscopic reviewof cases. all with thediagnosisconfimed bymicroanalysis(energydispersivexrayspectroscopyln thescanning electron microscope, infrared spectroscapywboth) revealed adistinctwefeathery or br"shlikeappearanceinal suchdeposits.thisfeaturewasapparentat lowpower, whileconvincingvisualisation of crystalswithinthedepositswasdiff~cuneven withanoil immersionobiective.thesignof birehingeneeof cppd crystals is more dificult to demonnrate i" tissue sections than synoviai fluids. due to iactois such as stain. fragmentation and heaping up of crystals, buf it can be more readily assessed in unstained sections or following microincineration of the section. in six of our c a m the deposits were exclusively within bone: demonstration depended on relatiyeunderdecalcification: deposits in this position have not previously been recognised. this study thus provides new information an the histological identdication of cppd deposns in tisues and implies heterogeneity in the pathogenesis of such deposits. in human knee osteoarthrosis (oa) overt unicornpartmental disease is frequently accompanied by a macroscopically normal second cornpartmsm. however. there are a number of light micmscopical changes within the latter which might amount to an "early osteoarthrosis". these are chondrocyte pmliferation and chondrone formation. decreased proteoglycan staining and disruption of the chandra-osseous interface with duplication of the tidemark and resorption ofthe cartilage by chondmclasts. tidemark duplication is also a feature of established oa re~resenti~gadistu~ancedfcabif~cat~oninrone ofarticularcartilage.slnceslwp~~t~i~~~~ knowncalciumion transponmoleculs.altsrationm~~sstainingpanernand~ntens~tym~grn beanlndexotearlyo~. sl~~protemstamng was studied in the tiblai plateau cartdage of cases: with unicornpartmental oa. with bicompartmental oa and controls. lntheianerall chondrocytesgavepositivemembrana and cytoplasmic staining (especially in zones e n d ) , d~u s e w e a k m a t~~c~l s t~~~~~g~~~~~e l , s t~o~g p a r i c e l l~i a~a~d~~t~~-t~~t o~~l s t~i~~~g~~~o~~s a~d . in overt and early oa the orderly malrlcal panem was disrupted with prominent patchy staining around chondrocyte clusters. the significance of the change in the matncal distribution ~n oa is unknown and unlikely lo be related to a disturbance of calcification in zone . road, london. wcl m e histogenesis at alveolar son part sarcoma (asps) is still unsenled: nonchromaffin paraganglioma. malignant granular cell myoblastoma, neuml tumour, myogenic tumour. habdomyosarcoma and renin-producing tumoui theories have been proposed lmmunohistochemical studies have yielded discrepant results we recentlyobsewed that the diastase resistant crystalline material in asps was strikingly reactive for vimentin by the immunoperoxidase method on paraffh sections one case. we then studied paraffin sections of seven other cases from our files between and ~ by routine light microscopy, immunohistochemistry and electron microscopy all eight cases were reactlve for vimentin. which highlighted the crystalline materlal as in the first case. vanabie numbers of immunoreactlve cells for desmin were found in three. and smooth muscle specific actin in four. all cases showed some reactivity for neuron specific enolase. and seven of the eight cases reacted for s- protein. the clinical and pathological lea lure^ of cases of synoviai sarcoma have been reviewed. these tumours are aggressive with a poor prognosis. typically affecting young aduns with a long history before presentation and detinltive diagnosis. a high index of suspicion is required if the diagnosis is not to be missed several features, including age, size. site. histological type (monophasicibiphasic), mitotic actlvity and necms~s have been investigated withaview toestablishing possible prognosticindicators. the bestguideto prognosisis assessment mitotic actlvity. size and histological type dld not affen the outcome. in this study the effecl of subcapsular orchidectomy on skeletal metastases from prostatic carcinoma was studied using bone histomorphomatnc paramsten. twenty-eight patients with bone metastases were studied immediately before and for seven months after orchidectomy. tetracycline labelled bone biopsies were taken from metastases and turnour free areas at the beginning and end of study. sixteen of the patients also underwent o~teocla~t inhibition tor six months using disodium pamidronate ( mg i.v. weekly for weeks then anemale weekly for five months) to enable differentiation of the skeletal response to castration. histamarphametnc anslysi~ tumour frse bonerevealed adrapin overall bonevolume. histological analysisofmetastasesshawedadecrease m osteoblastic activity but widespread osteoclast mediated ostealysis. tumour regression and manow recolonisation were present in most c~s b s but malignant foci remained in % of repeat metastatic biopsies, inducing a typical. lacalised disruption of bone metabolism. it is concluded that orchidectomy causes ostmblastic regression blrt induces increased ostboclast mediated bone destruction, which is most pronounced within metastases. although tumour regression and marrow recolonisation usually occur within metastases, active turnour foci anen perstsf. ( ) chairman s. g. silverberg, washington theeffectsafoncogeneson thedeveloping nelvoussystem havebeenstudiedinaneuraltransplant system ~ntats, taking advantage of the extraordinary capacity of fetal cns to differentiate in and fully integrate with, the adult host brain. gene transfer tnto tetal brain cells was mediated by in viiro infection with replication-defsctwe retroviral vectors. fetal rat brain suspensions were then stermtaxically injected into the caudoputamen of adult f rats.animals carrying transplants exposed in vim lo the polyoma medium tantigen developed endothellal hemangiomas in the graft which often led to fatal cerebral hemorrhage within - days alter transplantatm lntroductm of the viral sic gene caused astrocytic and mesenchymai tumors affer latency periods of months.following infection of fetal donor cells with a vector encoding the v-myc oncogene led to the development of only a single embryonal cns tumor whereas exposure to v-ha-ras and v-myc resulted in the rapbd induction of multiple malignant neoplasms. when injected intracerebrallyinto newborn ratsin vivo, complementation of theseongogenes led the development of malignant hemangioendotheliomas, undinerentiated neural tumors andlor leukmia.oncogene transferthus constitutes a challenging new model to assess the effects of translorm~ng genes on the developing nervous system. since the presentahon of molecular genetic evidence supporting the knudson hypothesis m the eariy s there hasbeen increasing interestinthe ideathatasimilartwo-hit mechanism mayaperateln sporadadlccancers therens now evidencbthatlassofonealleleandretentionottheother~nmutated/wildfarmaccuninsparad~ccalono. lung. renal and breast cancers in breast cancer lossof heterorygoslty(l h) has been demanstratedfarseveral alleleson chromosomes l p q and an the short arm of chromosome . loh has also been demonstrated on other chromosomes thegeneral viewisthat loh indicatesthatasuppressorgene may bepresentattheseloct. hawevet in interpreting lohthere sdifficulty indetermining when loh becomessignificant because itsincidencevariesfrom % / depending on the locus examined. in this presentation, knowledge about loh on chromosome p (and q where a new deletion has been recently discovered), and p will be presented and the pathqlenetic significanceand ciinicsl relevanceexamined. lnthe lattercontext anewrapid methodfordetemlnmg alleljc dosage will a l x be described which does not requlre rflp analysis. expelt systems - .e. computer software that can function as cansuhant, decision support system, or process contr~llerhas undergone a rapid development durlng the past decade. applications to histopathology have gnaw some ,ocqdiffuse maiignantmesotheliomas( mm) occurannuailymn. americaat thepresenttime.cumulat~~ely, substantial numbersot lesser-knownfarms of mesothelia turnourare also seen, including serous paptllary tumoun of the peritoneum, well differentiatedpapillarymesothellomas and benign cystic mesotheliomas, but incidencedata are not available.the relativeiyfrequent atypical reactive hyperplmasof w o s a membrane add further vanstytothe range of mesothellai lesions which may present diagnostic problems. the expenence of a u.s.-canadian mesotheliomapanel suggests that dmm vs. metastatic carcinomacontinues to bethe commonest mesatheliamarelateddiagnostieehaliengeandthatthedifficunyandalwayssolved bytheappiicationofspecialstains almostas common are le~ions in which the ditferential diagnosiscenters on dmm us. a reactive process (atypicai mesotheliai hyperplasia, fibrous pleurisy) within the laner group it is particulady difflcun lo obtain a strong consensus opinion, andfoilaw-uphasconfirmedthemaiorityopinioninonly %ofcases. sarcomatousdmmvs.sarcomanosisaiso a notable area of difficuny. most mesothelial lesions tn north amenca. including dmm. are reviewed within a short time of biopsy by a pathologist in a major teaching centre, only a small number ( - %) being subpct to further scrutiny by a panel or ~n the coune of epidemiological or clinical studies however, many cases resurface later ~n a legal sening when the material and clinl~al information s often available. the original diagnosis of the hosoital pathologist s usually endaned by the courts. sharon w. weiss llposarcoma is one of the most common forms of son tissue sarcoma and may be divided into several subwes well-differentiated. myxoid. round cali, phmwpha, and dedifferentiated. this presentation will discuss the ) diagnosticcnteriaof lipblasts ) d agnosisand behaviorof myxoidround cell iiposarcoma andthe ) behavior and incidence of "dedifferentiation" dwell-differentiated ihposarcoma. the diagnosis of lipsarcoma depends in part on the identdication of lipoblasts or pnmitive fat ceils. since iipoblast-l!ke cdls may be seen m a variety of conditions apart from iiposarcoma, strict diagnostic c!i tma must be applied in identlfving these ceiis. these cells contain a hyperchromatic. indented or scalloped, ecmtnc nucleus set in a cytoplasm containing one or more lipid-nch vacudes. these cells must also occur in an appropriate histaiqic background. vanous lesions which may contain ihpoblast-like cells and which may. therefore, mimic liposarcoma include fat necrosis, fat atrophy, silicone granuloma, signet nng carcinomas or melanomas, and a variety of malignant tumors with fixation artifact. myxold androundcell lipsarcomarepresentthemost commanfarmofsarcomaaccuningmearlytamidadunlifeandare commoniy located #n the region of the mlgh and popitteal fossa. although the designation of "myxold and "raund cely suggest two separateturnortypes. they represnt ends d a common h~stologlcspbctnm. myxaid liposeicoma represents the well differentiated end of the spenrum. whereas round cell represents the pcarly differentiated end.however, transitional or mixed forms ex~st. accounting for confusion as to how such tumors should be classlfled mymd liposarcomas are characterized by nodules of bland stellate or rounded cells set n hyaiuronic aod-rich strama. an intricate plexiform vasculature and numerous lipblasts are easily vdentaed. wlth progression to round celliiposarcomathecellsbecomelarger,moreatypicalandthestromalessmyxaid. lipoblastsaremoredlfticultto find and a plexiform vasculature s less apparent. recent work by evans suggests that evaluatlon of myxoidlfound cell ihpasarcomashould includethe percentage of round cell component wdhin atumorsincethisdirectclh/ mtluences prognosis. our policy is to carefully sampk such tmors, submining t section for each centimeter in greatest diameter of the tumor. a rough estimate of the percentage of a round cell component s made. we w a r d tumors havinglessthan faroundcellcomponentasgradei.thosehav,ngbetween & % roundcelicomdonent are considered grade . whereas those with more than % are considered grade . well dmerentiated liposarcoma is one of the most common sarcomas accuning m late adult life. characteristically affecting the deep musclesaftheextremities, theretropentonealspace. and the groin, this tumor ischaractenzed by varying amounts of mature fat interspersed with fibrous bands, atypical hyperchromaric spmdledceiis, and lipoblasts. these lesions are considered low grade sarcomas having a high rate of local recurrence but no abilny to metastaslze. recently evans and ammi eial suggested that well-differentiated liposarcomas occurring m the wbcutaneous tissue and muxles of the extremity cause so hnle morbidity that the use of the term "atypical lipma" should be used rather than ihposarcoma. in contrast. well-differentiated liposarcomas in the retropentoneum cause significant morbidihl and pose a significant risk of death from local disease. thus. nearly all pathologists agree that the term "weii differentiated lipasarcoma" should be retained for these tumors ~n the retroperitoneum. however. none of these recent studies have followed a large number of these tumors for a proianged period at time m order to assess the long term behavior and specihcalty risk that such es ons may progresswith time to a higher grade lesions ( . dedifferentiate). we have recently completed a follow-up study of cases of well ditferentiated iiposarcoma accurringmthemusc~oftheextremity,retropntoneum,andgroin. inalllmatlonsthenskof local recurrence was high. however,dedifferentiationoccurred~n % of casesandwasnotrestnnedtotumors~nanyparticularlacatlon. butcouldbe bestconelatedwiththedurationofthetumor. withmostcasesoccun~ngaflerl mmoreyean thus. dedifferentiation is not a site-dependent phenomenon, as has previously been suggested. but rather a time dependentphenomenon.anhough thesedatado notneee~~arily indicatethatwe should abandon theterm"atwca itpoma" they do indicate the need tor proianged foilow-up of patients wlth wei&-dlfferent,ated ddosarcoma and the small but deflnne risk of dedifferentiation as a long term complication of the disease. key: cord- -kjuc nqa authors: asiamah, nestor; opuni, frank frimpong; mends-brew, edwin; mensah, samuel worlanyo; mensah, henry kofi; quansah, fidelis title: short-term changes in behaviors resulting from covid- -related social isolation and their influences on mental health in ghana date: - - journal: community ment health j doi: . /s - - - sha: doc_id: cord_uid: kjuc nqa this study assessed the behavioral outcomes of coronavirus (covid- ) social distancing protocols and their influences on mental health. an online survey hosted by survey monkey was utilized to collect data from residents of three ghanaian cities of accra, kumasi and tamale. a total of surveys were analyzed, with a sensitivity analysis utilized to select covariates for the regression model. the average age of participants was about years. findings indicate that reduced physical activity time and a change in sexual activity and smoking frequency are some short-term changes in behavior resulting from social isolation during the lockdown. an increase in sedentary behavior had a negative influence on mental health. for the most part, changes in behaviors in the short-term were associated with lower mental health scores. the study implied that covid- social distancing measures should be implemented alongside public education for discouraging unhealthy changes in behaviors. coronavirus (covid- ) is a novel virus that was first detected in wuhan city, china (lewnard and lo ; lin et al. ) . over a period of < months (between december and april ), the virus grew from being a local epidemic in wuhan to a fear-inspiring global pandemic. as of september , , the virus had killed , out of , , people who tested positive for it (johns hopkins university ), making it one of the world's deadliest pandemics (lin et al. ; pung et al. ) . covid- can be considered a highly contagious virus not only because it has infected thousands of people but also because it has taken a relatively short time to spread to most regions of the world. without appropriate measures to contain it therefore, covid- could infect a third of the world in a year. as the case has been with previous pandemics, the nonavailability of a vaccine has allowed covid- to spread and grow into a global pandemic. since the testing and production of a potential vaccine for an infectious disease takes an average period of months (josefsberg and buckland ; anderson et al. ) , measures to cut the chains of infection are the only way to contain covid- in the short-term. one of such measures, which is considered the best way to contain an epidemic or pandemic in the absence of a vaccine (lin et al. ; pung et al. ) , is the enforcement of social distancing protocols in affected regions. with covid- , the adoption of this approach is a global shared goal that has brought about a complete or partial lockdown of affected countries. as demonstrated by china with its lockdown of wuhan, social distancing protocols are the ultimate weapon for fighting covid- in the short-term. this notwithstanding, their socio-economic impacts can be dire (brooks et al. ; armitage and nellums ) . economic impacts such as unemployment, the weakening of the foundation of the global economy characterized by the united states (us) and china, and a potential collapse of emerging economies, at least in the short-term, are frequently reported economic consequences of covid- (anderson et al. ; lewnard and lo ) . other consequences that are significant but seem underreported (lewnard and lo ; armitage and nellums ) are social implications such as public health decline that exacerbate the above economic losses. social distancing mechanisms, for example, could curtail individual physical activity (pa) trajectories. it is also possible that social distancing will limit access to food and public services, especially in developing countries where citizens may be unable to afford basic needs such as food if socially isolated. we argue based on the fogg behavior model (fbm) proposed by fogg ( ) that a pandemic such as covid- and its sudden lockdowns are extreme events that would cause fear and panic as people try to cope with them. per the disengagement theory of aging developed by cumming and henry ( ) , anxiety and mental health struggles may result from a sudden lockdown because social disengagement is a gradual process that would overwhelm people who try to achieve it instantly or in the short-term. we are, therefore, of the view that social isolation necessitated by a covid- -related lockdown would not only cause fear and panic in the short-term but could also lead to anxiety and consequently a decline in mental health in the general population. similarly, short-term social isolation can cause major changes in health behaviors that can increase the burden of disease and disability. this argument is corroborated by some researchers (malcolm et al. ; armitage and nellums ) who have opined that a decline in mental health is the most likely consequence of social isolation caused by an unexpected event such as the outbreak of a disease. if so, stakeholders need to understand how significant changes in behaviors and their influences on mental health are and roll out suitable programs for avoiding or at least reducing public health risks that could be predicted by a covid- lockdown. with some predicting the outbreak of a similar epidemic in future (lewnard and lo ; li and siegrist ) and others seeing the possibility of covid- spreading for a long time (li and siegrist ; pung et al. ) , stakeholders need to understand changes in behaviors that could result from a lockdown as a precursor to designing appropriate programs for discouraging unhealthy changes in behaviors. possible covid- -related changes in behaviors have been acknowledged in the literature. the most frequently acknowledged changes are reduced pa and increased sedentary behavior due to limited access to the built environment and community services during the lockdown ). on the other hand, the ability of exercise service providers to promptly move exercise classes online in response to social distancing measures have been reported jakobsson et al. ). as such, many individuals could exercise at home during the lockdown. conspiracy theories have also indicated that alcohol intake, smoking, and the use of some substances (e.g. garlic) can protect the individual against covid- . particularly in less educated populations, therefore, many individuals may take to substance use and smoking. because families including couples may spend more time together at home during the lockdown (fisher et al. ) , sexual activity and domestic violence are also likely to increase due to social distancing measures. this study aimed to examine these changes and their influences on mental health. we understand that the aforesaid changes can be affected by demographic and individual characteristics. education and income, for example, are likely to affect one's ability to utilize online exercise classes during the lockdown. for this reason, we adjusted for key covariates in testing the association between the said behavioral changes and mental health. our choice of mental health as an outcome variable draws on commentaries indicating that mental health is the aspect of health most likely to be affected in the short-term by social distancing protocols (serafini et al. ; vindegaard and eriksen benros ) . our investigation was based on this primary research question: do changes in behaviors due to covid- social distancing measures have a significant influence on mental health? this study employed the descriptive correlational approach and online surveys targeting the general population. a cross-sectional analysis technique was adopted. the exclusive use of an online survey was the only way to collect data during the lockdown. as this study was aimed at informing policy decisions for a specific region, the setting of this study was three cities (i.e. greater accra, kumasi, and tamale) affected by a covid- mandatory lockdown in ghana. the study population was individuals of the general population, preferably those aged years or more, who were socially isolated as they complied with the mandatory lockdown. participants were selected based on four inclusion criteria: ( ) currently living in any of the cities facing mandatory lockdown; ( ) having acquired at least a basic education instructed in english, the medium in which the survey was administered; ( ) being in social isolation owing to the lockdown; and ( ) willingness to participate in the study. the use of a powered sample (i.e. a sample determined based on a pre-determined statistical power and effect size) was not possible in this study for a couple of reasons. firstly, we did not find any existing study that was based on our context. secondly, we could not have used information from previous research to calculate a sample size because all existing studies applied substantially different methods. a deep look into the literature suggested that related webbased studies had utilized sample sizes ranging between n = and n = to reach credible findings (merolli et al. ; balhara and verma ; liang et al. ) . considering our research approach and the geographical scope of the study setting, we hoped to achieve a sample size between and . the survey was developed by the researchers and hosted on survey monkey, a free survey creation platform that allows data sharing and analysis between research team members. it was chosen because of the researchers' ample experience with it and the fact that it provides user-friendly data transfer and analysis tools. the survey was developed from scratch, as opposed to using a template, because no existing template was suited for our study. the survey comprised multiple-choice questions and a question introducing the mental health measure. the first question included the ethics statement and instructions for completing the survey. the next two questions (i.e. q and q ) screened for individuals who did not meet the inclusion criteria. questions - and captured demographic variables and covariates. changes in behaviors were measured with questions - as well as - . question presented the -item mental scale measure. the 'one question per page' design option that comes with the most legible text (regmi et al. ) was chosen. the survey was developed after the researchers discussed with two groups on what could be the ideal measures of mental health and changes in behaviors in the context of the study. the first group, which included four of the authors, was a whatsapp-based group made up of research fellows of a center of excellence. members, through the use of text messages and audio recordings, suggested potential measures for the study. over skype, the researchers then consulted with the second group, comprising two psychometricians and a statistician, to agree on an initial list of items for the survey. the lead researcher then developed a questionnaire of the items proposed. following this, copies of the questionnaire in sealed envelopes were sent through a private courier to individuals aged years or more who had agreed to complete it in the neighborhood of the lead researcher. this step was part of the survey piloting arrangement. over days, questionnaires were completed and returned by out of the participants through the courier. respondents commented on ambiguities and wording problems associated with the questionnaire. through a voice call, the lead researcher contacted the participants to confirm and better understand the issues reported, enabling the researchers to further improve the wording of the items. a major change made to the instrument was replacing the word 'self-isolation' with 'social isolation' in most of the measures. an online survey of the final items (including an ethics statement) was then developed and piloted online with different participants (whatsapp = ; facebook = ; twitter = ). with no issues identified in the second pilot study, we sent the survey back to the two psychometricians consulted earlier for approval. this study focused on possible short-term changes in behaviors resulting from covid- -related social isolation or fears. changes in smoking frequency, alcohol intake, and substance dependence were incorporated into the study owing to fake news about the possibility of smoking and the dependence on some substances (e.g. garlic, alcohol, marijuana) protecting against covid- . the other changes in behaviors were considered because they could be encouraged by social isolation. table shows a summary of all changes in behaviors and their operationalization. as table indicates, categorical variables were dummy-coded and one of their levels (categories) set as the reference. the table also shows underlying health conditions and demographic variables that, per existing studies (sederer ; lund et al. ) , can confound the primary relationships of interest. mental health was measured with a -item standard scale (with descriptive anchors strongly disagree- ; disagree- ; the individual's gross monthly income continuous --somewhat agree- ; agree- ; and strongly agree- ) from lukat et al. ( ) . this tool is a unidimensional scale that produced satisfactory psychometric properties (including a cronbach's α coefficient = . ) on a sample representing the general population. it was preferred to other mental health measures because it has been properly validated for the general population and is the most holistic mental health measure (lukat et al. ). in the current study, it produced a satisfactory cronbach's α coefficient of . . scores on the mental health measure were generated in harmony with the lukat and colleagues; items were 'parceled' by adding them up. appendix table shows items of the mental health measure used. this study received ethical clearance from an institutional ethics review committee (# -ace) after the research protocol and ethical statement were reviewed by the committee. in agreement with best practices, we ensured that the first question of the survey presented the ethical statement (merolli et al. ; balhara and verma ) , which means that only individuals who agreed to participate voluntarily (by ticking 'yes') completed the survey. the ethical statement indicated the purpose and importance of the study as well as the risk-free nature of our data collection process. the inclusion criteria and instructions for completing the survey were also presented as aspects of the ethical statement. we created different versions of the survey that could easily be completed on all social media platforms including whatsapp. we published the survey a week after the lockdown by sending a link of it to all our contacts using whatsapp and asking them to complete the questionnaire and share it with their contacts. thus, snowball selection was applied to distribute the survey. subsequently, the researchers, through their personal accounts, published the link on facebook, twitter, linkedin and other social media platforms. the shared link took the participant to a pop-up questionnaire that could be completed even with a relatively weak internet network. participants did not have to download the survey before completing it. the survey was distributed and completed over about weeks (april - , ) and was closed on april , . its average completion time was about min. we programed the survey at survey monkey to prevent multiple responses from the same participant. for further research purposes, we designed the survey to allow individuals outside the study setting to respond. we did not provide incentives for participation. data in a microsoft (ms) excel format were downloaded from survey monkey. coding was done in ms excel and the resulting data transported to spss version (ibm inc., ny, usa), which was used for data analysis. descriptive statistics (frequency and per cent) were used to summarize the data after five questionnaires with missing items were discarded in line with the recommendation of garson ( ) . the shapiro-wilk's test was performed to screen for outliers and confirm normality of data of mental health (garson ) . this test on the data confirmed normality (p = . ) and the absence of outliers. pearson's correlation test was then used to assess bivariate correlations between the variables. a multiple linear regression model was fitted to assess the influence of the changes in behaviors on mental health, with potential confounding variables adjusted for. before fitting the regression model, a sensitivity analysis was conducted to screen for relevant potential confounding variables in harmony with the procedure adopted elsewhere (rothman and greenland ; rezai et al. ) . in this analysis, univariate regression models were used to estimate crude coefficients (i.e. standardized and unstandardized coefficients and their % confidence intervals) indicating the influence of the covariates and changes in behaviors on mental health. covariates with p > . were removed and those with p ≤ . were kept for the second level of the sensitivity analysis. at this stage, only chronic disease status (cds) was removed. at the second level, multiple linear regression models were fitted to estimate coefficients (including their % confidence intervals) representing the influences of changes in behavior and each of the remaining covariates on mental health. any covariate that led to a % change (decrease or increase) in the coefficients of the behaviors from the first level was kept and incorporated into the final regression model (see table ) as a covariate. at this stage, age and income were removed. we achieved a survey completion rate of %, which means all participants (n = ) completed the survey. after applying the inclusion criteria, questionnaires were dropped. of the remaining questionnaires analyzed, % (n = ) were completed by residents of accra, % (n = ) by residents of kumasi; and % (n = ) by residents of tamale. as table indicates, about % (n = ) of participants were female and % (n = ) were male. about % of participants (n = ) had tertiary education, which means that most of the sample had a high education. the age of participants ranged between and years. table shows some dramatic changes in behaviors. that is, % of participants lost moderate physical activity time, with over similarly, more than % added at least h to their sedentary behavior time. about % (n = ) of participants were exercising during the lockdown, whereas there was no change in the frequency of smoking for % (n = ) of participants who were smokers. the frequency of alcohol intake increased for % (n = ) and did not change for % (n = ) of participants. the frequency of eating decreased for % (n = ) and increased for % (n = ) of participants. about % (n = ) of participants agreed they used substances to protect themselves against covid- and faced a higher risk of domestic violence. sexual activity decreased for % (n = ) and increased for % (n = ) of participants. appendix table shows a distribution of average mental health scores across categorical predictors. table shows some significant correlations at p < . and p < . . for example, mpatl and mental health are negatively correlated (r = − . ; p < . ; two-tailed). this result connotes that mental health decreases as moderate physical activity time lost increases. table shows regression coefficients resulting from this and other correlation coefficients in table . it can be seen that moderate pa time lost makes a significant influence on mental health (β = − . ; t = − . , p < . ), suggesting that mental health decreased with an increase in moderate pa time lost. sedentary behavior time added also made a negative influence on mental health (β = − . ; t = − . , p = . ). the influence of 'sf-unchanged' is about times lower compared with that of 'sf-non-smokers' (b = − . ; t = − . , p = . ), which indicates that the mental health of individuals who did not have a change in their smoking frequency was lower compared with non-smokers. the influence of 'ai-not applicable' on mental health is about times lower compared with that of 'ai-increased' (b = − . ; t = − . , p = . ), implying that individuals who did not drink alcohol at all reported lower mental health scores compared with those whose frequency of alcohol intake decreased. the influence of 'ai-unchanged' on mental health is about . times higher compared with that of 'ai-decreased' (b = . ; t = . , p = . ), indicating that individuals whose frequency of alcohol intake remained the same reported larger mental health scores compared with those who had a decrease in their frequency of alcohol intake. an increase in eating frequency (i.e. ef-increase) and a decrease in eating frequency (i.e. ef-decrease) were both associated with lower influences on mental health compared with 'ef-no change'. that is, compared with those whose eating frequency remained the same, individuals whose eating frequency increased or decreased reported lower mental health scores. those who used substances to protect themselves against covid- had better mental health compared with those who did not (b = . ; t = . , p < . ). finally, those with increased sexual activity (i.e. sa-increase) had better mental health compared with those whose sexual activity level did not change (β = . ; t = . , p = . ). the independence-of-errors and multicollinearity assumptions were met based on tolerance ≥ . (for each predictor) and durbin-watson = . (for the regression model) in table (garson ). a major change in behavior resulting from covid- -related social isolation is a reduction in physical activity and an increase in sedentary behavior time. ellingson et al. ( ) conducted in the us also backs our result with its evidence that a loss in moderate pa time and an increase in sedentariness is negatively associated with mental health in young adults. more so, several studies (walsh ; allen et al. ; shim et al. ; lund et al. ) reported a negative influence of reduced pa or increased sedentary behavior on mental health in the general population. unlike previous pieces of evidence however, our result is the first linked to social isolation driven by a pandemic. a noteworthy connotation of our result is that social distancing measures should be rolled out with pa promotion programs to encourage indoor pa during social isolation. it may thus be necessary for governments to intensify pa counseling via the media before and during a lockdown. as done in parts of the uk (mytton et al. ; anderson et al. ), gyms and parks should be considered essential service providers and allowed to operate during a lockdown. however, a strict observance of social distancing protocols at these pa centers is imperative. despite the strong force with which conspiracy theorists used the social media to promote smoking as a behavior that protects against covid- (li and siegrist ; anderson et al. ) , this study did not find any change in smoking among socially isolated participants. based on , we argue that this result may be due to the fact that over % of the sample were highly educated individuals who may not yield to unfounded claims. besides, smokers whose frequency of smoking did not change reported lower mental health scores than their colleagues who never smoked. this finding endorses previous studies that have found a negative relationship between smoking and mental health (lawrence et al. ; walsh ; allen et al. ; shim et al. ; lund et al. ) . coupled with other distressing conditions caused by social distancing during the spread of covid- , our result may be an indicator of an intensified consequence of smoking. in any case, campaigns discouraging smoking and related behaviors during a lockdown ought to be intensified. since the primary weapon for fighting many infectious diseases such as covid- is the individual's immunity, behaviors such as smoking that have the tendency of weakening or disabling the immune system (lawrence et al. ; lund et al. ) must be eschewed at the individual level. for this reason, there is no alternative to conscientizing smokers regarding the health risks of smoking, particularly for those socially isolated. a key change in behavior associated with covid- -related social isolation is a decrease and increase in the frequency of alcohol intake. interestingly, those who did not drink alcohol at all reported smaller mental health scores compared to those who maintained their frequency of alcohol intake. this outcome tends to support previous empirical studies (german and walzem ; kaplan et al. ; chiva-blanch and badimon ) confirming a positive table the association between mental health, changes in health behaviors, and covariates (n = ) b unstandardized coefficient, β standardized coefficient, ci confidence interval, s.e. standard error (of b), sbta sedentary behavior time added, mpatl moderate physical activity time lost, vpatl vigorous physical activity time lost, sf smoking frequency, ai alcohol intake, ef eating frequency, sa sexual activity, dvi domestic violence increase a dummy variable for smoking frequency with 'sf-non-smoker' as reference b dummy variable for alcohol intake with 'ai-decreased' as reference c dummy variable for eating frequency with 'ef-no change' as reference d dummy variable for sexual activity with 'sa-no change' as reference **p < . ; *p < . (kaplan et al. ; chiva-blanch and badimon ) have warned that excessive consumption of alcohol increases the risk of disease. this being so, an increase in the frequency of alcohol intake can cause a major public health concern, especially for those forced into social isolation owing to the spread of an infectious disease such as covid- . a change in the frequency of eating as a result of social isolation is a key finding of this study. while a fall in eating frequency may be due to poor access to supermarkets and supplies, an increase is possible for the working class or managerial elites with abundant food supplies. as social isolation during the lockdown compelled individuals to spend more time at home, an increase in the frequency of eating among those with enough savings is likely. this can be said of most of our participants who were highly educated and had a regular income. further to the above, an increase and decrease in the frequency of eating were associated with lower mental health scores, logically because an increase translated into abuse of food while a decrease resulted in malnutrition in the short-term. this thinking squares with studies (prentice ; fuhrman ) that have revealed that food can only confer its nutritional and health benefits when consumed in moderation. moreover, short-term side effects of over-and/or under-eating include mental health struggles that can compel individuals to poorly rate their mental health (prentice ; fuhrman ) . with these possibilities in view, programs for conscientizing residents facing a lockdown would have to be cognizant of potential changes in dietary behaviors. a segment of our sample used substances (e.g. garlic, ginger) to protect themselves against covid- during social isolation, which points to the likelihood that people were influenced by fake news regarding the protective properties of eating garlic, ginger, and other substances against covid- . more interestingly, those who used these substances reported higher mental health scores, an outcome that tends to add weight to claims that garlic, ginger, and similar substances have anti-inflammatory properties and therefore enhance the immune system and confer other health benefits (arreola et al. ; percival ) . we would want to reason that the foregoing result was possibly driven by participants' psychological reaction to using substances to protect themselves against covid- . that is, dependence on substances may have boosted the confidence of participants in their health and consequently made them to overrate their mental health. whether substance use was well-fated or a guise, it is understandable that people are likely to use unprescribed substances during the lockdown to protect themselves against covid- . regardless of its impact on mental health in this study, substance use could mar individual and public health, thereby causing disabilities that may cost governments a fortune to rehabilitate. over the years, empirical research has produced mixed findings regarding the influence of sex on mental health (bennett ; galinsky and waite ) , but researchers, from a psychology perspective, have reasoned based on different scenarios that mental health could improve with sexual activity (ganong and larson ) . congruent with this stance is our result indicating an increase in mental health in those whose sexual activity increased. a common explanation to a positive influence of sexual activity on mental health is that sexual intercourse and romance increase individuals' happiness and satisfaction with life if they satisfy the individuals emotional needs (bennett ; galinsky and waite ) . we opine based on this argument that sexual activity would make a positive short-term influence on mental health during covid- -related social isolation. we would want to premise this stance around the idea that sexual activity in the early days of the lockdown may have provided total emotional satisfaction possibly because most participants, who make up an elite working class, did not have enough time for sex before the lockdown. as such, the participants had unsatisfied sexual needs before they went into social isolation. psychologists explain that an increase in sexual activity to meet one's unsatisfied sexual needs always produces mental health benefits (galinsky and waite ) . drawing on the foregoing assertion, we admit that changes in behaviors and their influences on mental health may differ in the long run when people may successfully adapt to the lockdown or use up economic resources saved. moreover, the dynamics may differ from what was explained based on the disengagement theory and fbm in the shortterm, leading to a more or less compelling changes in behaviors. we, therefore, concede that focusing on short-term changes in this study is a major shortcoming that future researchers should address. this said, further studies may adopt randomized longitudinal designs to assess the impact of time on the changes considered in this study as well as their effects on mental health. we are also worried that our sample was not powered and may, as a result, not be representative of the general population. while we believe findings of this study may apply to some settings owing to the normal distribution of our data (garson ; yap and sim ) , it is important for future studies to use representative samples to enhance the generalizability of our results. the replication of this study in new contexts may suffice in situations where the use of a powered or representative sample is not possible. some segments of the population (e.g. older people) who did not use the internet were not included in the sample. with english serving as the sole medium of questionnaire administration, residents with poor english skills may have been underrepresented. despite these limitations, this study is novel for being the first to assess changes in behaviors that may result from self-isolation during the spread of an epidemic. it does not only provide a foundation for future research but also offers insights into what stakeholders could do to ensure that behavior changes do not compound public health issues accompanied by the spread of an epidemic or a related extreme event. at least, this study makes us to contemplate the need for covid- social distancing measures to be rolled out alongside public education programs for discouraging unhealthy changes in behaviors. the study confirms short-term changes in behaviors attributable to covid- -related social isolation, with key examples being a reduction in individuals' physical activity time and an increase in sedentary behavior time. sexual activity and eating frequency have changed in the short-term owing to covid- -related social isolation. an increase in sedentary behavior time has made the most compelling negative influence on mental health, which suggests that the biggest decline in mental health in our sample was due to increased sedentariness. the only change in behavior that has a positive influence on mental health is substance use. for the most part, changes in behaviors in the short-term attributable to covid- social isolation were associated with lower mental health scores. these changes in behavior are, therefore, potential public health risks that may compound over time. see table . social determinants of mental health how will country-based mitigation measures influence the course of the covid- epidemic? the lancet covid- and the consequences of isolating the elderly. the lancet public health immunomodulation and anti-inflammatory effects of garlic compounds a review of web based interventions focusing on alcohol use adolescent mental health and risky sexual behaviour the psychological impact of quarantine and how to reduce it: rapid review of the evidence. the lancet opinion wuhan coronavirus ( -ncov): the need to maintain regular physical activity while taking precautions benefits and risks of 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normality tests acknowledgements we thank members of the 'share research-ace' whatsapp team including dr samuel awuni azinga and mr wisdom mensah avor for their technical advice and guidance. we acknowledge hon. kojo yankah for proofreading this manuscript. author contributions na conceived the research project and wrote the manuscript. ffo coordinated data collection and survey administration. em analysed the data. swm contributed to survey design, validation, and data collection. hkm and fq contributed to the design of the survey and formatted the manuscript. all authors read and approved the draft manuscript.funding the researchers did not receive funding for this study. conflict of interest the authors declare that they have no conflict of interest.ethical approval this study was approved by the institutional ethics review board (with code # -ace). the board ensured that participation in the study was voluntary and the study was not harmful to participants.informed consent every participant provided informed consent before completing the survey. see table . key: cord- -cw dfos authors: reuben, jayne s.; crawley, william t.; webb, paris; den brok, koen; woodburn, elizabeth; montemayor, jennifer r.; roberts-lieb, sol; de jong, peter g. m.; dickinson, bonny l. title: iamse meeting report: student plenary at the th annual conference of the international association of medical science educators date: - - journal: med sci educ doi: . /s - - - sha: doc_id: cord_uid: cw dfos nan health professions are rapidly changing in response to advances in technology, modern approaches to information collection and processing, financial pressures, changes in society and demographics, and shifts in political climates. in response to these and other challenges, healthcare providers must become agile and modify their clinical practices to continue to provide exceptional patient care. importantly, these challenges also need to drive and empower educators to evolve in their approaches to training future providers. thus, educators not only need to prepare students to practice in current healthcare environments but also must provide a foundation for practice in the rapidly evolving healthcare environments of the future. while research that gathers students' thoughts, attitudes, and impressions about their education and training is useful, hearing their voices first-hand is essential to identifying and addressing their needs to inform curricular change. with this in mind, iamse held its first student plenary featuring students studying medicine and dentistry in the netherlands and the usa. the title of the session was student voices: envisioning the future of health sciences education across different healthcare professions worldwide. the goal of the session was to engage meeting participants in a discussion of how best to prepare students for the healthcare environments of the future. each presenter gave a -min presentation outlining their thoughts about the future of health profession education. an audience question and answer session followed the presentations. the student presentations are summarized below. william tyler crawley, ms student, rocky vista university college of osteopathic medicine, usa the field of medical education has long lagged behind other disciplines when it comes to teaching about diversity. as a result, medical providers are not being adequately taught how disease uniquely affects different populations or how to apply evidence-based medicine to treat diverse patient populations. implicit bias is another area requiring further attention in medical curricula. we all develop unconscious attitudes and stereotypes that impact our decision-making. these biases are responsible for the incorrect assumptions we hold that can narrow the options provided to our patients with the effect of limiting their ability to make informed decisions about their own healthcare [ ] . research has shown that interpersonal experiences and education can raise awareness of implicit bias, and that this may help to prevent negative impacts on patient care [ ] . to address implicit bias, students must partner with educators and institutional officials to change not only what is taught but also how it is taught. educators the iamse annual meeting introduced a new program feature: the student plenary. in this session, the student perspective on the meeting theme was highlighted by a panel discussion with four students from different health professions programs. in this meeting report, we provide a brief summary of their presentations. working with students and experts in the community should identify opportunities to integrate information about diverse patient groups into the medical curriculum. furthermore, a thorough review of curricula should be conducted to ensure that learning materials and assessments are free from historical, medical, and societal stereotypes so that these biases are not perpetuated. in summary, as universities seek to address diversity, an explicit commitment is needed to provide dedicated time in the curriculum for education on minority populations and diversity topics. these efforts should also extend beyond the classroom to address diversity issues in the faculty, staff, standardized patients, and university policies. opportunities should be sought for institutions to partner with experts in the community who can share their knowledge, serve as speakers, and provide learning resources. finally, these efforts can be facilitated as institutions provide students with a mechanism to report concerns about learning opportunities and to recommend suggestions for improvement. paris webb, d student, texas a&m university college of dentistry, usa in response to the covid- pandemic, many institutions were forced to switch from a traditional in-class lecture model to a virtual educational format. these efforts demonstrated that virtual learning is possible, although it cannot substitute for the requisite face-to-face learning required to master communication and hand skills that dentistry requires. thus, the pandemic has presented a unique opportunity to rethink how we educate health professions students. another driver of change at texas a&m college of dentistry occurred in response to changes in the national dental board examination [ ] . specifically, the institution introduced several curricular changes to provide new educational approaches and assessments to prepare future dentists to excel in the evolving field of dentistry. these changes included reorganization of a siloed first-year curriculum into an integrated systems-based human structure, function, and pathology course that included content from anatomy, histology, physiology, biochemistry, and pathology. in addition, the creation of an integrated practice of dentistry course enabled firstyear students to practice diagnostic techniques as part of the physical assessment of the dental patient, which facilitated early entry of these students into the dental clinics to assist upper-level students. third, a new longitudinal comprehensive pain management curriculum was developed in response to the ongoing opioid epidemic. a final driver of change in healthcare education is the growing diversity of the us population [ ] , which highlights the importance of teaching students about cultural competence and social determinants of health in all specialties of healthcare including dentistry. these changes in healthcare education necessitate collaboration between students, educators, and institutions to ensure that the next generation of dental health professionals are well prepared to provide individualized patient care. koen den brok, master ( th year student), radboud university medical center, the netherlands over the years, the practice of dental care in the netherlands has slowly changed due to an aging population with different healthcare demands [ ] . at the same time, the number of oral health professionals is declining as many of them are facing retirement in the next - years. in response to these developments, dentists have begun to collaborate in shared practices and group-clinics [ ]. to prepare the dental students of today for this new reality of the future, radboud umc dental faculty developed a master clinic program for - th year students. this master clinic creates an interprofessional workplace simulating a dental clinic. in the master clinic, both dentistry and oral hygienist students learn practical and theoretical dentistry together while providing care to the patients in their respective clinics. entrustable professional activities have been implemented as an effective tool to teach students selfreflection and to follow study progression on clinical and professional activities [ , ] . students also learn leadership skills as they are now involved in teaching their peers and managing the clinics. in the program, teachers have taken on the role of coaches and guides instead of lecturers. this novel approach is highly valued amongst students and staff and has proved to be a significant addition in preparing and motivating the students for life-long learning. this successful innovation could not have happened without synergy between student representatives and the faculty. finally, the covid- epidemic has been an accelerator for the development and implementation of new digital teaching modalities in the dental curriculum. these new teaching modes have enabled students to learn at their own pace and have opened up possibilities for making the study program more flexible. these and other curricular changes have the potential to decrease stress felt by healthcare professions students and should be carried forward into the future. the future of medical education should be forward-focused, patient-centered, and student-driven to graduate professionals who are engaged in their work and positioned to lead the field of healthcare forward. as students have access to an everincreasing amount of information in the form of texts and digital resources from which to learn the basic sciences, there is a need to refocus classroom time on collaborative learning and synthesis of information. with problem-based learning serving as an early foundation, students can translate learning to patient care and scholarship, so they are better stewards of healthcare resources [ ] . dedicated independent study time in the curriculum is needed for students to examine medicine from different perspectives and to devise potential solutions for clinical problems they encounter [ ] . these changes will encourage students to embrace a growth mindset and create positive change. to achieve these goals, health science educators must also adopt a growth mindset and be open to developing and using novel interventions, methodologies, pedagogies, and learning tools. conflict of interest the authors declare that they have no conflict of interest. informed consent na unconscious bias in academic medicine: overcoming the prejudices we don't know we have contact and role modeling predict bias against lesbian and gay individuals among early-career physicians: a longitudinal study integrated national board dental examination united states census curriculum development for the workplace using entrustable professional activities (epas): amee guide no. assessing trainees and making entrustment decisions: on the nature and use of entrustment-supervision scales problem-based learning and medical education forty years on. a review of its effects on knowledge and clinical performance introducing first-year medical students to product innovation and entrepreneurship publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord- -c mthxv authors: martin-garcia, e.; celada-Álvarez, f.; pérez-calatayud, m. j.; rodriguez-pla, m.; prato-carreño, o.; farga-albiol, d.; pons-llanas, o.; roldán-ortega, s.; collado-ballesteros, e.; martinez-arcelus, f. j.; bernisz-diaz, y.; macias, v. a.; chimeno, j.; gimeno-olmos, j.; lliso, f.; carmona, v.; ruiz, j. c.; pérez-calatayud, j.; tormo-micó, a.; conde-moreno, a. j. title: % peer review in radiation oncology: is it feasible? date: - - journal: clin transl oncol doi: . /s - - - sha: doc_id: cord_uid: c mthxv purpose: peer review has been proposed as a strategy to ensure patient safety and plan quality in radiation oncology. despite its potential benefits, barriers commonly exist to its optimal implementation in daily clinical routine. our purpose is to analyze peer-review process at our institution. methods and materials: based on our group peer-review process, we quantified the rate of plan changes, time and resources needed for this process. prospectively, data on cases presented at our institutional peer-review conference attended by physicians, resident physicians and physicists were collected. items such as time to present per case, type of patient (adult or pediatric), treatment intent, dose, aimed technique, disease location and receipt of previous radiation were gathered. cases were then analyzed to determine the rate of major change, minor change and plan rejection after presentation as well as the median time per session. results: over a period of weeks, cases were reviewed. median of attendants was six physicians, three in-training-physicians and one physicist. median time per session was ( – ) minutes. . % of cases presented in – min, . % in – min and . % in ≥ min. . % of cases were accepted without changes, . % with minor changes, % with major changes and . % were rejected with indication of new presentation. most frequent reason of change was contouring corrections ( . %) followed by dose or fractionation ( . %). conclusion: everyday group consensus peer review is an efficient manner to recollect clinical and technical data of cases presented to ensure quality radiation care before initiation of treatment as well as ensuring department quality in a feedback team environment. this model is feasible within the normal operation of every radiation oncology department. assuring quality of care is a safety challenge within radiation oncology related to the evolving growth of technology and the multistep planning process in this field. individual decision making among radiation oncology physicians and the complexity of radiation treatment plans can also contribute to potential errors in this process. target volumes and normal tissue delineations represent an important source of those errors as well as treatment prescription (total dose and dose fractionation), especially for hypofractionated schemes and sbrt plans. as such, an adequate peer-review process involving plan evaluation in a feedback environment from a multidisciplinary team is an effective strategy for assuring plan quality and patient safety recommended by professional organizations such as astro, acr and ranzcr [ ] [ ] [ ] . several reports have proven the benefit of peer-review process into facilitating treatment plans but also have enumerated a list of barriers to its effective implementation and efficiency such as scheduling, workload, time dedicated to peer review, equipment, repetition, the attendance by key personnel and distraction from clinic time with patients [ , [ ] [ ] [ ] . in addition, there is still little guidance and limited published research about the frequency, mechanics or metrics of peer review in radiation oncology from professional organizations [ , ] so nowadays, each radiation oncology department should identify and mitigate those barriers to an effective application of peer review to its individual process and workflow. currently in spain, [ ] centers provide radiation therapy services. although its delivery is relatively standardized in our country, there's no data published about peer-review system in those departments so the extent and structure of this process vary widely among centers and even remain uncertain at some point. in respect to our institution's peer-review process, we report the rate of plan changes as well as the amount of time and resources needed for this process. the aim of our study is to demonstrate the feasibility of performing peer review to every case in radiation therapy. a sample of typical results is presented just as illustrative purpose. historically, peer review was established at our institution as an audit procedure to review treatment indication, target/oars volumes, dvh analyses, treatment plan changes and any other remarkable aspects concerning patient's case, recorded in our quality guarantee department certification. these conferences occur daily at . am where all patient cases are presented prior to dosimetry and treatment initiation. the review process is performed by a multidisciplinary team that includes physicians, resident physicians and medical physicists. although daily attendance varies, typically at least six attending physicians (range - ) are present at each session. the minimum number of senior radiation oncologists required to run the meeting is two, but this situation only occurs when vacation period, attendance to several tumor committees, previous day on-calls, or special considerations such as medical leave or congress attendance meet. after this conference, radiophysics department has its own specific meeting to discuss cases presented at our peer review among other topics. this conference is attended by medical physicists, physic residents and dosimetrists. we balanced this approach to respect the workflow of both departments. during the conference, the treating physician or covering physician resident provides a brief review of the patient's history in addition to treatment intentions. patient's history, physical exam findings, radiological images and documents such as anatomopathological analysis or complete surgical reports are available within electronic medical record and linked via computer to live broadcasting during discussion if questions arise. simultaneously, all axial, sagittal and coronal slices of patient's simulation ct scan and any image fusions (pet/ ct or mri) are examined and projected streaming on a screen to review target volumes including gross tumor volume (gtv), clinical target volume (ctv) and planning target volume (ptv) as well as organ at risk (oar) contours/ constraints using aria ® . varian medical system. peer-review activity regarding patient's identification, main case's characteristics and decisions made in our conference is recorded in an official logbook. individual dvh analysis and dosimetry in relation of ptv coverage and oar sparing are reviewed only if case is considered complex (i.e., re-irradiation, cases involving re-planning or adaptive planning, pediatric, etc.). cases presented must accomplish our department's precise established dvh/constraints protocol on each pathology to facilitate workflow and to highlight those cases requiring thorough peer review to achieve efficiency compromise and make peer review % feasible. in cases of re-irradiation, composite plans with previous doses are examined during peer review. retrospectively, urgent treatments performed the previous day during on-calls are also presented. controversial cases discussed at pretreatment review might be presented again before treatment initiation focusing on final approved plan, dvh and any remarkable issue on treatment delivery. attendees are encouraged to make suggestions during and after presentation of the case regarding aspects of the radiation treatment plan such as target volumes and normal tissue contours in close proximity to them, total dose, dose fractionation, frequency, treatment intention, technique, igrt imaging or any other details with implication in treatment planning or dose calculation (i.e., skin affectation or not, prosthesis, setup margins, dibh, pacemaker and its relevance, photon/electron boost radiotherapy, registration quality, ptvs coverage and oar constrains…). cases requiring special procedures (brachytherapy, radiosurgery, total body irradiation, intra-operative radiation therapy) are currently being revised by groups with experience in those techniques prior to treatment delivery and only controversial treatment indications (i.e., salvage treatment with brachytherapy for irradiated patients) or cases in which boost with hdr-bt is going to be performed after ebrt (i.e., breast cancer, cervical cancer) go through our peer-review conferences before starting treatment. in addition, as a department with in-trainee physicians, peer-review conference serves as a forum for continuing education to collectively analyze every case to recognize proper diagnosis procedures, previous/concurrent chemotherapy, data from previous surgery, possible future oncologic procedures and indication or not for radiation therapy treatment. collectively, plans are either accepted or rejected with indication of new presentation. cases are classified into four categories: no changes, minor changes, major changes or plan rejection. parameters analyzed to determine change's nature are the same for minor/major: contouring, dose/ fractionation, technique or a combination. criteria to allocate a recommended change into minor or major takes into account the clinical impact of the plan on each patient. if approvement is achieved, then the plan does not have to go back through conference (i.e., no changes, minor/major change recommended), but if it was rejected for re-planning, then the case must be presented again according the agreed change level. changes can be done by the planner in real time (i.e., minor/major changes to total dose or dose fractionation) or can be performed later (i.e., minor/major changes in contouring). when a plan is rejected, usually a combination of circumstances might be present (i.e., more than one major change, a decision to present the case in tumor committee for neo/ adjuvant chemotherapy or immunotherapy, an indication to perform a new simulation ct scan for newly discoveries during peer review or for a better patient's immobilization, no treatment decision, a suggested confirmation of dose/ fractionation/constraints in a certain location for sbrt…). modifications made after our conference to radiation therapy plans are documented by the attendance medical physicist. dosimetry can only be assessed if changes suggested in peer review are made by the responsible radiation oncologist and therefore compliance with the conference recommendation before treatment initiation is ensured. based on our group peer-review process, we prospectively recollected some data on cases presented at our institutional conference to quantify the rate of plan changes, time and resources needed for this process. items such as time to present per case, type of patient (adult or pediatric), treatment intent, tumor type (primary, metastasis or both), dose, technique, disease location and receipt of previous radiation were gathered. cases were then analyzed to determine the rate of major change, minor change and plan rejection after presentation as well as the type of change (dose/fractionation, contouring, technique or combination) and also median time per session. over a period of weeks cases underwent peer review at our department prior to starting treatment. a total of conferences took place during this period. median of attendants was six physicians (range - ), three in-training-physicians (range - ) and one medical physicist. approach assessment was the most frequent reason for case presentation ( . %). the majority of cases presented were adult patients ( . %). in terms of type of tumor, . % were primary tumors and . % were metastasis. the most common technique proposed in the plans review was d-crt which was performed in approximately % of cases presented, followed by vmat ( . %), sbrt ( . %) and sr ( . %). . % of cases had a radical treatment intent, . % had a neo/adjuvant treatment intent and . % of cases were presented for palliation treatment. nearly % of presented cases had received previous radiation nearby the area of concern, in-field or at other location either at our institution or another institution. the most frequently reviewed sites were breast ( . %), followed by pelvis ( . %), bone/soft tissue ( . %) and central nervous system ( . %). a summary of characteristics of cases presented is found in table . as previously described, once the plan is reviewed at the conference it can be approved or rejected for re-planning if it undergoes a plan rejection. in our case, nearly % of cases were accepted without changes, . % with minor changes, % with major changes and . % were rejected with indication of new presentation. most frequent reason of change was contouring corrections ( . %) followed by dose or fractionation ( . %). peer review resulted in breast ( %), pelvis ( . %), bone/soft tissue ( . %) and h&n ( . %) cases requiring most of the changes recommended. . % of cases were accepted after presentation. overall, total rate of change including both major and minor changes and plan rejection was nearly %. a summary of the change rates is found in table . furthermore, median time per session was min (range - ). the majority of cases ( . %) were presented in - min, . % in - min and . % in ≥ min. of the sites presented, the longest median time of presentation was for abdomen cases ( min), followed by pelvis ( min) and central nervous system ( min). in terms of technique, sr ( min) and sbrt ( min) had the longest mean time of presentation. additionally, re-irradiation cases, no treatment decision cases discussed and those that had any type of change had slightly longer presentation times. a summary of presentation's length is found in both tables and . this study analyzes peer-review process at our institution and its feasible implementation to our daily clinical practice. a remarkable proportion of modifications have potential clinical implications as disease outcome could be directly affected by radiation therapy plan [ ] . introducing this 'audit and feedback' process among each center's protocol is a quality assessment whereby adherence to published guidelines is measured [ , ] . we found a total change rate of . % of all cases presented and a major change rate of %. previous studies identified similar proportions of recommended plan changes after peer review, accounting for . [ ] , [ ] , . [ ] , . [ ] and . % [ ] of total change rate and a major plan change rate of . [ ] , . [ ] , . [ ] , . [ ] and % [ ] respectively. in addition, some reviews also included changes in total dose ( . %) or fractionation ( . %) [ ] that did not resulted in re-planning and a total minor change rate of [ ] , . [ ] , . [ ] and . % [ ] respectively, which is in alignment with the . % rate of minor changes obtained at our institution. accordingly to a survey of north american teaching centers, % of respondents estimated that major changes occurred in < % whilst minor changes were estimated to be requested in < % of cases by % of respondents [ ] . contouring corrections ( . %) followed by dose/fractionation ( . %) resulted in most frequent reason of change, similarly to data reported in other reviews [ , [ ] [ ] [ ] ] . it is true that contouring plays an important role in our revision because it is known to be a major responsible for treatment outcomes and also isodose or dvh data are completely dependent on contour definition, especially when using imrt-vmat. to optimize peer-review process, ballo et al. [ ] highlighted the importance of reporting disease sites to identify those with a higher proportion of changes resulting in h&n, gi and gyn cases requiring a plan change more than % of the time. esophagus, uterus, upper/lower limb, cervix, h&n, bilateral lung, right supraclavicular lymph nodes, rectum and spine were cancer groups proposed to most necessitating mandatory peer review according to rouette et al. [ ] . we found breast ( %), pelvis ( . %), bone/soft tissue ( . %) and h&n ( . %) cases requiring most of the changes recommended from our peer-review members during our monthly recruitment period. several different models have been suggested for peerreview process within radiation oncology. a survey developed by astro to its physician members revealed considerable variations among centers in the timing and content of peer review requiring additional clear guidance and formal recommendations from expert consensus [ ] . some practices have established a multistep process with separate peerreview stages consisting of a first presenting case reunion followed by consensus planning conferences and final chart rounds to evaluate patients who have started a new treatment plan. other institutions have a dual-layer process with both early and late peer review evaluating different aspects. in addition, centers perform either prospective peer review (before radiation) or once the therapy has started (inside the first week) and mostly discuss only cases considered complex [ , , , ] . according to rd / [ ] , items such as treatment indication, contouring, isodoses, dvh and not expected reactions should be discussed in departmental clinical sessions. at our institution, peer-review process is unique: in a single conference we always gather the first two aspects of cases mentioned above and the third one when case is considered complex due to a strict established service protocol, and then modify plans when indicated based on peers recommendations always prior to treatment initiation, which is considered beneficial and easier to implement. the fourth aspect is not routinely assessed and we might contemplate including it henceforth as an additional quality control of the radiation therapy process complying with established protocols endorsed by scientific societies of recognized solvency such as astro and estro. notably, most centers focus on peer reviewing ebrt cases leaving special treatment modalities (brachytherapy, stereotactic radiosurgery) revision rates to a significantly lower proportion. in particular, lawrence et al. [ ] detected that % of institutions across the united states never peer review pre-implant or post-implant dosimetry for prostate brachytherapy, % never peer review gynecologic brachytherapy cases and only % consider rs. also, hoopes et al. [ ] pointed out that prospective review in brachytherapy cases accounted for % among physicians surveyed. it is remarkable to mention that, as a reference center for brachytherapy procedures at valencian community and at national level, we treat patients from different institutions who have already received ebrt or will be sent back to complete it after our brachytherapy treatment it's performed. although each brachytherapy procedure is unique for us, currently final treated plans are unfortunately not routinely presented at our daily peer-review conference. in agreement with lawrence et al. [ ] , brachytherapy is of potential concern because it involves high-dose radiation with curative intent in which spatial issues (applicator position, target definition) can have highly significant consequences. in our opinion, each brachytherapy procedure is unique, in the sense that depends not only on the patient conditions but also on the physician skills training, hence all brachytherapy cases might retrospectively be reviewed to evaluate the quality of the implants, fiducial marker placement, the problems and solutions found, etc. this should complete our quality control system protocol and a forthcoming event at our department along with other special treatment modalities (stereotactic radiosurgery, total body irradiation, intra-operative radiation therapy…) on which controversial treatment indications are presented but technical considerations and final treated plan should be retrospectively review for educational benefits and treatment planning consistency, reinforcing collective decision making and assuring quality control, with further investigation into its clinical impact. interestingly, the variability of parameters evaluated in the classification for minor/major changes and therefore its implication in the acceptation/rejection plan rates it's still an unsolved question as there's no specific guidance published from professional organizations [ ] . this inconsistency can be appreciated through previous articles. for example, to ballo et al. [ ] , events variations are classified in three types: radiation dose, target or major changes that changed treatment modality (i.e., surgery rather than external beam radiation) or added modalities (i.e., chemoradiation therapy rather than radiation alone). driving forward into changes' nature, rouette et al. [ ] analysis defines a minor change as a recommended change that did not meet the criteria for a "major" change and did not lead to significant repeat treatment planning whilst a major change is defined as a change requiring repeat planning and/or have a foreseeable effect on treatment toxicity or cancer outcomes in the view of the peer-review physician, which is more in alignment with our change's proposal. during our monthly prospective data base, we conducted major changes in nine cases ( %) and minor changes in cases ( . %). major recommendations at our multidisciplinary conference accounted for contouring (i.e., breast cancer case involving regional lymph nodes that have not been considered when contouring before peer review or modifications to gtv), dose/fractionation (i.e., changes in dose/fractionation for sbrt-vmat lung case from × gy to × gy or unacceptable and avoidable oar dose in a re-irradiation pelvic case) and technique (i.e., d-crt instead of vmat for soft tissue sarcoma of the limb) while a change in dose/fractionation for palliative bone metastasis from × gy to × gy due to patient's performance status was considered a minor change and adding prv to a certain oar was considered a minor contouring recommendation. from our point of view and awaiting for more official detailed guidance, the definition of recommendation magnitude to classify a change into minor or major is inherently subjective and should be appraise individually on each case and assigned to every radiation oncology department itself. arising from our experience, peer-review process brings to light unperceived considerations or additional viewpoints that could lead to changes resulting in the outcome of radiation plans that otherwise would not have been made without this process. the degree of compliance with peerreview recommendations arising from walburn et al. [ ] was reasonably good ( %) but it decreased as recommendation magnitude increased: % for minor and % for major recommendations respectively, suggesting that early rather than late peer review may enhance compliance and efficiency creating a safety-oriented culture. moreover, rouette et al. [ ] reported a rate of . % of major changes for plans presented after treatment had begun. fairchild et al. [ ] revealed that retrospective peer review resulted in higher failure rates and worse overall and progression-free survival after delivery of radiation that did not make compliant with protocol guidelines. consequently, along with astro [ ] we recommend performing peer review before the initiation of treatment for assuring plan quality and patient safety. additionally, an important overarching question remains concerning the optimal proportion of plans to review to optimize the utility of peer review. in our conferences, we present every single patient and not only those cases considered complex, explaining why breast cancer accounted for . % and therefore why d-crt was the most common technique utilized ( . %) due to the high volume of patients suffering from this pathology and to palliative cases. previous publications suggested that peer review might not be necessary on every breast cancer patient [ ] .from our point of view, breast cancer is of great importance as radiotherapy is considered a radical treatment in this pathology with significant curative rates and arising controversies among axillary management. although wide guidance recommendations published by experts help reducing practice variation, we have a high level of rigorousness when performing ct-scan-based contouring and, for patients with nodal involvement, the ct slice that will establish the separation between the beams treating breast (tangential beams) and the half beams treating nodes is also decided in our peer-review conference among radiotherapists and with the advice of the medical physicist. this decision has a huge impact in the reduction of lung dose, humeral head avoidance, spinal cord and medial extension of nodal ptv as well as an important reduction of the planning time. following on from this, lymberiou et al. [ ] showed that from breast radiation plans analyzed . % required major changes while rouette et al. [ ] identified a rate change of . % for left/right breast plans and . % for left/right chest wall. both studies noticed that plans involving the regional lymph nodes were more likely to have recommended changes suggesting that cases with locoregional nodal involvement should be a significant area of focus for peer review. moreover, from cases presented at our conference during that monthly period, seven accounted for h&n cases ( . %), with a reported change rate of . % from total changes. the majority of these changes were considered minor (dose/fractionation or contouring) and only . % were considered major enough to affect clinical care (i.e., one case was rejected with indication of presentation in the h&n tumor committee to considered neoadjuvant chemotherapy). as described in previous studies, h&n is a frequent revision's site mainly because it's inherent complexity in normal tissue contouring but also because there are always physician-specific decisions that are only exposed during a process of peer review [ ] . rouette et al. [ ] compiled peer-reviewed treatment plans among cancer centers in ontario including h&n cases ( . %) with changes recommended after peer review in . %. of the cases presented over a period of years of peer review at ballo et all analysis [ ] , were h&n patients with a reported change rate of . %, prompting that peer review should be mandatory for all h&n plans and prior to treatment initiation in favor of re-planning avoidance. similarly to our proportion of cases peer reviewed, albert et al. [ ] found h&n cases ( . %) from an overall of cases presented resulting in one of the most frequently reviewed sites and also with one of the longest mean time of presentation ( min). discussing all patients might seem to lengthen the time needed for peer-review process and also could led to an unnecessary effort, but when comparing our median time per session ( min) to other experiences [ , ] , the result is quite similar. based on site involvement, previous experiences reported differences in the time needed to present cases. albert et al. [ ] found that cases involving lung, cns and h&n required slightly more time whereas ballo et al. [ ] suggested performing peer review for all disease sites but focusing on those requiring changes more often (h&n, gi or gyn). for us, abdomen cases, pelvis (especially gu, gyn) and cns accounted for the longest mean time of presentation likely due to sbrt cases with critical structures nearby and higher number of oars reviewed for these sites. ultimately, radiation oncology departments should integrate peer-review system into their routine workflow, scheduling conferences at the optimal moment of the day and with a reasonable average of reunions per week to reassure staff that their time is well spent. furthermore, peer review serves for educational opportunities and creates an environment that fosters respectful questioning. resident physicians have the opportunity to take an active part in the process by asking questions and giving their own feedback, becoming that way proficient in plan evaluation. seasoned physicians can also benefit from peer review given the variety of cases presented, maintaining them updated for disease sites plans even if they are not their main sites of focus [ ] . topics arising from clinical cases on peer review are also adopted to create academic bibliographical sessions every weeks approximately reinforcing its importance to stay abreast of the latest updates in the oncology field. in addition, this system has also indirect quality benefits: reduces practice variation by promoting standardization, improves communication and reinforces knowledge sharing [ ] . it has also been documented that centralized plan review increases group consensus and consistency which is reflected in decreased treatment plan changes over time, suggesting that the act of peer review has an educational effect and the system directly change care by increasing adherence to institutional guidelines [ ] . all of this enhance the role and utility of peer review as part of routine clinical practices. nevertheless, despite evidences providing proof of its benefits, previous surveys showed that only approximately % of centers performed peer review of radiation plans in the united states [ ] and only . % in canada [ ] . in europe, harmonizing clinical care based on expert consensus agreement can improve clinical outcome in the face of uncertainty, with quality and safety playing a priority place in radiation medicine according to estro [ ] . recently, the working group on patient safety and quality of the spanish society of radiation oncology (seor) revised national and international recommendations for patient safety evaluating whether they are included in spanish legislation. they found that even though peer review plays an important part in maintaining quality standards and improving performance and safety of care, neither rd / nor rd / consider the peer-review process [ ] . nevertheless, a new rd / [ ] reinforcing mandatory oars and treatment volumes revision became effective a month ago so performing peer review will be a compelling reason henceforth. unfortunately, there's no data published about peer-review system in spain so far to our knowledge. this report is the first one providing information about the structure, operation and metrics analyzed in this process at national level. finally, advances in tumor genomic data and technology will enable more personalized radiation treatment based on patient-specific knowledge. in this context, artificial intelligence (ai) will be a part of our future by providing prognostic models built on radiomic features, creating automated segmentation of oars/tvs on contouring area so as to prediction of normal tissue complications and assuring quality by streamline physician peer-review process. even though potential benefits of ai in radiation quality assurance, challenges remain before its widespread implementation in clinical setting [ , ] . from our point of view, adopting contouring guidance similar to those used in clinical trials seems to be essential nowadays in the delivery of radiation treatment to assure quality as an established methodology exists. we believe peer review is a quality assurance procedure to guarantee patient safety and feasible within the structure of every radiation oncology department. emergently, due to covid- outbreak, drastic disease-control measures from official institutions have forced centers to adapt their usual clinical care and organization level [ ] . on this scenario, teleconferencing via webex ® has been established to allow staff to conduct peer review and, therefore, maintaining qa and patient's security in spite of the situation. there are several weaknesses to consider within our current analysis, but by publishing this review we would like to encourage other centers to implement peer review to their daily routine and to report their experiences proving its feasibility. one limitation is the low sample of plans compiled ( ) compared to other publications with thousands [ , , , ] . as we revise all cases, data collected represents an assortment of pathologies treated at our institution during a whole month period and, even though we assume it is not accurate to extrapolate results, we believe it gives the reader an idea of the amount of cases and disease's locations presented during a year ( ebrt cases in ). secondly, not expected reactions and retrospective peer review to other special modalities (brachytherapy, sr, tbi, iort…) are not routinely assessed and seems to be a compelling inclusion henceforth for assuring additional qa system and its impact in clinical outcomes. our findings show that everyday group consensus peer review is an efficient manner to recollect clinical and technical data of cases presented to ensure quality radiation care before initiation of treatment as well as creating a feedback team environment. it is also considered as a security system to avoid potential errors whereas serving for continuing education. despite the challenge nature of its implementation, our 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practices in radiation oncology the european society of radiotherapy and oncology (estro) european higher education area levels and postgraduate benchmarking document for radiation therapists (rtts) patient safety in radiation oncology in spain: a need to change real decreto / , de de julio, sobre justificación del uso de las radiaciones ionizantes para la protección radiológica de las personas con ocasión de exposiciones médicas artificial intelligence in radiation oncology treatment planning: a brief overview the future of artificial intelligence in radiation oncology covid- : global radiation oncology's targeted response for pandemic preparedness the impact of transitioning to prospective contouring and planning rounds as peer review authors declare that there is no conflict of interests regarding the publication of this paper. informed consent not applicable. key: cord- - hxnez authors: de kort, hanne; baguette, michel; lenoir, jonathan; stevens, virginie m. title: toward reliable habitat suitability and accessibility models in an era of multiple environmental stressors date: - - journal: ecol evol doi: . /ece . sha: doc_id: cord_uid: hxnez global biodiversity declines, largely driven by climate and land‐use changes, urge the development of transparent guidelines for effective conservation strategies. species distribution modeling (sdm) is a widely used approach for predicting potential shifts in species distributions, which can in turn support ecological conservation where environmental change is expected to impact population and community dynamics. improvements in sdm accuracy through incorporating intra‐ and interspecific processes have boosted the sdm field forward, but simultaneously urge harmonizing the vast array of sdm approaches into an overarching, widely adoptable, and scientifically justified sdm framework. in this review, we first discuss how climate warming and land‐use change interact to govern population dynamics and species’ distributions, depending on species’ dispersal and evolutionary abilities. we particularly emphasize that both land‐use and climate change can reduce the accessibility to suitable habitat for many species, rendering the ability of species to colonize new habitat and to exchange genetic variation a crucial yet poorly implemented component of sdm. we then unite existing methodological sdm practices that aim to increase model accuracy through accounting for multiple global change stressors, dispersal, or evolution, while shifting our focus to model feasibility. we finally propose a roadmap harmonizing model accuracy and feasibility, applicable to both common and rare species, particularly those with poor dispersal abilities. this roadmap (a) paves the way for an overarching sdm framework allowing comparison and synthesis of different sdm studies and (b) could advance sdm to a level that allows systematic integration of sdm outcomes into effective conservation plans. biodiversity is under threat across the globe, and its preservation requires transparent and effective guidelines for policy, conservation practitioners, and educators based on realistic assessments of biodiversity-environment relations (elith & leathwick, ; kok et al., ; pereira et al., ; titeux, henle, mihoub, & brotons, ) . species distribution modeling (sdm) has been a popular toolbox for studying relationships between environmental change stressors and spatial shifts in species' suitable habitat and for predicting the potential distribution of single species, communities, and ecosystems through user-defined environmental change scenarios (alvarado-serrano & knowles, ; peterson, ; weber, stevens, diniz-filho, & grelle, ) . the overall sdm framework is not just an interesting tool for identifying areas of local conservation concern or areas not yet occupied but potentially suitable; it has the potential to contribute substantially to the global protection of biodiversity and ecosystem services threatened by multiple environmental stressors, including land-use change and habitat fragmentation, climate change, invasive alien species, pollution, and overexploitation (franklin, ; kok et al., ; wiens, stralberg, jongsomjit, howell, & snyder, ). ignoring the joint effects of multiple environmental stressors can be highly misleading; they have been found to give rise to ecological outcomes unpredicted by single environmental stressors (bellard, leclerc, & courchamp, ; fournier, barbet-massin, rome, & courchamp, ; guo, lenoir, &bonebrake, ; marshall et al., ; peterson & nakazawa, ; segan, murray, & watson, ; visconti et al., ,) and can trigger evolutionary responses that differ from expectations assumed by single stressor evolution (kelly, debiasse, villela, roberts, & cecola, ; mcclanahan, graham, & darling, ) . accounting for multiple environmental changes and potential evolutionary responses can greatly improve model accuracy (bellard et al., ; titeux et al., ) , providing sdm outcomes that better represent the potential distribution and ultimately the occupied distributional area of the species or community under study ( figure ) . indeed, part of the suitable habitat of the potential distribution is often not reachable due to dispersal limitation and spatially variable habitat connectivity (e.g., physical barriers), sizing the potential distribution down to the accessible habitat (barve et al., ; peterson, papeş, & soberón, ; pulliam, ) . where dispersal of the focal species strongly depends on the distribution and dispersal of interacting species, the biotic context is an additional determinant of the occupied distributional area of the focal species (figure ). finally, decreased connectivity among suitable habitat patches may promote evolution toward reduced dispersal (cote et al., ; graae et al., ) , further reducing the size of the occupied distributional area (figure ). in this review, we pinpoint how joint environmental changes drive population, species, and community dynamics in comparison with single stressors, focusing on climate and land-use change as two of the most prominent threats to biodiversity. predominantly driven by the ongoing biodiversity crisis, a universal urge for largescale land-use restoration, and the existence of user-friendly implementation tools, scientists increasingly study the impacts of both climate-and land-use change on biodiversity redistribution using sdm (araújo et al., ; harrison & gassner ; milanesi, rocca, & robinson, ; titeux et al., ) . this ongoing sdm f i g u r e schematic summary of the elements contributing to species distribution modeling (sdm) reliability and utility (a), the eco-evolutionary processes underlying distribution dynamics and sdm performance (b), and a hypothetical representation of the predicted occupied distributional area of a species (phengaris arion) that relies on the presence of host plants (c). colors represent the accessible and suitable habitat (purple), habitat unsuitable due to the predicted absence of host plant (gray), habitat with host plant but inaccessible (dark purple), inaccessible habitat without host plant (dark red), and environmentally unsuitable habitat (white background). patches occupied by larger butterflies (representing better dispersers) are predicted to be accessible due to dispersal evolution (after dekort, prunier, et al., ) boost opens the door for comparing and synthesizing published sdm studies for answering taxon-wide and large-scale research questions, including the role of species' traits and evolutionary potential in driving general species distribution shifts in response to land-use change. the lack of an overarching, streamlined, and widely adoptable sdm framework containing all tools required for model preparation, parameterization, and selection, however, likely strongly refrains sdm from its full potential (araújo et al., ) . moreover, a standardized and science-based sdm toolbox would pave the way for practitioners to more systematically use the main outcomes (predicted range maps) of the whole sdm framework are frequently misleading and sometimes misused (see carlson, chipperfield, benito, telford, & o'hara, ) due to several main sources of uncertainty that the user, especially the beginner user, needs to have in mind when performing sdm. first, the correlative nature of sdm inherently prevents this toolbox from building on causal relationships between environmental input variables and the occurrence of a species. this causality limitation is unavoidable because sdm inevitably requires predictors able to reflect or capture presumed causal mechanisms from natural history knowledge. the lack of information about the causal link between the predictor variables and the response variable, usually a binary variable representing species presence-absence or presence-background data, is clearly the main limitation that the user needs to constantly keep in mind when running sdm. for instance, every single sdm method, and especially the most advanced ones such as machine learning methods, will almost always be able to find a statistical link between spatially structured predictors and the response variable used to measure species distribution. even when predictors are known to be completely unrelated to the species distribution, such as the caricatural but very eloquent use of paintings to predict species distributions (fourcade, besnard, & secondi, ) , sdm will allow the user to draw nice-looking maps with high prediction accuracy according to the metrics used by state-of-the-art sdm methods. this suggests another important limitation hampering the use of sdm techniques which is the misuse or overconfidence in metrics used to measure the performance of sdm outcomes (lobo, jiménez-valverde, & real, ) . indeed, typical sdm model performance metrics such as the area under the receiving operating characteristic curve (auc), the kappa statistic, and the true skill statistic (tss) have been heavily criticized, as they tend to overpredict model performance under the influence of sample prevalence, consequently compromising model validation accuracy and model comparability (e.g., leroy et al., ; morán-ordóñez, lahoz-monfort, elith, & wintle, ) . hence, the reason why these metrics are misleading measures of the performance of predictive distribution models partly relates to one last limitation to have in mind when running sdm which is the data quality of the response variable. unless reliable and ground-truth distribution data are available, such model validation parameters are extremely questionable. in situ validation of the presence or abundance data at locations for a range of predicted probability estimates therefore is highly recommended for an unbiased perspective of model performance. the final but equally important issue regarding data quality revolves around absence data. all the metrics used to measure the performance of sdm outcomes somehow rely on absence information, whether obtained from field observations or from a random selection strategy of background data, also known as pseudo-absences. clearly, collection of field-validated true absence data across the species range is highly encouraged over pseudo-absences selection strategies to improve sdm reliability (leroy et al., ; lobo, ) . the sampling strategy used to select pseudo-absences, such as considerations on the spatial extent to be used, is known to highly influence sdm outcomes (barbet-massin, jiguet, albert, & thuiller, ; lobo, jiménez-valverde, & hortal, ; van der wal, shoo, graham, & williams, ). even absence data recorded during field surveys are tricky to use and should be carefully handled depending on the aim of the undertaken sdm exercise (hattab et al., ) : mapping the occupied distributional area or modeling the potential distribution. if the user aims at mapping the occupied distributional area, which is more or less a spatial interpolation of the suitable and accessible locations for the focal species, then all absence data recorded during field surveys are useful for model calibration and validation: whether it represents true environmental absences or dispersal-limited absences reflecting sites out of dispersal reach but potentially suitable regarding the abiotic and biotic conditions. in such cases, absence data bearing dispersal limitation information are very important and should be incorporated in the sdm framework together with predictor variables reflecting dispersal constraints, such as speciesspecific dispersal kernel, so as to improve the mapping of the occupied distributional area (lobo et al., ; meentemeyer, anacker, mark, & rizzo, ; václavík & meentemeyer, ). however, if the user aims at modeling the potential distribution, which implies spatial extrapolations beyond the actual occupied distributional area, then it is recommended to exclude dispersal-limited absence from both model calibration and validation (hattab et al., ) . indeed, using dispersal-limited absences to validate the potential distribution predicted from sdm will inevitably and mathematically lead to low auc or tss values, which results in models misclassified as having poor predictive performance. hence, one needs to carefully think about the meaning of the available absence data and how to use it for sdm calibration and validation steps. modeling the potential distribution or mapping the occupied distributional area are two very different sdm exercises requiring a different thinking on the use of absence data (hattab et al., ) . sdm for conservation decision making while being aware of the potential pitfalls (box ). strikingly, however, while land use has begun its debut into sdm, scientists do not generally quantify or map the amount of habitat that will likely be colonized by the species under study. to meet this concern, we specifically elaborate on the impacts of reduced habitat connectivity, a major side effect of various environmental changes, on sdm outcomes. we also investigate how dispersal is limited both by the effects of the landscape structure and configuration, and by changes in biotic interactions within communities. we then discuss why evolutionary processes should be incorporated to increase the reliability of sdm (bush et al., ) , and finally propose an sdm roadmap that incorporates multiple environmental stressors, dispersal, and evolution into a feasible, more reliable, and streamlined modeling framework. we stress that even for data-insufficient systems, it is possible to incorporate the processes described above. for rare and poor-disperser species in particular, realistic scenarios that take into account the various drivers of population and range dynamics can have crucial conservation implications. an sdm roadmap could also motivate nonexperts in the modeling field and young scientists to apply sdm to their target species, thereby facilitating the implementation of sdm in applied sciences and conservation practices. we stress, however, that expert evaluation of any sdm study is vital to a correct interpretation of sdm outcomes (see box for potential pitfalls). the majority of sdm studies still relies on a single environmental stressor (or group of related stressors), with bioclimatic stressors (e.g., warmer and dryer conditions) representing, by far, the most popular environmental predictor used to forecast species' distribution changes (titeux et al., ) . these studies observed drastic reductions in the modeled climatic suitability of currently occupied habitat for macroinvertebrates (up to %, domisch et al., ; parmesan et al., ), vertebrates (up to %, warren, wright, seifert, & shaffer, , and plants (up to %, aguirre- gutiérrez, van treuren, hoekstra, & van hintum, ; kane et al., ) . the effective impact of climate change on biodiversity goes beyond direct climate-occurrence relations, also involving disruption of habitat connectivity and of species interactions within communities (bertrand et al., ; garcia, cabeza, rahbek, & araujo, ; walther et al., ) . first, through reducing the amount of suitable habitat, climate change increases isolation between remaining patches, consequently inhibiting gene flow across the landscape and impairing population dynamics (graae et al., ; inoue & berg, ; razgour et al., ) . reduced gene flow increases local inbreeding risk and extinction rates, and reduces the exchange of adaptive variation (razgour et al., ; slatkin, ) . second, climate change renders habitat more prone to alien species' invasions (bellard et al., ; hulme, ) and can alter community composition and ecosystem processes (carroll et al., ; garcía molinos et al., ; pearson et al., ; perring et al., ; sheldon, yang, & tewksbury, ; sunday, bates, & dulvy, ) . global change may therefore not only alter species' distributions through direct abiotic environment-occurrence interactions but also indirectly through shaping the biotic context (e.g., prey, competitors, and pollinators) (carroll et al., ; gonzález-varo et al., ; warren & bradford, ; wisz et al., ) . over half of the terrestrial, ice-free surface is transformed through ongoing land-use change and habitat loss and fragmentation, considerably adding to the impacts of climate change on terrestrial biodiversity (aguiar et al., ; hansen et al., ; newbold et al., ; reino, beja, araújo, dray, & segurado, ; titeux et al., ; vitousek, , but see warren et al., for antagonistic effects). assuming unchanged (static) land use in sdm thus renders future projections questionable (ay, guillemot, martin-stpaul, doyen, & leadley, ; fournier et al., ; perring et al., ; titeux et al., ), yet despite the continuous recognition that land-use change constitutes a major threat to global biodiversity (perring et al., ; titeux et al., ) , the translation of this awareness into sdm keeps lagging behind. given the extent and magnitude of climate and land-use change, their combined impact on natural ecosystems is expected to be complex (guo et al., ) , urging for a transparent framework allowing the identification of areas susceptible to combined global change threats. co-occurrence of climate and land-use change has been shown to have interactive and often synergistic effects on biodiversity and species redistribution (jetz, wilcove, & dobson, ; marshall et al., ; pereira et al., ; visconti et al., ; zwiener et al., ) . first, land-use change reinforces climate warming when it is associated with livestock breeding and deforestation, which considerably boost greenhouse gas emissions (naudts et al., ; reisinger & clark, ) . second, land-use change increases the amount of suitable habitat edges that are sensitive to climate change due to the absence of a protective microclimate (brook, sodhi, & bradshaw, ; lembrechts, nijs, & lenoir, ; suggitt et al., , vanneste et al., . typical examples are the increased risk of forest fires due to increased wind exposure of forest remnant edges and inward desiccation of natural grasslands due to conversion of surrounding habitat into intensively managed agricultural land (e.g., alencar, brando, asner, & putz, ; tuff, tuff, & davies, ) . third, deforestation can accelerate the rate of upslope range shifts in tropical regions whereas the opposite has been demonstrated for higher latitudes (guo et al., ) , indicating the confounding effects of land-use change on climate-driven range shifts. fourth, reduced gene flow across the remaining suitable landscape jeopardizes the exchange of adaptive genetic variation that could otherwise allow evolutionary adaptation to a changing climate. inhibited gene flow also compromises genetic diversity and fitness within remaining populations, increasing their vulnerability to environmental stressors (frankham, ballou, briscoe, & ballou, ; markert et al., ; schrieber & lachmuth, ) . a global study assessing the interactive impacts of climate and habitat loss on vertebrate diversity predicted that nearly half of the ecoregions worldwide, mainly including tropical forest, savannahs, and wetlands, will be impacted by a synergy between habitat loss and climate change during the st century (segan et al., ) . species distribution modeling studies increasingly include variables representing both climate and land-use change in their models and have found varying support for land-use change impacts on predicted species distributions, depending on the thematic resolution of the land-use variables and the species under study (brodie, ; marshall et al., ; martin, van dyck, dendoncker, & titeux, ; scheller & mladenoff, ; zamora-gutierrez, pearson, green, & jones, ) . with the availability of a -m resolution regional land-use map and iucn distribution maps for mammal species, brodie ( ) was able to study the interactive effects of climate change and land-use change (through oil palm plantations) on mammal diversity in south-east asia. mammal species were found to be robust to changes in climate alone, but responded dramatically to the combined effects of climate and land-use change, with median habitat suitability losses of . % (low carbon emission scenario) up to . % (high emission). in a similar vein, sdm and conservation prioritization testing based on , woody plant species of the brazilian atlantic forest showed that contrasting climate change scenarios did not shape conservation prioritizations, while management strategies aiming to reduce habitat fragmentation were found to be indispensable for the long-term conservation of atlantic forest diversity (zwiener et al., ) . we stress that while the ma- the potential distribution as predicted by sdm unconstrained by dispersal limitations is fundamentally different from the occupied distributional area, that is, the distribution actually occupied by the species, requiring the integration of species' dispersal abilities and dispersal barriers into sdm. in other words, the occupied distributional area of a given species (also referred to as the realized distribution) can be seen as a spatial interpolation of the suitable and accessible locations occupied by the focal species at a given moment in time: an instantaneous map of the real spatial occupation of the focal species. by contrast, the potential distribution can be interpreted as a spatial extrapolation, albeit not an environmental extrapolation beyond the species environmental niche, of where the species could find suitable environmental conditions to occur if it would be able to reach that location, that is, unlimited by its own dispersal abilities or by dispersal barriers. many studies reporting poleward or upward range shifts in the occupied distributional area also show that actual expansion rates of the studied organisms lag behind the displacement of their climatic envelopes (i.e., the potential distribution) (e.g., bertrand et al., ; bertrand et al., ) , most likely due to dispersal and establishment lags at the leading edge (alexander et al., ) . two nonexclusive factors can explain dispersal and establishment lags: (a) the displacements of individuals are slowed down by low habitat connectivity, and (b) individuals are struggling to settle viable populations in new habitat due to their dependence on biotic interactions. a basic understanding of these processes is required to assist with the implementation of dispersal-informed sdm (alexander et al., ). habitat connectivity describes how dispersal of individuals across the landscape is facilitated or impeded by landscape structure and configuration (taylor, fahrig, henein, & merriam, ) . dispersal, that is, movements potentially leading to gene flow among populations (ronce, ) , is thus key for species to track suitable habitat shifts (berg, julliard, & baguette, ) . the study of dispersal in ecology and evolution is a swiftly evolving field of investigation since almost two decades (bowler & benton, ) , generating findings that are crucial for understanding the role of dispersal in sdm. changes in landscape structure and configuration entail high dispersal costs and hence strongly affect the fitness of dispersing individuals (bonte et al., ) . accordingly, theoretical models predict that dispersal will be most generally counterselected if its costs increase along habitat fragmentation gradients and exceed its expected benefits (cote et al., ; duputie & massol, ; heino & hanski, ; mathias, kisdi, & olivieri, ; travis & dytham, ) . empirical studies confirm that habitat fragmentation can decrease dispersal propensity (the probability that an individual leaves a habitat patch) and increase dispersal efficiency by reducing dispersal costs either through a reduced search time and/or through the selection of safer dispersal routes (baguette, blanchet, legrand, stevens, & turlure, ; baguette & van dyck, ) . however, under particular conditions of landscape configuration and habitat suitability, theory also predicts the emergence of dispersal polymorphism within populations, in which high dispersal phenotypes with a generalist strategy coexist with low dispersal specialist phenotypes (mathias et al., ) . taken together, habitat connectivity can have various ecological and evolutionary consequences, and fully ignoring this important component of the accessibility to suitable habitat may therefore strongly interfere with sdm outcomes. to increase our understanding of the mismatch between the potential distribution (as modeled through sdm unconstrained by dispersal limitations or habitat connectivity) and the effectively occupied distributional area, the integration of dispersal and habitat connectivity metrics into sdm studies was put forward as a priority a decade ago (araújo & guisan, ; hirzel & le lay, ) . although a growing number of studies discussed or implemented the effects of dispersal in sdm, the proportion of sdm studies implementing dispersal has remained steady for years (bateman, murphy, reside, mokany, & vanderwal, ; holloway & miller, ) . one of the most striking examples of the spatial lapse between potential distribution and the occupied distributional area was provided by an sdm study on bornean orangutans (struebig et al., ) . the authors predicted a loss of ca. % (within current range) and % (outside current range) of the potential orangutan distribution by s due to both climate change and direct habitat loss. however, given the sedentary lifestyle of the females, it is unlikely that the species would shift its distribution toward all suitable habitat (not all suitable habitat is accessible, even within the current range) with the predicted pace of environmental change. hence, conservation corridors or assisted translocation is required to merge suitable and accessible distributions and to ensure long-term persistence of this endangered species (struebig et al., ) . such extreme examples of jeopardized dispersal clearly show the urgency of a paradigm shift in conservation biology distinguishing suitable from accessible. in the same vein, albert, rayfield, dumitru, and gonzalez ( ) evidenced that accounting for connectivity in spatial prioritization of protected areas for focal vertebrate species strongly modified conservation priorities. how climate change interacts with landscape features to affect dispersal is another key question, and the fine-scale consequences of the interplay of climate and land-use change on the spatial distribution of suitable habitat may explain dispersal lagging behind climate change (lembrechts et al., ) . mestre, risk, mira, beja, and pita ( ) accordingly showed that range shift predictions consecutive to climate alterations were overly optimistic when using sdm disregarding habitat connectivity. in a very interesting study, fordham et al. ( ) showed that models incorporating both habitat connectivity and climate suitability provided better predictions of the range shifts observed between and for british bird species, than do models based on climate suitability changes alone. such integrated modeling scenarios could be greatly simplified by translating climate changes directly into habitat connectivity changes after assessing how changes in climatic conditions modify both habitat suitability and resistance to individual movements (see also inoue & berg, ; razgour et al., ) . the end point of this procedure is a single model that incorporates the effects of climate change as one of the drivers of changes in habitat suitability and connectivity. accordingly, this parsimonious approach is biologically more relevant than the production of competing models that consider the effects of either climate change or land-use change on the accessibility of suitable habitat, whereas these two factors are clearly not independent. (alexander et al., ) . in general, effective dispersal of specialists depends on dispersal abilities of their least dispersive interactor (see also supporting information s ). for instance, the expansion rate of specialist butterflies to accessible and suitable habitat patches under contrasted scenarios of habitat connectivity has been shown to be severely curbed by the dispersal rates of their only host plants (dekort, prunier, et al., ; schweiger, settele, kudrna, klotz, & kühn, ) . conversely, the dispersal lag between a weakly mobile parasitoid and its highly dispersive butterfly host provides the latter with enemy-free accessible and suitable habitat patches in highly fragmented habitats (bergerot et al., ) . the vast majority of sdm studies implicitly considers the relationship between an organism's ability to survive and the environmental conditions as fixed in time and space. however, the probability for a species to overcome environmental changes increases, by definition, with its adaptive potential. the ecological niche of a given species can thus evolve (visser, , wasof et al., , and local adaptation can be seen as an alternative or complementary solution for dispersal under changing environmental conditions (graae et al., ) . correspondingly, phenological shifts were reported for a variety of organisms, ranging from plants (e.g., franks, sim, & weis, ) over birds (e.g., charmantier & gienapp, ) to arthropods (e.g., van asch, salis, holleman, van lith, & visser, ) . other traits, including color morphs (karell, ahola, karstinen, valkama, & brommer, ) , dispersal ability (travis et al., ) and the thermal niche (rolland et al., ) have been shown to evolve under climate change. the key elements to phenotypic evolution and thus the evolution of the ecological niche under climate change are local genetic additive variance (underlying interindividual variation), life history (affecting both the pace of natural evolution and the feedbacks among traits), and the interplay between dispersal and the landscape structure (balancing genetic mixing and drift). evolutionary potential may thus differ considerably within and between species. using a dynamic eco-evolutionary model coupled to correlative niche projections, cotto et al. ( ) showed that evolutionary adaptation is unlikely to prevent the predicted range contraction of long-lived perennial alpine plant species under predicted climate change. on the contrary, evolutionary rescue was reported for insects with short generation time and fast growth (kearney, porter, williams, ritchie, & hoffmann, ) . similarly, using a hybrid sdm approach that incorporates dispersal and evolutionary dynamics into correlative sdm (see supporting information s ), bush et al. ( ) showed that evolutionary adaptation reduces projected range losses up to % for species of drosophila. although evolution will probably not be adequate to ensure population persistence for most species under the current pace of climate change, slowing down the pace of climate change is expected to promote evolutionary rescue (cotto et al., ) . evolution of phenotypic traits, including dispersal evolution (see supporting information s ), thus plays a major role in shaping and conserving the potential distribution of many species. environmental predictor maps (figure . ) are often freely downloadable and can be merged with occurrence points using a basic gis application. global climate data are widely available for researchers to model past, current, and future climate projections (e.g., worldclim and chelsa), rendering climate niche modeling an attractive approach for global change research relative to land-use modeling. yet, also regional and global land-use maps have become accessible (e.g., globcover, modis , corine) and can be converted from a vector to an sdm-friendly raster format using any geographical information system (gis rasterizing) to model land-use and habitat connectivity. moreover, while the short-term nature of land-use maps and land-use change may complicate land-use change sdm, simple land-use change scenarios could be tested in a sensitivity analysis for a given near-future climate change scenario. where land cover maps generally lack resolutions beyond the dominant land cover types (e.g., "grasslands" and "urban area"), there are several ways that allow more ecologically relevant land-use mapping. information on the protection level of areas across the globe (e.g., through the protected area network) (kremen et al., ) can be merged with a land cover map to indicate the level of management of specific land cover types (rodrigues et al., ) . grasslands and forests are, for example, more likely to be intensively managed or exploited when unprotected. alternatively, human population density maps and road maps can be used to fine-tune the intensity of land disturbance (e.g., newbold et al., ) , while a forest cover map can be integrated to model high-resolution forest change for species strongly depending on the absence or presence of forests (e.g., hansen et al., ) . finally, inclusion of microclimatic variables such as topography not only allows increasing the spatial resolution and accuracy of sdm predictions, it also has been demonstrated to play an underappreciated role in shaping the trajectories of species evolution and redistribution (de kort et al., ; suggitt et al., ) . (see also box ). when soil water availability, for example, has been suggested to be deterministic for the presence of the species under study, a wetness index map or equivalent, should evidently be incorporated during modeling. although the technical aspects underlying model evaluation lie beyond the scope of this review (see, e.g., aiello-lammens, boria, radosavljevic, vilela, & anderson, ; muscarella, galante, soley-guardia, & boria, ; phillips & dudík, ; radosavljevic & anderson, , for techniclal sdm considerations), we highlight the importance of model tuning and parameterization as a key criterion for realistic modeling (box ). the predictive performance of sdms is usually evaluated through cross-validation, using training data to fit models and a set of testing data that is spatially independent of the training data to evaluate these models (hijmans, ) . this model evaluation approach is, however, highly sensitive to spatial autocorrelation of environmental variables between training and testing data, and only tests the set of known occurrence data. these drawbacks artificially inflate cross-validated model statistics (hijmans, ; morán-ordóñez et al., ) and stress the importance of in situ validation (see below). we recommend sdm users to consistently model at least three scenarios: climate only, land use only, and climate and land use f i g u r e circular roadmap presenting the necessary steps toward science-based species distribution modeling (sdm) that is feasible for most species and implementers, with the emphasis on poor dispersers as a particularly vulnerable and valuable group of species. note that step has been given priority over step because of its direct conservation implications. step represents the completion of the suitable and accessible species distribution as predicted by the sdm framework. see figure for color scheme of hypothetical distribution. we refer to the roadmap steps in the main text using bold references (e.g., figure . refers to step of the roadmap), where specific directions are provided for the respective steps. please carefully read box to understand the importance of the "further considerations" presented in the blue rectangle of figure together, in addition to a full model that incorporates potential additional predictors such as soil moisture and biotic interactions ( figure . ) . such a standardized sdm framework allows (a) addressing model complexity issues (see brun et al., ; gregr, palacios, thompson, & chan, ) and (b) comparing the contribution of major environmental change stressors to predicted shifts in species distributions between independent sdm studies. although more efforts are needed for true cross-species and cross-study comparison of sdm outcomes (e.g., the development of an overarching, decision tree-based sdm environment that systematically informs users about parameter and predictor choice and more detailed land-use scenarios), we emphasize the promise that standardizing sdm holds (a for uncovering overall effects of dispersal, evolution, life history, and anthropogenic stressors on the projected distribution of many species and communities, and (b) for framing each study into a much larger and significant context. integrating evolution into sdm without assumptions on mutation and demographic rates (supporting information s ), which can be particularly challenging for rare species, can be achieved if the evolutionary potential of populations across the study area is quantified (gotelli & stanton-geddes, ; ikeda et al., ) . quantitative genetic screening of phenotypic traits has long been thought to provide the most accurate information on the evolutionary potential of traits and life-history syndromes. such quantitative genetic research is now, however, questioned due to drawbacks related to limited statistical power, high time consumption, unrealistic assumptions on the genetic architecture underlying adaptive traits and poor representation of natural conditions (hoffmann, sgrò, & kristensen, ; wood, yates, & fraser, ) . more recently, the use of genomic markers representing neutral and/or adaptive genetic variation has been proposed and tested for modeling local adaptation and adaptive potential in sdm (fitzpatrick & keller, ; ikeda et al., ; marcer, méndez-vigo, alonso-blanco, & picó, ) . as a consequence of local adaptation, many populations develop a local genetic signature shaped by historical and current environmental conditions (watanabe & monaghan, ) . variation in population responses and environmental change is thus expected, and shown, to affect the ability of sdms that do not incorporate genetic structure to predict future species' distributions (ikeda et al., ; marcer et al., ) . in addition to the implementation of neutral genetic structure, explicitly incorporating genetic variation underlying adaptive traits can be achieved through ecology-informed genome screening (e.g., de kort & honnay, ) . as phenotypic traits respond to environmental change through shifts in the underlying genes, associations between genetic and environmental variation are assumed to result from local adaptation (manel, schwartz, luikart, & taberlet, ; storfer et al., ) . even without a sequenced reference genome, landscape genomic analysis of genetic markers, allowing the identification of genetic patterns associated with environmental variation (e.g., climate or land use), is a feasible strategy for any species (frichot, schoville, bouchard, & françois, ; manel & holderegger, ; rellstab, gugerli, eckert, hancock, & holderegger, ; sork et al., ) . high variation at genetic variants associated with temperature or habitat fragmentation may therefore indicate a high potential to adapt to future climate and land-use change. while the integration of this adaptive potential into sdm is under development (e.g., peterson, doak, & morris, ) , simple correlations between neutral and adaptive genetic diversity on the one hand, and sdm habitat suitability estimates on the other hand, indicates the extent to which adaptive evolution may affect sdm projections. high adaptive potential at the rear edge, for example, may prevent local extinctions despite a projected northward shift with climate change (erichsen et al., ; exposito-alonso et al., ) . low genetic diversity at suitable locations, on the other hand, may be indicative of imminent local extinction and poor connectivity, and/or of relatively recent colonization after a period of reduced habitat suitability (e.g., gutiérrez-rodríguez, barbosa, & martínez-solano, ; dekort, baguette, et al., ; dekort, prunier, et al., ) . conservation actions focusing on expanding or connecting sdm-based suitable patches holding populations of low genetic diversity may consequently increase options for dispersal and evolution. we therefore recommend conservation prioritization based on a combination of sdm and genetic marker assessment (figures . and . ) . although the collection of genetic material for genetic marker analysis may be particularly challenging for rare species, noninvasive sampling such as fecal, hair, and eggshell sampling may overcome this issue (e.g., depending on the scale of the study, land-use change scenarios ( figure . ) could rely on local managers that are aware of ongoing land-use developments, regional storylines related to demands of arable production, livestock number, urbanization, and/or international socioeconomic parameters (e.g., dekort, prunier, et al., ; dullinger et al., ; la sorte et al., ; marshall et al., ) , or global land-use scenarios as outlined by the intergovernmental panel on climate change (ipcc). land-use variables can subsequently be manipulated (e.g., partial conversion of extensive grasslands into forest, or forest into built-up area) using basic gis applications to generate sdm scenarios integrating both climate change and habitat fragmentation (e.g., lehsten et al., ; marshall et al., ; martin et al., ) . climate change scenarios have been outlined by the ipcc and are freely available at chelsa-climate.org and worldclim.org. at this point, all data are available for modeling, using dismo or another sdm framework (figure . ). models are first trained and tested using the occurrence points and (cropped) environmental maps, finally providing a habitat suitability map that reflects the present distribution. the model results are then extrapolated to predict future distributions under the provided scenarios. these projections do not account for dispersal and evolution, and require fine-tuning and contextualization based on species' life-history traits (through partial dispersal modeling, figure . ) and genetic markers (through discussion of the adaptive potential, figure . ). stevens et al., ) . these life-history traits can therefore be considered to define a partial dispersal sdm approximating the occupied distributional area to a more accurate extent than a sdm assuming no dispersal (bateman et al., ) . alternatively, it is possible to create a species-specific dispersal kernel and use it as a predictor variable capturing the impact of dispersal limitations on the occupied distributional area (hattab et al., ; meentemeyer et al., ; václavík & meentemeyer, ) (see box ). even relatively simple partial dispersal models, where the potential distribution has been clipped down to accessible distribution based on estimates of maximum dispersal distances, have been shown to improve distribution projections under environmental change (dekort, prunier, et al., ; fitzpatrick, gove, sanders, & dunn, ; meier et al., ; midgley et al., ) . the implementation comfort and the limited number of assumptions related to demographic rates make this type of partial-dispersal sdm the preferred option for many species (bateman et al., ) . the most reliable and informative partial dispersal sdms are expected for species with poor dispersal capacities, because poor dispersers (a) are often of high conservation concern, (b) facilitate integration of dispersal into sdm through assuming limited dispersal between suitable patches (figure . ) , and (c) provide conservative estimates of patch accessibility for associated species. although we do not specifically recommend to focus on poor dispersers, we do believe that this important target group should receive particular attention in future sdm studies aiming to develop dispersal-and evolution-informed conservation strategies. although model accuracy improves considerably in partial dispersal sdm, they still not fully reflect real conditions. a more mechanistic approach, for example, through hybrid models integrating both correlative and mechanistic principles, could further increase model reliability (see supporting information s ). in silico model parameterization and validation should be complemented with in situ model evaluation in unsampled regions, through extracting a set of suitable and unsuitable habitat coordinates from model output and empirically evaluate occurrence in the field (araujo, pearson, thuiller, & erhard, ; dekort, prunier, et al., ) (figure . ) . among the rare examples of studies using in situ sdm validation, williams et al. ( ) were able to find new localities (out of checkpoint sites) shared among four rare plant species across the rattlesnake creek terrane in california. area under the curve (auc) of the receiver operating characteristic (hanley & mcneil, ) , a commonly used model validation statistic retrieved from the cross-validation approach, ranged between . and . , commonly interpreted as nearly perfect predictive performance (but see box for pitfalls related to this model validation metrics like the auc statistic). two important conclusions can be drawn from this study. first, there can be considerable inconsistency between in silico (simulated) and in situ (real) model validation, which may reflect (a) the drawbacks of in silico model validation methods and (b) the (in)accessibility of suitable habitat patches. second, sdm studies can be highly suitable for conservation purposes, given that (a) validation was performed both in silico using independent calibration and testing data and in situ, (b) relevant environmental maps and scenarios are generated, and (c) dispersal and evolution are implemented. we finally recommend projected shifts in distributions to be followed up in situ at regular time intervals (figure . ) (see, e.g., areias guerreiro, mira, & barbosa, ; barbet-massin, rome, villemant, & courchamp, ; west, kumar, brown, stohlgren, & bromberg, ). this has two major advantages, including the ability to test the power of sdm approaches to predict distribution shifts and to assess the impact of conservation actions on projected shifts in sdm. we thank the referees and editor for handling this manuscript. all data used were accessed through referenced literature. https://orcid.org/ - - - delimiting the geographical background in species distribution modelling land use change emission scenarios: anticipating a forest transition process in the brazilian amazon crop wild relatives range shifts and conservation in europe under climate change spthin: an r package for 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in nature? meta-analysis and conservation implications forecasting the combined effects of climate and land use change on mexican bats failure to migrate: lack of tree range expansion in response to climate change planning for conservation and restoration under climate and land use change in the brazilian atlantic forest toward reliable habitat suitability and accessibility models in an era of multiple environmental stressors key: cord- -udokbcki authors: lilitsis, emmanouil; stamatopoulou, vaia; andrianakis, eleftherios; petraki, adamantia; antonogiannaki, elvira-markela; georgopoulos, dimitrios; vaporidi, katerina; kondili, eumorfia title: inspiratory effort and breathing pattern change in response to varying the assist level: a physiological study date: - - journal: respir physiol neurobiol doi: . /j.resp. . sha: doc_id: cord_uid: udokbcki aim: to describe the response of breathing pattern and inspiratory effort upon changes in assist level and to assesss if changes in respiratory rate may indicate changes in respiratory muscle effort. methods: prospective study of patients ventilated on proportional assist ventilation (pav+). at three levels of assist ( %- %- %), patients’ inspiratory effort and breathing pattern were evaluated using a validated prototype monitor. results: independent of the assist level, a wide range of respiratory rates ( - br/min) was observed when patients’ effort was within the accepted range. changing the assist level resulted in paired changes in inspiratory effort and rate of the same tendency (increase or decrease) in all but four patients. increasing the level in assist resulted in a % ( - %) decrease in inspiratory effort and a % ( - %) decrease in respiratory rate. the change in respiratory rate upon the change in assist correlated modestly with the change in the effort (r = . ). conclusion: changing assist level results in changes in both respiratory rate and effort in the same direction, with change in effort being greater than that of respiratory rate. yet, neither the magnitude of respiratory rate change nor the resulting absolute value may reliably predict the level of effort after a change in assist. assisted mechanical ventilation aims to assure adequate ventilation by assisting and partially unloading the respiratory muscles (pierson, ) . ideally, the level of assist should not only result in normal or near-normal arterial blood gases but also to normal or near-normal levels of inspiratory effort, to prevent both over-and under-assistance induced injury of the diaphragm . setting the level of ventilator assist in everyday practice relies mostly on the clinical estimation of inspiratory effort, as indicated by the breathing pattern -tidal volume (vt) and respiratory rate (rr)-and clinical signs of respiratory distress (boles et al., ; hansen-flaschen, ; hess, ; ray et al., ) . however, the relationship between ventilatory assist and inspiratory effort, tidal volume, and respiratory rate is complicated and multifactorial, affected by the patient's respiratory drive, ability to generate alveolar ventilation, and the mode of support (vaporidi et al., ) . therefore, although tidal volume and respiratory rate are commonly used in everyday practice to titrate the level of assist, the scientific evidence guiding assist titration to the patient effort is limited, derived from studies with a relatively small number of patients (berger et al., ; j o u r n a l p r e -p r o o f ] carteaux et al., ; giannouli et al., ; marantz et al., ) and do not consider the random/normal variability in breathing pattern, unrelated to the level of assist. the aim of this study was to ) characterize the responses of respiratory drive, respiratory effort, and breathing pattern to changing levels of ventilatory assist in critically ill patients and ) assess if changes in respiratory rate may indicate changes in respiratory drive and effort. to this end, during proportional assist ventilation with adjustable gain factors (pav+), noninvasive measurements of respiratory drive, effort (as indicated by inspiratory muscle pressure) were obtained at different levels of assist, using a validated prototype monitor (pvi) (kondili et al., ; younes et al., ) . contrary to conventional modes of assisted mechanical ventilation, pav+ permits the patients to select their desired breathing pattern as determined by feedback mechanisms of control of breathing (younes, ) . this study was conducted in the medical-surgical icu of the university hospital of heraklion between january and july after approval by the hospital ethics committee ( ). since the study protocol does not involve a therapeutic intervention and clinical or diagnostic interventions, it was considered as carrying no more than minimal risks, and informed consent was waived. patients were included in the study after at least hours of mechanical ventilation, when they fulfilled standard criteria for initiation of weaning from controlled mechanical ventilation (hemodynamic stability, reversal of cause of respiratory failure). exclusion criteria were: age under years, pregnancy, severe muscle weakness or cervical spine injury, uncontrolled pain, fever, or delirium, readiness for spontaneous breathing j o u r n a l p r e -p r o o f ] trial, and when pvi monitor was unavailable. the study was interrupted if the patient could not trigger the ventilator, developed respiratory distress, or became for any reason unstable (increase in vasopressor requirements, deterioration of oxygenation, etc.). at the time of the study, all patients were ventilated on assisted modes (pressure support or pav+). all patients were studied on pav+ at three different levels of assist %, %, and % at the peep set by the treating physician. each level of assist, applied randomly, was maintained for min. moreover, recordings were obtained for minutes at the mode selected by the treating physician (ps or pav+) before and immediately after pav+. arterial blood gases were obtained during the last minute of low ( %) and high ( %) level of assist. the pressure generated by the inspiratory muscles per breath (pmus) was evaluated as an index of effort and calculated on a breath-by-breath basis by a research prototype monitor (pvi monitor, yrt limited, winnipeg, canada) using a method described in detail previously. briefly, the inputs required by the monitor to calculate pmus are the airway pressure (paw) and flow (v′), whereas volume (v) was obtained by v′ integration. at least two points during expiration that satisfied passive conditions (i.e., flow was driven by the elastic recoil pressure) were automatically identified by the monitor. at these points, the equation of motion was applied, and elastance (erspvi) and resistance (rrspvi) of the respiratory system were calculated. using these values and the equation of motion, pmus was calculated in each breath. provide non-invasively measurement of pmus, as well as of tidal volume and respiratory rate in breath by breath basis (kondili et al., ; younes et al., ) . patient mechanical inspiratory time (ti) was measured as the interval between the beginning of pmus increase and the point at which pmus started to decline rapidly. patient mechanical expiratory time was measured as the remainder of the respiratory cycle. the rate of rise of pmus (dp/dt,) was calculated as the change in pmus during inspiration divided by ti and was used as an index of respiratory drive. the level of peepi during the different levels of assist was measured as the positive deflection of pmus from the onset of mechanical inspiration to the point of zero flow. additionally, the following data were collected: severity score on admission, etiology of respiratory failure, days on mechanical ventilation, icu, and hospital length of stay and outcome. the output of pvi monitor data was processed before analysis to optimize data quality (e.g., artifact rejection). from each -min period, the th to th min of the recording was selected for analysis. in each patient, for each variable and a given level of assist, the distribution of values was evaluated. means and sd were calculated after examining for normal distribution. the calculated means in each variable were used for comparisons among patients. between groups of patients or levels of assist, differences in continuous variables were as determined by the study design, at the end of the study period ( min), patients were placed again on the same ventilator settings used by the treating physician. thus, in each patient, recordings were available with the same ventilator settings at approximately min apart, while the patient's condition was considered stable (pre-and post-study). an analysis of ventilation variables in the pre-and post-study recordings was used to determine the variability (range of difference) of each variable in the 'stable' critically ill patient. we used ibm spss statistics for windows version (armonk, ny) for analysis. during months, critically ill patients were included in the study. the patients' characteristics are presented in table . patients were studied on the ± day of mechanical ventilation; the baseline measurements from the ventilator and the pvi monitor are presented in table . in the analysis of single patient data ( -min breath by breath analysis), and for all pvi variables, a normal distribution was found in more than % of cases, and thus, the mean values were used for comparison among patients. results were qualitatively similar when median values were used (data not shown). ventilatory variables in the pre-and post-study period were not different (table ) . this was true when the two ventilation modes (ps, pav+) were examined independently (data not shown). arterial blood gases were also not different before and after the study. the variability in tidal volume and respiratory rate, indicated by the coefficient of variation (tobin et al., ) , was % and %, respectively. the - range of change in each variable was calculated as an indicator of the variability in breathing pattern that can be expected in a relatively stable critically ill patient when ventilation settings have not been modified (table ) . we subsequently analyzed, independently of the level of assist, the correlation between indices of respiratory drive (dp/dt), effort (pmuspeak), and breathing pattern (tidal volume -vt and respiratory rate -rr). a very strong linear correlation was observed between respiratory drive and pmuspeak at each level of assist (r= . ). respiratory drive correlated weakly with respiratory rate (r= . ), but not with tidal volume. when respiratory drive and pmuspeak were within the accepted 'target' range for mechanically ventilated patients , a broad range of respiratory rate (r - : - br/min) was observed (figure ). finally, we examined the patterns of change of respiratory drive, pmuspeak, rr, and vt in response to changes in the level of assist. with increasing assist, small but statistically significant changes were observed in all variables (table ). the change in effort, in response to change in assist, was greater than the change in respiratory rate ( figure ). respiratory drive decreased as assist increased, and this change correlated strongly with the change in the effort (r= . ), but moderately with the change in respiratory rate (r= . ), and not with the change in tidal volume. the patterns of response to assist increase were characterized by a concomitant increase in tidal volume and decrease in respiratory rate, yet, these changes were mostly within the expected range of variation, and not specific for the change in effort (figure ) ). a significant change in respiratory rate, opposite to that of effort, was observed only in four patients ( %). the sensitivity and specificity of a decrease in respiratory rate to predict a decrease in effort was % and %, respectively, with a positive predictive value of %. yet, the change in respiratory rate correlated only modestly with the change in effort (r= . ) and the resulting inspiratory effort at high assist (r= . , figure ). in some patients (n= , %), effort did not decrease with increasing assist. the development of intrinsic peep due to increase in tidal volume ( cases) and the presence of high respiratory drive due to metabolic acidosis ( cases) were the most common causes of this phenomenon. this study in critically ill patients in the post-acute phase examined the changes in breathing pattern, respiratory drive and effort and their correlations, induced by changes of the level of assist. patients were studied using pav+, a mode that permits patients to choose their breathing pattern and monitored with pvi, a prototype monitor validated to estimate inspiratory muscle pressure (kondili et al., ; younes, ; younes et al., ) . the main findings of the study are: ) patients respond to changes in ventilatory assist mainly by changing effort per breath; ) when respiratory drive and/or effort are normal, a wide range of respiratory rate is observed; ) although respiratory rate changes towards the same direction as effort, neither the magnitude of change, nor the resulting value of respiratory rate are related to the level of effort, suggesting the limited role of respiratory rate in titrating assist to a target level of effort. in this study the correlation of indices of respiratory drive (dp/dt) and effort (pmus) was, as expected, very strong (de vries et al., ; mauri et al., ; vaporidi et al., ) . respiratory rate correlated weakly with respiratory drive, and most importantly, a wide range of breathing frequencies was observed in patients with relatively normal respiratory drive and effort. tachypnea was present in several patients with normal respiratory drive and pmus, and, although no patient exhibited hypoxemia, hyperactive delirium or fever at the time of the study, other causes of tachypnea, such as systemic inflammation, receptor stimulation or cortical stimulation could not be excluded (telias et al., ; vaporidi et al., ) . in addition, in our study, respiratory drive was estimated using the rate of increase of pmus, which in the presence of neuromuscular weakness underestimates the actual respiratory drive (the rate of increase of electrical activity of the respiratory center output). therefore, at the presence of neuromuscular weakness, respiratory center activity during inspiration may be high causing an increase in respiratory rate despite normal inspiratory pressure during inspiration. this observation is in line with several studies showing a higher than normal respiratory rate in critically ill patients (akoumianaki et al., ; giannouli et al., ; marantz et al., ) . moreover, a low respiratory rate was not uncommon, despite a high respiratory drive and effort, highlighting the inadequacy of respiratory rate as an indicator of respiratory drive and effort. this is not an unexpected finding as the respiratory rate in critically ill patients may be affected by several factors, most commonly sedation and opioid analgesia, which were also present in these patients (grap et al., ; mcgrane and pandharipande, ; vaporidi et al., ) . the response of critically ill patients to changes in assist was characterized overall by a decrease in respiratory drive, effort and respiratory rate, and an increase in tidal volume. patients changed their effort more often and to a greater extent than their respiratory rate upon change of assist and drive. this pattern of response is the same as the one observed in healthy subjects (duffin et al., ) . in our study in most patients, these changes of rr and vt were within the range of variability observed without a change in ventilator settings. moreover, this variability, for both rate and tidal volume, was found higher in the critically ill than in healthy individuals (tobin et al., ) . in mechanically ventilated patients a decrease in the respiratory rate in response to the increasing level of assist is usually considered as indicative of respiratory muscle unloading (esteban et al., ; esteban et al., ; sellares et al., ; yang and tobin, ) . this study identified that, indeed, a decrease in respiratory rate has a positive predictive value of % to predict a decrease in the inspiratory effort. however, this study also identified that it is only the direction of change and not the magnitude of the effect that can be predicted. thus, these results indicate that, although a change in effort in response to a change in assist can be predicted by the change in respiratory rate with reasonable accuracy, neither the absolute value of respiratory rate nor its change can indicate if the resulting effort is low, normal or high. some methodological issues and limitations of this study should be considered in the interpretation of results. first, the changes in breathing pattern were studied while the patients were ventilated only in pav+ mode. therefore, different changes in breathing pattern in response to changes in ventilator assist may be present during ventilation on psv. yet, a previous study has shown similar changes in patients' neural respiratory rate in response to varying the assist level between pav and psv (giannouli et al., ) . second, the estimation of inspiratory effort and respiratory drive was performed using the measurement of pmuspeak, instead of the gold standard method of trans-diaphragmatic pressure (pdi). however, we have previously shown that pmuspeak derived by pvi monitor provides an accurate estimation of inspiratory effort (kondili et al, ) . at different levels of assist during ventilation on pav+, pmuspeak was compared with transdiaphragmatic pressure (pdi), and the pressure developed by all respiratory muscles (pmus) -calculated with the campbell diagram and using chest wall mechanics measured at active and passive ventilation. no significant difference between pmuspeak, and pdi and pmus, in terms of timing, magnitude, shape, and the rate of pressure increase. setting the level of ventilator assist aims in adequate unloading of the respiratory muscles. in everyday clinical practice, the titration of assist usually aims to achieve a tidal volume of - ml/kg and a respiratory rate of - breaths/min. moreover, automated systems have been introduced to facilitate titration of assist, relying on measurements of tidal volume and respiratory rate. it is widely believed that high breathing rate may be indicative of excessive work of breathing, and that a decrease in respiratory rate with increasing the level of assist indicates unloading of respiratory muscles and thus adequate assist level. however, it has been shown that frequently tachypnea may be due to factors unrelated to respiratory load. the results of this study emphasize that respiratory rate is not a sensitive indicator of the patient's effort and that neither the magnitude nor the absolute change in the respiratory rate in response to varying the level of assist reflects the changes in inspiratory effort accurately. it is, therefore important, in clinical practice to avoid oversimplifications and assumptions such as that a relatively normal respiratory rate assures a normal level of inspiratory effort. during the j o u r n a l p r e -p r o o f ] recent covid- epidemic, it has been reported that patients with severe disease 'surpisingly' did not develop tachypnea and respiratory distress, an observation which is in line with the findings of this study, and not at all unexpected. in conclusion, the present study, in mechanically ventilated patients on the recovery phase of acute illness, showed that changing the assist level results in changes of inspiratory effort in more patients and to a greater magnitude, than of respiratory rate, though both effort and rate change almost always in the same tendency (either increase or decrease). yet, neither the magnitude of change nor the absolute value of respiratory rate can be used to quantitatively estimate the resulting respiratory effort after a change in the level of assist. the injurious effects of elevated or nonelevated respiratory rate during mechanical ventilation mechanism of relief of tachypnea during pressure support ventilation weaning from mechanical ventilation comparison between neurally adjusted ventilatory assist and pressure support ventilation levels in terms of respiratory effort assessing breathing effort in mechanical ventilation: physiology and clinical implications a model of the chemoreflex control of breathing in humans: model parameters measurement extubation outcome after spontaneous breathing trials with t-tube or pressure support ventilation. the spanish lung failure collaborative group effect of spontaneous breathing trial duration on outcome of attempts to discontinue mechanical ventilation. spanish lung failure collaborative group response of ventilator-dependent patients to different levels of pressure support and proportional assist sedation in adults receiving mechanical ventilation: physiological and comfort outcomes dyspnea in the ventilated patient: a call for patient-centered mechanical ventilation ventilator modes used in weaning estimation of inspiratory muscle pressure in critically ill patients response of ventilator-dependent patients to different levels of proportional assist oxygenation patients recovering from severe acute respiratory distress syndrome sedation in the intensive care unit indications for mechanical ventilation in adults with acute respiratory failure acute respiratory failure in the elderly: etiology, emergency diagnosis and prognosis diaphragm-protective mechanical ventilation lung-and diaphragm-protective ventilation in acute respiratory distress syndrome: rationale and challenges predictors of weaning after acute respiratory failure is my patient's respiratory drive (too) high? variability of resting respiratory drive and timing in healthy subjects respiratory drive in critically ill patients. pathophysiology and clinical implications a prospective study of indexes predicting the outcome of trials of weaning from mechanical ventilation proportional assist ventilation, a new approach to ventilatory support. theory a method for monitoring and improving patient: ventilator interaction the authors declare that there is no conflict of interest. this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. key: cord- - jbehcb authors: nan title: confronting the pandemic superthreat of climate change and urbanization date: - - journal: orbis doi: . /j.orbis. . . sha: doc_id: cord_uid: jbehcb nan the grand illusion. . . is that the collectivity of states is capable of agreeing upon the taking of decisive action. -colin s. gray uch has been written about climate change and its causes and effects. while some deny that climate change is happening or that humans have any mitigating role to play, most reasonable people would concede that climate change poses real problems that must be addressed. regardless of one's skepticism on the matter of climate change, it is essential to consider its worst-case outcomes when developing u.s. security strategies and policies. as the department of defense (dod) has reported to congress, "the effects of a changing climate are a national security issue with potential impacts to [dod] missions, operational plans, and installations." of the projected negative effects of climate change, from severer storms to more extensive droughts, probably the threat with the most devastating potential is a pandemic. from disrupting trade to decimating populations, a pandemic could potentially cause more destruction than a nuclear strike; thus, it is essential to understand how changing climate could unleash such destruction-with little to no advance warning-and how we can best manage the consequences. most experts agree that the world is heading into a potentially severe warming phase, with average temperatures expected to rise - degrees celsius (c) by the end of this century. as a result, weather events such as heat waves and storms, and the attendant consequences of droughts and floods, are expected to increase in frequency and severity. and due to these climatic and weather changes, a significant increase is expected in the potential for infectious diseases transmitted via insects, rodents, and contaminated water. deadly diseases carried by insects, such as malaria, thrive at higher temperatures and so are expected to be a more active threat in the years ahead. in addition, waterborne infectious diseases, such as cholera, will also likely increase due to poor sanitation caused by water scarcity in times of drought and by "overwhelmed sewage lines or the contamination of water by livestock" during times of flooding. as an example of how bad a cholera epidemic can become, consider the recent outbreak in yemen, which erupted in april and has since led to over one million suspected cases and over , deaths. the ongoing civil war in yemen and the resulting scarcity of water have exacerbated the epidemic. pandemic disasters caused by climate change are not new; there are plenty of examples in world history. one especially tragic example is the "black death" of the mid-fourteenth century, which killed upwards of million people in europe and asia and likely was caused by climatic changes that stimulated the growth of the rodent population. certainly, this example involved a complex interaction of factors over an extended period of decades. a mild climate in eastern kazakhstan and southwest china "fostered plant-food abundance and hence wild rodent proliferation." as a subsequent cooling took place, "plant growth declined" and catastrophic floods "displaced and drowned many people." the environmental changes "likely also displaced wild rodents and increased rodent-human contact." at the same time, in western china "encroaching nomadic mongol pastoralists" and "han chinese farmers" engaged in increased conflict, and this also displaced people and likely increased rodent-human contact. this chain of climatic effects could well have potentiated a deadly plague epidemic. "before long, trade caravans or (more probably) horse-borne mongol armies, with 'companion' black rats, carried the disease westward shuman, "global climate change and infectious diseases." into europe . . . via the black sea port of kaffa." within ten years, europe's population was decimated. this pandemic starkly illustrates the dark side of fourteenth-century globalization. it demonstrates all too clearly that climate changes in one location can result in cascading effects that ripple around the world and eventually wreak havoc far removed from the source of initial disaster. those forces were the same ones that we face today: natural disasters, migrations of people, conflict over scarce resources, and ultimately a pandemic transmitted around the world through trade and war. at present, with billions more people and modern means of transportation, it is clear the same situation today would be exponentially more disastrous. lest one think that modern society is safer due to advanced technology and capabilities, consider that many experts believe the opposite: modern societies, though better resourced and more interconnected, are also more densely populated and infrastructure-dependent, and hence less flexible and more vulnerable to a sudden disastrous shock. one of the starkest differences between now and the mid-fourteenth century is how populated the earth is at present, and how much of that population has crowded itself into urban centers. the current world population is over . billion people, approximately percent of whom live in urban environments. compare that to a world population of approximately million before the onset of the "black death," most of whom lived in rural, sparsely populated areas. looking ahead, the trend to urbanization is only expected to continue: experts estimate that roughly percent of the population will live in dense urban environments by the year . while the movement of rural populations to cities is caused by several factors, including economic and educational opportunities, it is being exacerbated by the increased incidence of droughts, floods, and other consequences associated with climate change. these climate-related disasters hurt agricultural production and serve as "push" factors from rural to urban, just as jobs and education serve as "pull" factors. experts predict that climate-induced migrations potentially will accelerate after . most of the urban growth is projected to occur in asia and africa, where developing countries dominate the landscape and where basic healthcare, sanitation, and other infrastructure is sorely lacking in rural areas. as a result, cities in developing countries that are already at, or near, the limits of the population they can adequately support are being slowly overwhelmed by at-risk migrants who are overtaxing already weak infrastructure and security systems. this problem is exacerbated by the rapid development of megacities, defined as having a population in excess of million. "in , developing nations boasted megacities compared to seven in developed countries." this trend toward expansion in developing countries is set to continue, especially in africa. for example, luanda, predicted to become a megacity by , "is expected to undergo a percent increase in its population between and , placing further strain on housing, transport, public utilities and sanitation." one critical problem with the expanding urban landscape is that the growth is largely uncoordinated. densely populated shanty towns and slums ring the edges of otherwise modern and ordered square blocks of skyscrapers and malls. the uncontrolled development leads to increased human interaction with rats and other rodents, thereby greatly increasing the likelihood of zoonotic infectious diseases. also increasing the potential for zoonotic contagions is that the new populations push cities out into previously undisturbed ecosystems (e.g., adjacent deserts and forests), and as a result contact between animals and humans increases, generally in unsanitary environments. the overcrowded conditions heighten the risk of vector-borne diseases, such as malaria and dengue fever. basic sanitation and water systems become overwhelmed, resulting in contaminated water supplies and increasing the potential for cholera and other water-borne diseases. diseases that were formerly confined to rural areas accompany the migrants into the new urban environment, where they have a much greater chance to thrive. security services are often unable to properly police the expanded slum areas, which can become lawless and less secure. in addition, where order has disappeared, other city services generally will not go. most critically, existing healthcare services cannot properly serve the increased population. if a pandemic takes root in such an environment, chances are high that it will spread rapidly before it can be detected. to understand how severe the problem can become, one can look at mogadishu, somalia. mogadishu's devastation has been caused by many factors, including poverty, violence, and lack of effective governance. climate change has been a significant contributor to all of this. as scholars giovanna kuele and ana cristina miola have noted, "over the past decade climate change-related desertification has expanded in somalia, greatly increasing the vulnerability of the local population." this change has sharpened disputes over scarce water resources and has led to "high rates of malnutrition, disease outbreaks, and food insecurity." cholera epidemics have been recurrent in somalia, the most recent one starting in late , and thousands have died as a result. with disease and violence rampant, over one million somalis have been displaced; with mogadishu in utter chaos, many of them have migrated to nearby kenya, thus putting a strain on kenya's resources. in the yemen cholera outbreak noted above, scientists have determined that the strain causing the outbreak "came from eastern africa and entered yemen with the migration of people in and out of the region." thus, it is likely that somalia's dysfunction has contributed to yemen's. combined, these problems have enabled easier recruitment of warlords, reyes et al., "urbanization and infectious diseases," p. . intent on controlling the capital city mogadishu through violence and terror. mogadishu is the prime example of what richard j. norton has termed "feral cities," where degradation and insecurity create conditions where disease and violence flourish. be realistic. more aggressive action is required to resolve, or at least mitigate, the immediate consequences and near-horizon disasters looming as a result of climate change and increasing urbanization-especially regarding the possibility of a pandemic. several arms of the u.s. government, including the department of state (dos), the u.s. agency for international development (usaid), and the centers for disease control and prevention (cdc), are actively combatting disease and climate change around the world. however, none of these agencies can match the global presence and financial heft of dod. this realization is especially true in the current political environment, as the trump administration seems intent on cutting foreign assistance and health budgets while increasing dod's. for example, president trump's fy budget "requests $ billion for dod, a $ billion or five-percent increase from the enacted level"; at the same time, it "requests $ . billion for the department of state and usaid, a $ . billion or -percent decrease from the estimate." congress is unlikely to agree with the president's overall budget proposal, now or in the future, but it seems safe to predict that military resources will increase. due to its global reach and resources, the u.s. military is better placed than "the relationship between the proliferation of illegal armed groups and the severe droughts in somalia is evident in the case of al-shabaab. the group has been successful in attracting young people who are affected by famine and food insecurity and who face no job prospects; those youth end up joining the armed group in a bid to survive, finding no other option other than to submit to the extremist group. nearly any other organization and should take an aggressive leading role in preventive efforts. unfortunately, to date, the international community has not demonstrated the will to act decisively on climate change. while many nations voluntarily have signed onto international agreements, such as the kyoto protocol and the paris agreement, few of these countries adhere to the agreed standards. and, the agreed standards themselves have been called into question as not being sufficient. in addition, some of the greatest contributors to pollution and environmental degradation flagrantly ignore climate concerns. indeed, both india and china, two of the biggest polluters in the world, were exempted from the kyoto protocol, and the paris agreement contains no formal enforcement mechanisms-one of the glaring weaknesses of internationally engineered solutions to climate change. at the recent climate gathering in katowice, poland, participants agreed to a new rulebook to improve the paris agreement; however, details of the "rulebook" are not clear, and key countries did not support the creation of a global carbon market, seen by many economists as the key to reducing carbon emissions worldwide. the gathering appeared to promise more of the same: heavy polluters will continue to pollute, while certain developed countries will continue voluntarily to reduce emissions (or not). this is not to say that international government organizations (igos) bring no benefit to the discussion. the united nations and other igos serve a useful role in raising awareness, providing a forum for debate, and providing resources in some david g. victor, keigo akimoto, yoichi kaya, mitsutsune yamaguchi, danny cullenward, and cameron hepburn, "prove paris was more than paper promises," nature, aug. , , https://www.nature.com/news/prove-paris-was-more-than-paper-promises- cases. for these reasons alone, the united states should stay actively involved in international efforts to combat climate change and ensure as much as possible that u.s. interests are promoted. however, a realist understands that due to the inefficiencies of international consensus-building, igos' actions generally will be longer-term solutions with slow and clumsy implementation. as divya srikanth has written: "developed states are highly reluctant to voluntarily stunt their economic growth by adhering to the kyoto protocol, and developing states are unwilling to compromise on their new-found economic successes. this has led to a stalemate in terms of tackling the impact of climate change." though the stalemate shows no positive signs of breaking, due to the severity of the stakes involved, america cannot abdicate leadership in this long-term effort. at the same time, it must lead efforts to combat the near-term consequences of climate change. engage partners, including china. fighting pandemics is one issue on which nations should share strategic goals and could therefore use the opportunity to build better relations. the united states should utilize this common interest and leverage alliances and partnerships in the most at-risk locations. for example, one of the current hotspots for a future pandemic to break-out is southeast asia, specifically the sulu-sulawesi sea area. the u.s. military currently works closely with indonesia, malaysia, and the philippines-the countries that ring the sulu-sulawesi sea-on counterterrorism and anti-piracy actions in the area. these relationships could be leveraged to take additional actions to prevent a pandemic from breaking out there, such as increased coordination and construction of research, detection, and healthcare facilities along the sea's edge. sulu-sulawesi sea and the adjacent south china sea, could be pulled into a multilateral partnership to fight against a pandemic. there are multiple challenges to a u.s.-china partnership, including lack of trust and a competition for influence, especially in southeast asia. as author robert kaplan recently wrote, u.s-china differences are "stark and fundamental" and "can barely be managed by negotiations and can never really be assuaged." however, there are opportunities to exploit, one of which is china's experience dealing with its own deadly disease outbreak in - , when severe acute respiratory syndrome (sars) took root in guangdong province and eventually infected over , people and killed more than . according to one chinese public health official, "the sars crisis provided an enormous boost to the development of china's public health system." china is also considered by some to be a likely origin of a future pandemic, especially from h n bird flu, which chinese and western scientists have been studying closely in hopes of preventing a disastrous spread. presumably, china has significant knowledge and resources to contribute in the war against pandemics. china also is keen to build roads, ports, and other facilities as part of its belt and road initiative (bri), especially in developing regions such as africa, and it has significant experience managing the growth of megacities. through effective partnering, china's infrastructure investments and its planning experience could be harnessed toward mitigating the negative consequences of climate change and urbanization. to consider an indirect approach, china is realizing that its rapidly aging population will not accommodate the country's current plan of sustained economic growth as a means to sustain internal stability and increase global influence. as china envisions negative population growth as early as , it will need to partner creatively with other countries and organizations to attain the necessary prosperity to support its changed demographics. the united states should leverage china's changing domestic politics to pull it into constructive partnerships on health and climate-change (and other) matters, and thereby, it is hoped, increase its stake in an international rules-based order. this becomes especially important as china seeks to build-up its navy and spread its influence. as bernard-henri levy has written, "one may hope, though without illusions, that china will send its steel junks in the wake of the fleets of the wise zheng he, china's admiral of the western seas and leader of expeditions bound for glory and not for conquest." active and constructive engagement on human-security problems is one way to help make this happen. research and model. one of the most effective defenses against a future pandemic is to undertake research now in the hopes of discovering the potential pandemic before it discovers us. several governments and ngos are actively engaged in this type of research; however, they often lack adequate resources or cohesion to be truly effective. the u.s. military could fill a serious need by bringing its resources to bear. the naval medical research unit (namru) is an entity that the military could employ to establish laboratory facilities to conduct research and support that of others, and to help develop and maintain an organized database of useful information. usaid's emerging pandemic threats program is one effort that this unit could support more deeply, and an example of an effective component of this program is the predict project, based at the university of california, davis. these organizations and others are researching contagions that potentially could infect humans, especially from animal hosts, and possible vaccines to combat them. researchers perform their work in areas where contagions are most likely to take root, such as the sulu-sulawesi sea described above. it is areas like this where the u.s. military should concentrate its efforts. as part of its research, the u.s. military can engage in predictive modeling, trying to determine based on weather patterns, people movements, socioeconomic factors, and potential contagions-what this author collectively refers to as pandemic intelligence-where the next pandemic might break out. an effective partner would be the national oceanic and atmospheric administration (noaa), which has programs and technology in place to monitor weather patterns and climate activity. the military could harness noaa's capabilities and knowledge, and that of others, to consider the potential cascade effects that would occur should a pandemic take root and spread. as chad michael briggs has noted, "environmental systems often exhibit feedback and multiplier effects, where a smaller change in one area leads to a cascade of impacts with much greater shifts elsewhere." for example, a heatwave in russia caused a food shortage and price spikes in north africa and the middle east and thus likely contributed to the arab spring revolts. cascade predictions could help to predict both near-and long-term consequences and would thus be critical in ensuring up-to-date operational plans to be used by the military when responding to pandemic outbreaks, and for efforts to prevent or contain outbreaks when responding to natural disasters. surveil and detect. it is also critical to conduct surveillance of possible threats from weather events, sudden migrations of people, and detected contagions. many developing countries lack modern and reliable warning systems for incoming severe storms. one of the reasons more than , people died when a tsunami hit sulawesi, indonesia, in september , was that the populations in its path were not properly forewarned of the seriousness of the danger. the u.s. military should help to ensure that essential warning systems are in place at critical spots, especially in southeast asia. in addition to working with local stakeholders to assess critical vulnerabilities, the military should coordinate with agencies such as noaa, which has models and equipment in place to warn of natural disasters. for example, noaa has tsunami detection buoys placed throughout the world's oceans and seas, most heavily in the pacific. however, despite these buoys and noaa's predictive capabilities, indonesia and other countries continue to rely on their own systems, with oftentimes disastrous results. in indonesia's case, its warning system was no longer operational in september due to lack of maintenance. because of its worldwide presence and resulting unique relationships and influence, the u.s. military is well-positioned to ensure coordination between developing countries and u.s. agencies such as noaa, who have capabilities to help but sometimes lack the resources to effectively coordinate. when natural disasters do occur, the u.s. military should maximize its strengths to complement the work of other relief organizations. for example, drone and satellite technology can monitor sudden migrations of people and detect infectious diseases that might be accompanying a group. scientists have experimented with using drones to take samples of mosquitos and other potential carriers of disease, and this could be an effective way to discover contagions before they hit a large city, or to determine which parts of a city might be at risk once a contagion has taken root. in addition, drones could be used to deliver self-spreading vaccines and other countermeasures to remote or highly infected locations. drones and satellites could also be used to monitor at-risk environmental ecosystems. scientists are developing techniques to use these technologies to monitor the health of crops, and the same ideas conceivably could be used to research the potential for zoonotic diseases to take root, especially in areas where human-animal interactions are unexpectedly increasing. the pandemic intelligence that results could be valuable not just for local predicting, but also for the cascade modeling described above. due to its reach and resources, the u.s. military is ideally situated to assist in this type of research and should work with the governments of at-risk nations, other u.s. government agencies, and allies who share common interests in the at-risk areas. as cities greatly expand in the coming decades, many predict that only a small percentage of them will be healthy and properly regulated; most will contain large areas of at-risk populations, where a pandemic could easily take root and spread. many cities in developing countries lack proper sanitation and sewage facilities and have insufficient healthcare infrastructure. to help correct this, the u.s. military should increase its partnering of civil-affairs and engineering resources with at-risk cities and development organizations to upgrade and build necessary infrastructure. because a significant reason for increasing urbanization is migration from rural areas, at-risk governments should take steps to improve rural infrastructure and services as well. again, the u.s. military has reach and resources that others cannot match. it could expand ongoing efforts, such as the pacific environmental security forum, and make them more frequent and routine. it is essential to expand partnerships with and support the world health organization (who), usaid, and others to add facilities, provide training, conduct services in the most vulnerable areas, and help cities better plan for expansion and a proper balance of interaction between humans, animals, and johns hopkins, "technologies to address global catastrophic biological risks," p. . sonia shah, "how cities shape epidemics," the atlantic, feb. , , https://www.theatlantic.com/health/archive/ / /urbanization-pandemicexcerpt/ /. neiderud, "how urbanization affects the epidemiology or emerging infectious diseases," p. . "between and , the group in urban settings which lacked sanitation actually significantly increased from million to million, which could be explained by population growth." based on this author's experience as economic officer in iraq, the u.s. military civil affairs teams were better at enacting effective and pragmatic changes than were usaid and other non-military organizations. this author worked closely with civil affairs team members who had agriculture, finance, and engineering expertise and who effectively shared it with iraqi government officials and business managers to bring about positive change. reyes et al., p. . for a description of the program and ongoing projects, see, "pacific environmental security forum," u.s. indo-pacific command, http://pesforum.org/. delicate eco-systems. as noted earlier, an effective partner could be china, whose focus on public infrastructure could be strengthened by america's traditional focus on policy reform and technical assistance in sectors such as healthcare. the u.s. military should also upgrade its own capacity in line with the new threat environment. while we place a renewed focus on great-power competition with china and russia and continue hedging against terrorism and other intermediate threats, we should also factor in human-security challenges such as pandemics. our force structure should change to reflect this. for example, we should add more hospital ships and invest more heavily in health-related artificial intelligence (ai) capabilities, even if that means cutting warships. in addition to providing essential medical services, the hospital ships could be redesigned and updated to include extensive laboratory facilities and drone and ai platforms to assist with research, surveillance, and detection. the drones could also be used to deliver medical supplies to hard-to-reach locations. some would argue that this change in force structure would cut against the grain of the national defense strategy's emphasis on traditional "lethality" and its focus on great-power competition, diverting money from much-needed warfighting capabilities. however, it is reasonable to argue that the american homeland and overall global security face a deadlier threat from a pandemic than from chinese or russian aggression. as an example, one should consider the spanish flu that broke out in , and by the spring of , it "had sickened an estimated one-third of the world's population and may have killed as many as million people," including approximately , americans. this black swan attacked a world completely unprepared for it, and its death toll was higher than that of the great war that immediately preceded it. a more recent example is the swine flu pandemic of , which, according to cdc, killed an estimated , people. security against this kind of non-traditional threat involves a different kind of lethality, one in which we invest in capabilities that are designed to preempt and destroy contagions rather than people and infrastructure. adding hospital and laboratory ships, and the associated drone and ai capabilities, would greatly improve our ability to provide medical services and research and surveillance support at the most at-risk locations. it would also demonstrate a positive u.s. presence to huge segments of the developing world's population. from a budgetary perspective, consider that "the cost to the global economy of sars is estimated to have been $ billion, according to the world bank, while the organization estimates that a 'severe flu pandemic' could cost over $ trillion, nearly five percent of global gdp." even if a newly designed hospital and laboratory ship carried a price tag of $ billion or so, the potential of catastrophic loss from a pandemic dwarfs the investment in human security that would come with the procurement of a fleet of these ships. when one considers that the majority of the world's growing urban population resides "in a coastal zone or a zone with distinct coastal influence," the argument to update the navy's human-security capabilities becomes even more cogent. as climate change and urbanization progress, pandemics are likely to occur, and american national-security professionals must consider this worst-case scenario as they develop strategies and policies. if we actively pursue preventive and mitigating actions, the outcome of a pandemic could be a disaster that can be managed, contained, and learned from. the alternative will be more mogadishus, or worse. igos play an important role in encouraging international debate and in providing limited resources; however, to address the looming threat of deadly contagion, we must take aggressive and pragmatic action. due to its reach and resources, the u.s. military is well-placed to lead. it should partner with governments and ngos, leveraging existing alliances and building new ones. primary actions should include conducting research and creative predictive modeling; ensuring effective surveillance and detection efforts; and upgrading and building infrastructure in high-risk areas. the military should also revise its capabilities to better address the growing pandemic threat. it is appropriate to close with an overarching statement of purpose from the u.s. national biodefense strategy: "the united states will use all appropriate means to assess, understand, prevent, prepare for, respond to, and recover from biological incidents-whatever their origin-that threaten national or economic security." due to the interconnectedness of today's world, it is hard to imagine a pandemic that would not have potential to threaten american security. we must approach the fight against pandemics aggressively, detecting, containing, and destroying a pandemic when it attacks, and the rest of the time working to prevent and prepare for the next pandemic. as the climate warms and eco-systems change, new and deadlier contagions will enter the fight, and we must stay engaged. groundswell: preparing for internal climate migration here's what you need to know about the megacities of the future here's what you need to know about the megacities of the future urbanization and infectious diseases: general principles, historical perspectives, and contemporary challenges how urbanization affects the epidemiology or emerging infectious diseases suburban sprawl into previously unpopulated areas is also a significant cause of new interaction between humans and other species how urbanization affects the epidemiology or emerging infectious diseases urbanization and infectious diseases: general principles, historical perspectives, and contemporary challenges," p. ; see, also, thayer, darwin and international relations, p. a new cold war has begun outbreak-changed-mainland-china. for infection and death figures, see, anmar frangoul how the sars outbreak changed mainland china is china ground zero for a future pandemic? how china sees the world: han-centrism and the balance of power in international politics one of them is its showcase city, shanghai, with a population of . million. see, sharon omondi china lowered its economic growth target "to between % and . %, bowing to a deepening slowdown that can't be quickly arrested without aggravating already-high debt levels the empire and the five kings: america's abdication and the fate of the world many governments of developing countries do not have the capabilities to organize effective response efforts, and igos, usaid, and others often do not have sufficient resources. "review of the dod-geis influenza programs: strengthening global surveillance and response usaid, emerging pandemic threats for example, see, noaa's following sites on climate and exploration tools: noaa, climate climate security, risk, assessment and military planning climate security, risk, assessment and military planning how urbanization affects the epidemiology or emerging infectious diseases surveillance is of primary importance to monitor the burden of disease and will give both local authorities and the global community a chance for a quick response to public health threats the art of predicting unpredictable tsunamis indonesia tsunami early-detection buoys haven't worked for six years due to 'lack of funding climate security, risk, assessment and military planning the art of predicting unpredictable tsunamis how tsunami early warning systems work, and why indonesia's system failed from self-spreading vaccines to d drugs: the tech that will stop a pandemic for a thorough review of research and technologies that could be used to prevent and respond to pandemics, see how urbanization affects the epidemiology or emerging infectious diseases adequate city planning and surveillance can be powerful tools improve the global health and decrease the burden of communicable diseases navy is considering upgrades to its hospital-ship capability, but the thinking seems too constricted for the human-security challenges, such as pandemics, that we face. the navy's focus is more on care and not enough on research and prevention. megan eckstein with the first line of effort being lethality, the undersecretary said, 'make no doubt about it-we are about warfighting the central challenge to u.s. prosperity and security is the reemergence of long-term, strategic competition by what the national security strategy classifies as revisionist powers why was the influenza pandemic called the 'spanish flu for an analysis of world war i deaths, see, david stevenson, cataclysm: the first world war as political tragedy counting the cost of a global epidemic retire-one-of-its-two-hospital-ships. this author has seen the huge impact that hospital ships can have on u.s. diplomacy. when he served in cambodia, the usns mercy conducted a humanitarian visit as part of pacific partnership . the doctors and nurses on the ship treated thousands of cambodians for health problems. this left an impact that will stay with those people-and their families, friends, and villages-for the rest of their lives. overall, the pacific partnership mission visited six southeast asian countries and treated , patients. for an overview of the mission, see counting the cost of a global epidemic the navy eyes replacing its hospital ships with a fleet of smaller medical vessels megacities and large urban agglomerations in the coastal zone: interactions between atmosphere, land, and marine ecosystems for a good discussion on the need for continuous vigilance, see, gray, fighting talk as noted above, the increased interaction of humans, animals, and previously untouched environmental ecosystems will likely lead to new contagions infecting humans. there are also unknown viruses lurking in the arctic. for a good discussion on the discovery of previously unknown viruses buried beneath permafrost, see key: cord- -o vm vdh authors: giordono, leanne; boudet, hilary; gard-murray, alexander title: local adaptation policy responses to extreme weather events date: - - journal: policy sci doi: . /s - - - sha: doc_id: cord_uid: o vm vdh at a global level, climate change is expected to result in more frequent and higher-intensity weather events, with impacts ranging from inconvenient to catastrophic. the potential for disasters to act as “focusing events” for policy change, including adaptation to climate change risk, is well known. moreover, local action is an important element of climate change adaptation and related risk management efforts. as such, there is a good reason to expect local communities to mobilize in response to disaster events, both with immediate response and recovery-focused activities, as well as longer-term preparedness and adaptation-focused public policy changes. however, scholars also note that the experience of disaster does not always yield policy change; indeed, disasters can also result in policy inertia and failure, perhaps as often or more often than major policy change. this study poses two key research questions. first, we ask to what degree policy change occurs in communities impacted by an extreme weather event. second, we seek to understand the conditions that lead to adaptation-oriented policy adoption in response to an extreme weather event. our results suggest two main recipes for future-oriented policy adoption in the wake of an extreme weather event. for both recipes, a high-impact event is a necessary condition for future-oriented policy adoption. in the first recipe for change, policy adoption occurs in democratic communities with highly focused media attention. the second, less expected recipe for change involves republican communities that have experienced other uncommon weather events in the recent past. we use a comparative case approach with cases and fuzzy set qualitative comparative analysis methods. our approach adds to the existing literature on policy change and local adaptation by selecting a mid-n range of cases where extreme weather events have the potential to act as focusing events, thereby sidestepping selection on the dependent variable. our approach also takes advantage of a novel method for measuring attention, the latent dirichlet allocation approach. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. at a global level, climate change is expected to result in more frequent and higher-intensity weather events, with impacts ranging from inconvenient to catastrophic (allen et al. ). the potential for disasters to act as "focusing events" that introduce windows of opportunity during which political actors compete to define the problem, identify solutions, and promote (or suppress) major policy change (birkland (birkland , kingdon ; rudel ) , is well known. in the face of a changing climate, local communities, which are heavily engaged in emergency and disaster preparation and often bear the brunt of losses associated with disaster events (crow et al. ; reinhardt and ross ; vogel and henstra ) might be expected to respond to extreme weather events with immediate response and recovery-focused activities, as well as longer-term climate change adaptationand mitigation-focused activities manifested as distinct policy changes. however, policy change is context-and event-dependent, and scholars note that the experience of disaster does not always yield policy change. indeed, disasters can also yield policy inertia and failure, perhaps as often or more so than major policy change (birkland (birkland , boin et al. ; nohrstedt nohrstedt , plein ) . while few studies have examined local policy responses to extreme weather events, those that do find both that extreme events can lead to policy change, and that community-and event-specific conditions matter. for example, mullin and rubado ( ) find that local problem conditions and institutional capacity influenced the adoption of water usage restrictions in the context of local drought. crow et al. ( ) find that disasters associated with colorado floods acted as "windows of opportunity for learning," especially in the context of lower fiscal capacity and high disaster-related damage. and yet, not all catastrophic events lead to policy change, as shown by the policy inertia that characterized response to the catastrophic flooding in southern west virginia (plein ) . a larger body of literature is focused on individuals' climate change beliefs, discussion, and policy preferences in the wake of extreme weather events, though results are mixed. despite broad evidence of political polarization on the issue of climate change (dunlap and mccright ; marquart-pyatt et al. ) , researchers have shown that personal harm from an event can change behavioral intentions and increase support for climate change policies (demski et al. ; zanocco et al. zanocco et al. , . however, recent research also suggests that community action may not be as sensitive to climate change beliefs and discussion as previously imagined javeline et al. ; orlove et al. ) . extreme weather events therefore provide fruitful ground for learning more about the role of focusing events in local climate change adaptation and mitigation efforts. this study explores the conditions under which an extreme weather event, with a strong potential to function as a focusing event, yields local policy change. weather events are exogenous at the local level, even when they are broadly attributed to human activity, strengthening our ability to infer a connection between the event and policy outcomes. this study poses two key research questions. first, we ask to what degree communities impacted by an extreme weather event enacted post-event policy change. second, we seek evidence of the conditions that lead to adaptation-oriented policy change in response to an extreme weather event. we selected cases where extreme weather events have the potential to act as "focusing events," on the basis of birkland's ( birkland's ( , birkland's ( , definition: events that are sudden, relatively rare, harmful and made known simultaneously to the public and policymakers virtually simultaneously. we expect that these cases will enable us to draw connections between extreme weather events and community-level adaptation-focused policy change. our approach fills several gaps in the literature. while the focusing events literature has contributed to our understanding of change at the national level, we know less about the conditions under which such events yield local policy change (crow et al. ; vogel and henstra ) , especially after extreme weather events, which may or may not yield catastrophic consequences. in contrast, the growing interdisciplinary literature linking extreme weather with climate change has focused primarily on individual attitudes and actions, yielding fewer insights about local policy actions resulting from such events. we take advantage of overlapping policy process and local climate change adaptation frameworks to develop a conceptual model of policy change in the wake of extreme weather events, contributing to the burgeoning adaptation (palutikof et al. ). moreover, while most studies select a small number of cases based on their responses to weather events, we select a mid-n number of cases ( ) based on their exposure to extreme weather events, rather than their policy response, offering more variation in our cases and avoiding the problem of selection on the dependent variable (king et al. ). finally, this study uses two novel methodological approaches: ( ) fuzzy set qualitative comparative analysis, a set-theoretic approach for identifying combinations of conditions that lead to an outcome; and ( ) latent dirichlet allocation, a computational topic modeling approach that uses text-based analysis to measure attention and problem definition. we find a moderate level of policy change in response to extreme weather events. the largest share of policy measures was focused on anticipating and responding to future risk, followed by responses to current risks, and building risk management capacity. we find scant evidence of policies focused on mitigation that were proposed after an event. enacted policies were relatively modest in scope, with few examples, if any, of pioneering policy innovations. we note two recipes for policy change. for both recipes, a high-impact event is a necessary condition for adaptation-oriented policy adoption. in the first recipe for change, policy adoption occurs in democratic communities with relatively focused median attention. the second, less expected recipe for change occurs in majority republican communities that have experienced other uncommon weather events in the recent past. moreover, we also find two recipes accounting for policy stasis (i.e., not policy change), both of which reflect the experience of predominantly democratic communities in the wake of low-impact and low-attention events. these results underscore previous observations about the power of focusing events, but importantly, suggest that political polarization and avoidance of climate change talk may not prevent communities from taking adaptation-oriented policy action after an extreme weather event. thus, while there is substantial evidence of political polarization about climate change, our work aligns with recent media coverage (flavelle ; smith and schwartz ) and scholarship javeline et al. ; orlove et al. ) highlighting the potential for communities to take action even while refraining from, or even avoiding, discussions about climate change, especially in the context of high-impact events. policy process scholars have long been interested in the determinants of policy change and conditions under which policy change occurs, in a variety of substantive arenas, including environmentally focused and disaster-inspired policy change. birkland's ( birkland's ( , development of the focusing events concept, inspired by kingdon's ( ) seminal work on agenda-setting in the policy process has demonstrated the power of catastrophic events to act as focusing events (birkland ; birkland and warnement ) . while it is widely accepted that some events have the potential to act as focusing events, generating significant attention, mobilization, and policy change, less is understood about the conditions under which some events gain attention and yield community-level change, while others do not (birkland and warnement ; nohrstedt and weible ; vogel and henstra ) . concurrently, a growing body of interdisciplinary literature seeks to better understand the link between the experience of an extreme weather event and individual beliefs, policy preferences, and local action in response to future disaster risk in the face of a changing climate. we synthesize the insights from these complementary literatures to develop a broad conceptual framework, which we use to inform our analysis and interpretation of results. specifically, we expect that combinations of event characteristics, features of the post-event debate, and community characteristics are likely to yield unique recipes for policy change, as shown in fig. . we expect that varying configurations of the three main conceptual elements from fig. -event characteristics, community characteristics, and agenda-setting-will influence the degree of policy change focused on adaptation. for the purpose of this study, we are primarily focused on formal policy changes that are adopted at the local or regional fig. conceptual framework (i.e., sub-state) level. we define policy change as local resilience and adaptation policies that are expected to: ( ) reduce community vulnerability to negative impacts of climaterelated change and/or ( ) improve community capacity to adjust to climate-related change (tiernan et al. ; vogel and henstra ) . motivation for the three main conceptual elements (event characteristics, community characteristics, and post-event attention) follows. both scholarship and anecdotal observation suggest that the characteristics of focusing events are critical to the policymaking process. birkland's ( ) own definition of potential focusing events-events that occur suddenly, rarely and with actual or projected harm-suggests that we should expect these characteristics to matter in the policy making process, compared with everyday events that do not exhibit such features. birkland and warnement ( ) further distinguish between events that are ( ) normal; ( ) novel; or ( ) normal with uncommon circumstances. normal events include those events that are likely to occur over the course of time, but are nonetheless unpredictable (e.g., tornadoes, hurricanes, industrial accidents). these events are distinguished from novel events, which are both unpredictable and new in nature or influence (e.g., / attacks; covid- pandemic). normal events with uncommon circumstances gain attention primarily due to unique or unusual features of the event (e.g., sandy hook elementary school shooting). accordingly, we anticipate that the impact of an event, with respect to losses and/ or government response, is likely to influence policy change. moreover, impact may be mediated by the timeframe of event onset, with discrete and rapid-onset events and resultant impacts being more influential than ongoing or slow-onset events and impacts (howe et al. ) . while particular types of extreme weather events are not likely to be wholly novel in nature, we may expect that, as the climate changes, some types events may be either new to a specific geographic area, result in a remarkable amount of damage or affect a previously unaffected population. furthermore, in the context of extreme weather, sisco et al. ( ) find that the "abnormality" of events, in comparison with the historical record, is a significant predictor of climate change attention, while boudet et al. ( ) find that events of unusual type or timing can lead to community climate change dialogue. moreover, our interview findings suggested that in the cases we examined, a series of multiple extreme events in a short timeframe, especially "opposite" types of events (e.g., flood and fire; drought and flood) were connected to discussion of climate change and future risks. some evidence suggests that repeated threats of disaster can yield public complacency (wang and kapucu ) and that repeated personal (negative) experience with disaster relief can undermine government authority (darr et al. ). however, cutter et al. ( ) note that "subjectively, people see an while we are aware that current scholarship is in the midst of theoretical distinctions about differences between resilience and adaptation, engaging in that discussion is outside the scope of this paper. furthermore, some scholarship suggests that local climate change initiatives themselves make little distinction between the terms (meerow and neuner ) , while other research suggests that the terms themselves reflect framing efforts by interested stakeholders (mcevoy et al. ) . as such, we use the terms interchangeably. our approach to operationalizing these concepts is described in a separate section (operationalization of outcome and causal conditions). initial event as discrete, but by the second or third event the initial disaster no longer is understood as singularity but becomes intertwined in its effects and historical meaning with other events" ( , ) . accordingly, we expect that extreme weather events that have a strong impact on human lives and/or property, exhibit unusual patterns and/or co-occur with other unusual or extreme events, are necessary for policy change, but not sufficient in isolation. by nature, disaster-focused research rarely examines non-disaster events, making it difficult to assess the influence of these characteristics on policy change. however, we can take observations of substantial variation in policy change in the wake of crises (nohrstedt and weible ) as suggestive of the insufficiency of extreme events alone in yielding policy change. while recent research substantiates the expectation that extreme weather events often lead to policy change, such changes are varied and far from inevitable. crow et al. ( ) , for example, describe changes in fiscal policy and related lessons learned among local governments in the wake of regional flooding in colorado. plein ( ) documents changes made in response to west virginia flooding, although most policy changes were focused on recovery, rather than adaptation and resilience. the ipcc report on local climate change adaptation efforts highlights the importance of contextualized adaptation strategies that take advantage of local culture, acknowledge cultural strengths and challenges, and build on existing local capacity as needed (cutter et al. ). accordingly, we expect two community characteristics to be relevant to the policy change process: political orientation and capacity. research consistently finds that political orientation plays a role in attitudes toward and beliefs about climate change, with self-identified liberals and democrats more likely to believe in anthropogenic climate change (albright and crow ; hamilton ) , perceive increasing risks related to climate change (hamilton et al. ; marquart-pyatt et al. ) and mobilize in information-seeking or activism lang ) , suggesting that party identification and/or political ideology are likely to influence support for policy change. however, the influence of political orientation on disaster response and policy is less clear. recent media coverage suggests that some communities that avoid discussing climate change may simultaneously engage in actions intended to build capacity for risk management and/or adapt to future risk (flavelle ; smith and schwartz ), an observation that has been borne out in recent research. indeed, javeline et al. ( ) find that actions by coastal homeowners are not associated with climate change beliefs, suggesting that post-event policy action may be equally likely among both democratic and republican communities. recent reviews of the adaptation and resilience literatures pay less attention to the influence of political orientation, instead emphasizing the importance of community capacity, including the presence of community leadership and community organizations (tiernan et al. ; vogel and henstra ) . moreover, while the impacts of extreme weather events are often felt most dramatically at the local level, adaptation is shared both horizontally and vertically among multiple jurisdictions, such that local policy decisions are nested in a broader policy system (nohrstedt and weible ; vogel and henstra ) . as such, we expect that a high community capacity for advocacy and mobilization to be an important condition for adaptation-oriented policy adoption, regardless of political orientation. agenda-setting concerns the process by which policy communities mobilize to frame policy failures, problems, and solutions. in the post-crisis context, the nature of the agendasetting process is expected to be highly contextualized, with variation reflecting differences in policy domains and relevant policy communities (birkland and warnement ) , as well as the degree to which the policies have "publics" (may ) . moreover, local contexts may differ substantially in the degree to which various groups have non-decisionmaking power, or the power to set the policy agenda and limit discussion of contentious issues (lukes ) , such as climate change. vogel and henstra ( ) suggest that climate adaptation policy is likely to be relatively low-salience without a "public" to seek and sustain attention. salience may be further inhibited by difficulties in communicating the relevant scientific knowledge; see tangney and howes ( ) . however, disasters may yield a sudden increase in salience and attention that open "windows of opportunity" (kingdon ) during which policymakers and entrepreneurs have a chance to influence policy discourse. indeed, recent studies demonstrate that disasters have "focusing power" (vogel and henstra , ) at the local level, prompting both dialogue and action. for example, henstra ( ) finds that policymakers used a severe storm in toronto, canada, to motivate adaptation-focused dialogue and action. we expect that highly focused media attention on a particular topic in the wake of an extreme weather event to be a condition that leads to policy change, while more diffuse media attention across a wide range of topics to limit policy change post-event. similarly, framing has been shown to play an important role in policy changes by drawing on collective beliefs and influencing both discourse and outcomes (boin ; boin et al. ; mcadam ; mcevoy et al. ) . for example, the argument for policy changes following a devastating storm in sweden was framed in terms of a challenge to the crisis response system (nohrstedt and parker ) . similarly, orlove et al. ( ) find that some glacial mountain communities draw more heavily on a community frame than a climate change frame when discussing adaptation projects, perhaps allowing them to bypass politically driven objections to adaptation policy. and in some cases, using a climate change frame to promote mitigation policy may be seen as exploiting event-related suffering shepard et al. ) as such, we expect that dialogue about climate change may offer an important path to policy change, but that it is unlikely to be the only path. vogel and henstra ( ) highlight comparative case study approaches as a promising method for gaining insight into local climate change adaptation and related policy change. we conducted a fuzzy set qualitative comparative analysis (fsqca) of cases of extreme weather events that occurred from to . fsqca is a set-theoretic approach that identifies configurations of causal conditions that are associated with observed outcomes of interest. the approach uses in-depth qualitative data to produce quantitative "scores" for outcomes and causal conditions associated with a mid-n set of cases and to identify necessary and sufficient configurations via boolean algebra algorithms (ragin (ragin , b . we used the "case-oriented" approach to fsqca, which emphasizes "the substantive interpretability of results" ( , ). one advantage of fsqca stems from its use of both qualitative and quantitative data to score and calibrate outcome and causal conditions, which offers distinct advantages compared to both traditional statistical approaches. first, fsqca permits equifinality, or the possibility that multiple paths can lead to the same outcome. second, fsqca accommodates causal asymmetry, allowing the positive expression of the outcome to result from a distinct set of causal conditions, compared with negation of the outcome. finally, conjectural causation, a hallmark trait of fsqca, allows multiple causes to act in combination to influence an outcome. the approach also offers advantages relative to traditional comparative qualitative analysis (qca), by introducing calibration of outcome and causal conditions that permits the analysis to move beyond the traditional qca binary scoring approach. unlike traditional qualitative comparative analysis, which uses similar techniques with binary ( , ) conditions, fsqca accommodates scores along the continuum from to , providing more opportunities to capture the rich variation available from qualitative data (ragin (ragin , b . we used the spatial hazard events and losses database for the usa (center for emergency management and homeland security ), also known as sheldus, as the main sampling frame for the study. the sheldus database provides county-level hazard data for us natural disasters; measures include event timing, location, property losses, crop losses, fatalities, and injuries. we limited our sheldus search to counties with weather events that caused fatalities and/or injuries between and (n = ) and then supplemented these data with weather events reported by noaa's billion-dollar climate and weather disasters (noaa national centers for environmental information (ncei) n.d.). to avoid over-sampling wealthy communities and under-sampling heatwaves, which yield little property damage (tierney ), we did not use property or crop damage as a selection criterion. after reviewing the initial sample, we selected events using a multi-step process. first, we selected events that resulted in at least countywide fatalities (n = ), with the intent of striking a balance between community impacts and the likelihood of media coverage. from among those cases, we further selected cases based on accessibility of a local (municipal or county) newspaper of record from the date of the event to months after the event, with coverage of the identified event in sufficient detail ( or more articles) (n = ). from the resulting list of potential cases, we then selected cases to cover different types of weather hazards: flooding, tornado, wildfire, landslide, winter weather, heat, and hurricane/tropical storm and all regions: northeast, southwest, west, southeast, and midwest. for multiple, co-located counties experiencing the same event as part of the same weather system, we typically included the most impacted county in terms of fatalities in our analysis. additionally, if the same weather event occurred in the same county years in a row, only one of these years was selected as a case-again typically the event with the highest number of fatalities. when weighing inclusion of competing cases, a diversity of hazard types and geographic areas sometimes superseded the number of fatalities from the event. figure displays the selected cases with relevant characteristics. online appendix a includes selected information about each case, including location, event, and number of injuries and fatalities. we began with a systematic search for articles and editorials about the event published in the year after its occurrence, following mcadam and boudet's ( ) advance preparatory fieldwork strategy. we supplemented the initial search with a second systematic search for the terms "climate change" and "global warming" in the year prior to the event and within the articles and letters focused on the event in the year after. we coded all relevant newspaper articles (n = ) and editorials (n = ) to identify leaders and key policy actors, groups and organization, events and meetings, and proposed policies. secondary data for county-level socioeconomic, demographic, organizational capacity, and event-related indicator were collected from multiple sources, including the us census bureau's american community survey (n.d.), the national center for charitable statistics (national center for charitable statistics n.d.), yale climate opinion maps (howe et al. ) , national academies report on event attribution (national academies of sciences, engineering, and medicine ). the newspaper and secondary data served as the primary sources for development of a preliminary case narrative, systematically structured to highlight our understanding of the weather event, related mobilization, and policy responses and to identify potential site visit and telephone interview contacts. we conducted a site visit to each case location, where we conducted interviews with key policy actors, including local and state elected officials, county/city staff, newspaper reports, leaders of local community organizations, and religious leaders. we logged a total of h over the course of interviews, averaging min per interview. while on site, we also attended relevant public meetings and conducted additional interviews, as suggested by our initial contacts. online appendices a through a include detailed information about newspaper and interview data by case, including sources, counts, and interview duration and protocols. for each case, we developed a detailed research memo write-up that systematically synthesized information from interviews, newspaper articles, and secondary sources (online appendix a ). we used the case data in these write-ups, as well as the original articles, interviews, and secondary data, to develop the outcome and selected causal condition. our primary research objectives were to identify ( ) the degree of local adaptation-focused policy change occurring in response to extreme weather events and ( ) the combination(s) of conditions leading to the presence or absence of adaptation-focused policy change. we operationalized our outcome and causal conditions based on both theoretical considerations and our empirical evidence derived from the cases. see online appendices a -a for detailed information about the development of conditions, including a codebook associated with the outcome conditions, as well as underlying data and calibration methods associated with all conditions. our primary outcome of interest, adaptation-and/or mitigation-focused policy change, which we refer to simply as "policy change," represents the degree to which the community enacted post-event public policies intended to address disaster-related risks. we searched newspaper articles and interview transcripts to compile evidence of local (and non-local) policy change and categorized each identified policy according to its proposal date (pre-or post-event), venue (private, administrative, legislative, ballot initiative, judicial), and jurisdiction (local/regional, state, national). for the purpose of this study, only policies that were local, public, and proposed after the event were retained in the data set for scoring and analysis. the ipcc report (cutter et al. ) identifies three main categories of potential policy change: ( ) coping with current data risk; ( ) building capacity to manage risk; and ( ) anticipating and responding to future data risk. we used insights from the ipcc report (cutter et al. ) to categorize each policy according to one of sub-categories, representing three major risk management categories, as shown in fig. . the first group of policies, "coping" measures, reflect how local places modify the impacts of disruptive events, features of the events themselves, and/or share or reduce the burdens of disaster risk. they include measures intended to improve the immediate capacity to respond to the event or similar events (e.g., equipment purchase, coverage of reliefrelated expenses, measures to prevent interference with emergency response), move people in response to an event (e.g., relocation) and/or rehabilitate and rebuild in response to the event (e.g., investments in new housing, changing building codes to encourage rebuilding). the second group, "capacity-building" policies, includes those that build the capacity of a community to manage future risk, such as long-range planning and knowledge production that is intended to improve the community or region's ability to respond to future events. the third group, "adaptation" policies, include land use and ecosystem protection measures (e.g., zoning changes, land acquisition, and resource management), structural measures (e.g., seawalls, measurement systems, and building codes, etc.), risk communication measures (e.g., warning systems, information channels, etc.), and resource storage, rationing protection (e.g., water rationing, medical triage systems, security measures). the last group, "mitigation" policies, include those target energy production and/or use at the local level (e.g., municipalization of energy production facilities, adoption of energy use reduction strategies) with an eye to limiting the rate or magnitude of climate change. see online appendix a for a description of each of these categories and codebook. each policy measure was independently assigned to one (mutually exclusive) ipccbased sub-category by two coders. after one reconciliation and independent recoding process, our coding efforts yielded a cohen's kappa of . across individual policy categories and a cohen's kappa of . across the four broad groups, indicating strong reliability (cohen ; mchugh ) . following ragin ( ) , we aggregated across these categorizations to create a macrocondition that ranged from (fully out of the set) to (fully in the set), prioritizing ( ) land use/ecosystem protection and structural measures; and ( ) policy change portfolios with a selection of new policies across all three categories. for example, for a case to be considered fully in the set of post-event policy change, we required evidence of either a new land use or structural policy, plus evidence of at least one recovery-focused policy and one capacity-building policy. we chose to privilege land use/ecosystem protection and structural measures based on the recognition that such policies, which tend to require broad support, political will, and considerable resources, can be an indicator of community investment in future-oriented adaptation. cases fully out of the set of post-event policy change included those without any evidence of land use or structural measures, and no evidence of any new recovery or capacity-building policies. see table for detailed information about the fuzzy set scoring process. we operationalized three types of causal conditions, based on the literature, including ( ) event-specific characteristics; ( ) context characteristics; and ( ) post-event attention. we briefly discuss the operationalization of each of these categories in turn below. operationalization of each causal condition is presented in table , with each causal condition scored along a continuum from to , in standard fsqca fashion. the underlying data and calibrated score for each causal condition are provided in online appendices a -a . while all of our cases can be described as non-typical weather events, on the basis of our fieldwork, we identified three characteristics for which we expected variation to influence the degree and type of event-related attention: ( ) event impact; ( ) event unusualness; and ( ) exposure to multiple extreme events. event impact was scored using a combination of the total number of fatalities, the number of fatalities experienced in a -h period, and the issuance of a presidential disaster declaration, with the highest impact events being those with or more fatalities in day and issuance of a presidential declaration. we used a combined measure of unusualness and exposure to multiple extreme events to measure events likely to represent an uncommon event, above and beyond its extreme nature. the unusualness of the event was scored on the basis of the event type and timing; events with the highest score are those that were described as not normal for the area in terms of both type and timing. we scored recent exposure to multiple extreme events with a combination of the number and type(s) of extreme events that occurred within a year prior to the event of interest. cases with the highest score on this causal condition included those that had experienced at least two events of opposing type (e.g., flood and fire, drought and flood); communities that had experienced at least multiple extreme events of the same type during a - years period were still in the set of multiple-occurring, but at lower scores. as suggested by our conceptual framework, we posited that two community characteristics, political orientation and environmental civic capacity, would be relevant to our analysis. for each county where our events took place, we measured political orientation as the percent of voters who voted democratic in the most recent pre-event presidential election ( or ), following levandusky et al.'s ( ) finding that presidential voting patterns are a good proxy for local partisanship. we measured local civic capacity as the pre-event number of nonprofit organizations per capita in the county, using data on the presence of nonprofits from the national center for charitable statistics (national center for charitable statistics n.d.) and aggregate population data from the american community survey (us census bureau n.d.). both measures were then calibrated using ragin's ( a) direct method of calibration for conventional interval-scale indicators. our theoretical expectations for community civic capacity was positive; we expected to see cases with either of those characteristics also present in the set of cases exhibiting post-event policy change. we did not have a theoretical expectation for percent democratic; while substantial literature points to political orientation as an important determinant of attitudes toward climate change, our knowledge of the cases and selected literature suggested that we might find majority republican cases exhibiting policy change, but without framing their action using climate change language. we measured post-event attention with two main indicators: ( ) attention focus and ( ) climate change discussion. both indicators were developed by applying latent dirichlet allocation (lda), a form of computational topic modeling, to local newspaper articles covering the event in the year after its occurrence. lda identifies the topics being discussed across a group of documents (blei et al. ) . lda operates by clustering words that appear in a group of documents based on their co-occurrence within individual documents. words that often appear together are (by assumption) more likely to be part of the same topic. though relatively new, lda has recently begun to be applied to studying climate change discourse (cody et al. ) . to measure attention focus, we used the kurtosis of the distribution of topics within each newspaper's coverage. this is a standard statistical measure which represents whether a distribution is more or less concentrated. lower kurtosis indicates a narrow-tailed distribution, meaning that coverage is focused on a relatively small number of topics. higher kurtosis indicates a heavy-tailed distribution, with more diffuse discussion of topics. in our data, attention focus ranged from a low of (relatively focused) to a high of (relatively diffuse). see online appendix a for a more detailed account of the lda method used in this study. we inverted the scale and normalized it to vary from to via calibration (ragin a) , so that a high fuzzy score would represent cases with focused attention. our measure of climate change discussion was the share of newspaper coverage clustered into the lda-generated topic that we identified as being devoted to climate change. while a fairly small proportion of coverage was devoted to climate change, the percentage ranged from less than to %, allowing us to calibrate the measure following ragin ( a) . we used ragin's ( ragin's ( , b method of fuzzy set qualitative comparative analysis (fsqca), a set-theoretic approach used to identify necessary and sufficient combinations of causal conditions, to explain variation in post-event policy response in the selected communities. according to standard fsqca methods, a necessary causal condition (or combination of conditions) is identified when the outcome is shown to be a subset of the condition. in other words, there are no cases of the outcome being present with the necessary condition absent, and for all cases where the outcome is present, the necessary condition is also present. consistency and coverage are the two standard measures for assessing the strength of the causal relationship between causal conditions and the outcome of interest. set-theoretic consistency reflects the degree to which cases with the same condition or combination of conditions also share the outcome of interest (ragin ) . strong evidence of necessity is indicated by the standard statistic, consistency of necessity (incln) greater than . , especially in the context of a high relevance of necessity (ron). the standard measure for assessing sufficiency is proportional reduction in consistency (pri); a pri score above . provides strong evidence of sufficiency. (ragin (ragin , a schneider and wagemann ) coverage represents the degree to which a specific condition or configuration of conditions explains occurrences of the outcome of interest among the selected cases. coverage is typically measured by raw coverage score (covs); a higher covs typically indicates that the given causal condition is more empirically relevant (ragin ) . following (ragin a) , we began by conducting tests of necessity for all hypothesized causal conditions, followed by tests of sufficiency. per schneider and wagemann ( ) , we conducted analysis of necessary and sufficient conditions as separate analyses and used standard metrics to assess the consistency and coverage of various models. the intermediate solution from all tests of sufficiency is presented in the text. finally, we conducted extensive sensitivity testing to assess the robustness of our findings, following schneider and wagemann's ( ) exhaustive enumeration approach. all fsqca analyses were conducted in r . . using the 'qca' package . described by duşa ( ) and thiem and duşa ( ) . we documented our analytic choices in extensive detail, as recommended by schneider and wagemann ( ) , and in alignment with recent studies conducted by boudet et al. ( ) and van der heijden ( ) . see online appendix a for a detailed description of our analytic approach and technical information about fsqca statistics used to assess necessity and sufficiency. see also online replication files. among the cases, we identified distinct policies that were formally proposed and adopted at the local level following these extreme weather events, ranging from to policies per case. as shown in fig. , the highest prevalence policy measures included emergency preparedness and response ( policies) and planning and partnership initiatives ( ), followed by structural measures ( ) and land use/ecosystem protections ( ). by major policy category, the largest share of policy measures included efforts to anticipate and respond to future disaster risk ( ; shaded gray), followed by policies to cope with current disaster risks ( ; horizontal lines). finally, a little over one-quarter of all policy measures were devoted to capacity-building activities ( ; dots). for our fuzzy set scoring, ten of the cases scored in the set of cases that enacted policy changes post-event (i.e., score > . ). those cases showed evidence of structural and/ or land use/ecosystem protection policy change(s), and most of them also adopted current-, future-or capacity-oriented policy. three cases, including lake county (ca), richmond county (ny), and travis county (tx), were fully in the set of events facilitating adaptation-focused policy change (i.e., score = ), having enacted both structural and land use/ ecosystem policies, as well as policies in all of the three main categories. these top scoring cases, which included a wildfire, flood, and hurricane, were diverse with respect to event type. despite evidence of policy change, it is important to note that qualitatively, few of these policy changes represented a full-blown shift in goals and values, or even in the means by which communities hedge against disaster risk. nor were most policies particularly innovative in nature. instead, as anticipated by policy change theory, they represented incremental changes to existing systems, sustaining status quo power structures and validating prevailing policy beliefs. indeed, evidence from our interviews suggests that some communities had already made relevant changes in response to events that occurred two to three decades earlier. that said, some communities took steps to reassess and adopt new goals, especially in relation to climate change and disaster risk management. for example, in richland county (sc), disaster recovery chief mike king noted that in the wake of floods "we are now looking at replacing at the level that fema recommends, not just… replac[ing] the same old culvert with the same old culvert. what we want to do is…to mitigate in every step forward now, try to mitigate future events because, it's going to happen again, with climate change, it's going to happen again." (interview ) other communities found that the experience of a major disaster opened the way to discussions about policy change. in richmond county (ny), the widespread devastation associated with hurricane sandy yielded extensive attention to the topic of climate in both local and national media, resulting in a wide variety of policy changes focused on adapting to future risks. one of the newspaper articles, for example, states, "as much as new yorkers would like to believe hurricane sandy is a once-in-a-lifetime event, the city is not taking any chances, especially as the science of climate change points to more such superstorms in our future" (article ). this sentiment was echoed by several interviewees, such as reverend karen jackson, director of recovery and community initiatives at project hospitality, who stated "…new york city is vulnerable, and therefore we need to be prepared for the next sandy, which will happen" (interview ). not surprisingly, we found that the impact of the event was an important motivating factor for policy change. indeed, our results indicate that a high-impact event was a necessary condition for policy change. in other words, most cases of policy change were in the subset of cases that experienced a high-impact event, while there were no cases of policy change that were in the set of low-impact events. in our test of necessity, the high-impact condition yielded a incln (consistency of necessity) score of . . see online appendix a for more detailed presentation of the results from these tests and our interpretation. in the set of communities that enacted policy change, articles and interviews indicated that the community had been devastated by the high number of fatalities and/or populations affected. for example, in cleveland county (ok), several children died during school hours when a tornado struck moore, ok (hampton ; kinney ). these deaths were described by interviewees as emotionally devastating and directly connected to subsequent policy change. robert romines, the superintendent of moore public schools stated, "buildings are easy to rebuild, but the loss of life and the seven kiddos that we lost, that is very hard to stomach," and went on to describe significant public interest and engagement related to a ballot measure funding storm shelters in every school (interview ). in contrast, in most of the communities that did not enact adaptation-focused policy measures, the human impact was low and the event did not yield a strong sense of urgency with respect to government intervention. all of these cases represented extreme temperature events that occurred in the context of an effective emergency response system, in part due to extreme weather events experienced decades earlier. in st. louis (mo), for example, all interviewees referred to the impacts of the "big daddy" heatwave that hit st. louis in the summer of , when more than st. louis metropolitan area residents died of heat-related causes and which motivated a series of key policy changes. such changes likely lessened the impact of the heatwave yielding policy changes that were more limited in scope and focused more on "tweaking" past changes. in the one community, suffolk county (ny), that experienced a high-impact event (blizzard), with a presidential declaration but a low number of fatalities; the majority of the resulting policy changes were focused on managing current disaster risk (e.g., snowplow purchases). our analysis of sufficient conditions yielded two distinct configurations, or "recipes," of other event-related conditions, community characteristics, and features of the agendasetting process, associated with post-event policy change. the overall sufficiency test associated with these causal combinations yielded both high consistency (pri = . ) and coverage (covs = . ), as shown in fig. and described in more detail below. see online appendices a -a for the truth table and results from our sufficiency analysis, as well as the results from our sensitivity testing process. the first causal combination yielded post-event policy change with moderate consistency ( . ) and coverage ( . ). this recipe to policy change was marked by a high-impact event occurring in a democratic county with relatively high post-event media attention. cases in this recipe included flooding events in boulder county (co), richland county (sc), and travis county (tx), as well as the snohomish county (wa) landslide and the lake county (ca) wildfire, all of which were in the set of policy change cases (i.e., fuzzy set score > . ). three of the five cases were fully in the set of adaption-focused policy adoption, with policies focused on land use/ecosystem protections or structural measures, as well as policies focused on building capacity and coping with current risks. lake county (ca) the "valley fire" that occurred in lake county, california, in autumn was the third most destructive wildfire in california's history at the time (lake county record bee a). governor brown declared a state of emergency for lake county the day of the fire, and president obama declared it a major disaster a week later (krohn ; lake county record bee b). the fire, which occurred in the midst of a major -year drought, burned , acres, caused the evacuation of nearly , residents, and destroyed structures (faries a, b; lake county record bee a, c; quirino ) . ultimately, the fire displaced residents, nearly % of lake county's population. lake county voted majority democratic ( %) in the presidential election. in response to the fire, lake county enacted as many as distinct policy measures across all three categories of adaptation policy. most policies were oriented toward adapting to future disaster risk, ranging from installation of a -foot fire break, plans and grant requests for activities to reduce fuel buildup, and the distribution of disabilityspecific warning equipment. lake county also implemented several changes to human resources procedures, including a shift in the organizational location of the office of emergency services to ameliorate coordination and information challenges experienced during the fire, as well as a grief program for public safety professionals. the county also began a broad initiative to revise its emergency operations plan, which had not occurred in over years. our topic analysis indicated that post-event local media coverage in lake county was relatively focused (kurtosis = ), with climate change making up a relatively high percentage of topical coverage ( %) compared to other cases. interviewees tended to associate the fire and the preceding drought with climate change, while also indicating that climate change was not a direct cause of the fire. at the state level, california governor jerry brown's acted as a statewide opinion leader, connecting wildfires throughout california to climate change: after meeting with fire officials to receive an update on the rocky fire, which has burned almost , acres of wildland and destroyed dozens of homes, the governor used the opportunity to tie this year's fire season to climate change, calling it an existential threat that could potentially spark similar large-scale destruction in the future. he attributed the now four-year drought california faces to the extreme weather conditions brought by climate change. (article ) these views were noted by local interviewees, including project coordinator for north coast opportunities traci boyle, who commented on the connections being made between the drought and climate change, "…because that's the conversation. particularly, that's what the governor on down is [saying and] all the state agencies are telling you" (interview ). not everyone agreed on the connection between climate change and the wildfire. greg giusti, for example, director of university of california cooperative extension for lake-mendocino county and advisor on forests and wildland ecology, described the fire as "a result of years of fire suppression and a weather event" (interview ). moreover, lake county also enacted at least one measure focused on achieving a rapid and comprehensive recovery, a relaxation of size requirements for lots and homes, highlighting the tension between human (re)development and disaster risk management in high-risk areas. most pointedly, will evans, director of the clear lake environmental research center, noted the distinct opportunity for change stemming from the wildfire, "it creates a policy window…the policy window has been open for a lot of stuff since the valley fire and then the floods this past year, like the whole oroville dam thing, another policy window opening up for more funding and changes to make things better." (interview ) ultimately, interviewees described the resulting policy changes as having occurred in response to the risk of future disasters and related discussions, even though the community had not reached consensus on climate change's role in the fire. the second causal combination yielded post-event policy change with a high degree of consistency ( . ) and moderate coverage ( . ). cases representing this recipe included the three tornados in our sample-cleveland county (ok), laurel county (ky) and winston county (ms)-as well as the yavapai county (az) wildfire and the richmond county (ny) hurricane. the affected communities represented by this recipe were majority republican counties that experienced high-impact and uncommon events. the yavapai county (az) stands apart as an example of a case with a high number of emergency responder fatalities (a hotshot crew), while the tornados resulted in a loss of citizen lives. moreover, all the events stood out to those interviewed as being uncommon, either because the event was unusual for the area in terms of type/timing or because the event occurred in sequence with other extreme events in a recent timeframe. the ef tornado that touched down in winston county was part of a series of tornadoes that swept across mississippi on april , -the beginning of days of severe weather in the state. all told, tornadoes caused deaths and damaged or destroyed approximately homes and businesses. in the wake of the tornado, the communities in and around winston county enacted two adaptation-focused policies, including application for and receipt of a federal grant for safe room construction at a local school and a new county policy to require every county building be insured. these policy measures were accompanied by ongoing efforts to map community structures, update emergency plans, and practice emergency operations drills. winston county is predominantly republican ( %). there was little evidence that the tornado led to climate change attention or increased awareness about climate change. among all the cases, winston county had one of the lowest levels of local media attention focused on the climate change topic ( %), and less than a third of interviewees described community dialogue about climate change in the wake of the event. interviewees indicated that climate change was not discussed in relation to the storm and attributed this to the fact that mississippi is simply known for experiencing tornadoes. indeed, joseph mccain, editor of the winston county journal, indicated that, "i think most people here, they explained [the tornado] with religion not science" (interview ). moreover, media attention was not singularly focused in the months after the event; the kurtosis measure was relatively high ( ), indicating fairly diffuse attention. that said, interviewees described a shift in the likely path of tornadoes, from tornado alley in the midwest to dixie alley in the southeast, and the need for new levels of awareness about potential risks, without explicitly linking the shift to climate change. for example, buddy king, the winston county emergency management director, discussed how technology has improved our ability to predict severe weather and increased awareness of severe storms, without attributing the observed increase to climate change: people are aware that this is happening. mississippi has now become the leader in tornadoes. and even though mississippi [has] a very rural nature…we're still having deaths because [of] the size and significance of the tornadoes. i don't know that it's new, but as our information increases in society, people are much more aware. (interview ) similarly, lacy vowell, administrator of the winston medical center nursing home, indicated that "i think things are changing and i think the intensity of any storm hitting anywhere in the world… you're just seeing bigger and worse" (interview ). some interviewees indicated that after the series of tornados, there was a shift in community members' perceptions of future weather risks, noting that people are much more aware of the weather and its potential impacts. mayor hill shared the following thoughts: "you can see these things on the weather channel. you can see them on your regional or local news where it hits someone else. but when it hits you or hits your neighbor and hits your community, you realize the destruction." (interview ). the evidence suggests that in the face of a high-impact event and shifting and unusual weather patterns, winston county mobilized to address future risks via adaptation-oriented policy, despite a reluctance to frame those risks in terms of climate change and political orientations that eschew the science of anthropogenic climate change. the fsqca process explained the occurrence of all ten cases of adaptation-oriented policy change. however, our analysis yielded one contradictory case, suffolk county (ny), which was scored as being out of the set of cases that adopted adaptation-oriented policy, but which exhibits the conditions represented by the first recipe (democratic and climate change attention). suffolk county experienced "winter storm nemo" in , yielding several fatalities and a presidential disaster declaration. policy changes were adopted across multiple towns, most of which were focused on purchasing new snow removal equipment, although instances of updated planning and efforts to protect an incoming supply line were also recorded, yielding a fuzzy set score of . , just below the cutoff score of . . the storm, however, came just on the heels of hurricane sandy (october ), which devastated the region and was readily linked to climate change, and indeed we observed that much of the climate change discussion was closely tied to that event. while our primary research question was focused on the conditions that lead to policy change, we used the same fsqca process to examine the conditions that led to policy stasis. five cases in our sample exhibited little to no policy change post-event, with two showing no evidence of post-event adaptation-oriented policy change: the heat events in cook county (il) and clark county (nv). minor policy measures were enacted after the cook county (il) cold event in the form of public transportation scheduling and notification policy, as well as disability-focused policy changes, while officials in st. louis (mo) conducted post-event research on respiratory issues related to climate change. as noted above, changes enacted in suffolk county (ny) included authorization for snow equipment purchase in multiple townships, as well as a plan to ensure a post-event supply line. while these policy changes were not irrelevant to these communities' needs, they did not meet our criteria for adaptation-oriented policy change. we hypothesized that the same underlying conditions would be relevant for policy stasis. our tests of necessity indicated no necessary conditions, although political orientation (democratic) was a close contender. as such, we included all six conditions in our main analysis, yielding two recipes for policy stasis, as shown in fig. . the recipes share three conditions, suggesting that a democratic community experiencing a non-impactful event that yields little attention is unlikely to show adaptation-oriented policy change in the wake of the event. the recipes differ only according to the last condition: ( ) relatively low attention to the topic of climate change; or ( ) low civic capacity. these recipes are aligned with the qualitative evidence. the fifth case, suffolk county (ny), remains unexplained, again likely due to the occurrence of that event within months of hurricane sandy, yielding high levels of lingering attention in affected communities. see online appendices a - for results from our tests of necessity, directional expectations, tests of sufficiency, and sensitivity testing. we found substantial evidence of local adaptation-focused policy change in most caseson average, measures per event-as well as distribution of those changes among the three major categories of policy change inspired by cutter et al.'s ( ) report on local opportunities for advancing climate change adaptation. almost half of the enacted measures were focused on anticipating and responding to future disaster risks, suggesting that these communities were able to orient themselves toward adaptation, even in the wake of significant disasters. moreover, a considerable portion of policy efforts were directed at building local and regional capacity, especially among cases representing communities with conservative political orientations. however, none of our cases passed mitigation-focused policies, the overall number of policies remained fairly modest, and the policies themselves were heavily oriented toward the type of event that had been experienced (e.g., safe rooms for tornados). furthermore, we find little evidence of policy change in communities that experienced low-impact events with low attention levels. in those cases, most of which were extreme temperature events, prior preparation levels may have contributed to both the low level of impact, as well as a strong sense of resiliency, thus alleviating the apparent need to adopt new policies. there are admittedly limits to our analysis. our cases-which were purposively selected on the basis of impact, event type diversity, and media availability-are a non-representative sample of extreme weather events, which limits generalizability. that said, the use of fsqca is not predicated on a representative sample; indeed, the method is intended as a case-based approach that takes advantage of existing diversity in the sample, yielding analytically generalizable results. finally, our operationalization of the primary elements from our conceptual framework is undoubtedly imperfect; another fsqca feature enables us to address this challenge, using our knowledge of these cases to calibrate our measures. all told, our findings substantiate two enduring propositions for the policy change literature, including: ( ) extreme events and disasters motivate policy change; and ( ) agenda-setting matters. however, our findings also underscore the complexity and diversity of recipes for change highlighted by the disaster and risk management literature and suggest an unexpected path to adaptation among conservative communities that resist (or at least remain silent about) attributing extreme events to climate change. in those communities, the use of alternative framing may yield more adaptation-oriented change than climate change frames. furthermore, policy adoption alone does not necessarily mean effective implementation or use of best practices (cutter et al. ) . we note the fairly narrow scope of the observed policy changes, suggesting that extreme events may trigger discovery of new policy tools and designs, or "instrumental learning," (may ) without yielding deeper social and political change. finally, while all of the communities represented by these cases were committed to recovery in the face of potential future risk, some adopted policies that were explicitly intended to facilitate rapid rebuilding, with little attention to longer-term risks, highlighting a paradox of policy change in the wake of an extreme weather event. adoption of adaptation-focused policy measures 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the contributions made by our interview participants. we also express our thanks for the data collection contributions made by n. downing, c. flathers, j. knobloch, r. nilson, h. satein, s. shepard, h. whitley, i. widiyasari, and c. zanocco, as well as guidance from j. flora and d. mcadam. this work was supported by a grant from the national science foundation, sociology program (no. ). leanne giordono · hilary boudet · alexander gard-murray oregon state university, corvallis, usa key: cord- - e ok authors: howarth, candice; bryant, peter; corner, adam; fankhauser, sam; gouldson, andy; whitmarsh, lorraine; willis, rebecca title: building a social mandate for climate action: lessons from covid- date: - - journal: environ resour econ (dordr) doi: . /s - - - sha: doc_id: cord_uid: e ok the covid- imposed lockdown has led to a number of temporary environmental side effects (reduced global emissions, cleaner air, less noise), that the climate community has aspired to achieve over a number of decades. however, these benefits have been achieved at a massive cost to welfare and the economy. this commentary draws lessons from the covid- crisis for climate change. it discusses whether there are more sustainable ways of achieving these benefits, as part of a more desirable, low carbon resilient future, in a more planned, inclusive and less disruptive way. in order to achieve this, we argue for a clearer social contract between citizens and the state. we discuss how covid- has demonstrated that behaviours can change abruptly, that these changes come at a cost, that we need a ‘social mandate’ to ensure these changes remain in the long-term, and that science plays an important role in informing this process. we suggest that deliberative engagement mechanisms, such as citizens’ assemblies and juries, could be a powerful way to build a social mandate for climate action post-covid- . this would enable behaviour changes to become more accepted, embedded and bearable in the long-term and provide the basis for future climate action. there has been no shortage of commentary on what we can learn from the covid- pandemic for climate change. the pandemic has caused misery, loss and hardship across the world, and should emphatically not be seen as a model for climate action. however, early polling suggests that cleaner air, less traffic, a less frantic pace of life and a less wasteful relationship with food are seen as positive side-effects of the lockdown policies (pritchard ) . thus, there may be opportunities to bed-in and maintain certain types of behaviour changes that would be positive low-carbon steps. there are also tentative signs that the public recognises the need for a response to climate change that mirrors the ambition of our response to the pandemic (stone ) . but there are also serious risks that after having had a 'taste' of restrictions on travel and consumption choices, many people will recoil from the idea that (even on a lesser scale, and with careful planning) some of these restrictions or adjustments should continue. with the tangible prospect of a global recession that could dwarf the impact of the global financial crisis of , countries and citizens alike may find it harder to justify investing financially in low-carbon choices, even if the economic logic of making the right low-carbon choices is unquestionably robust (hepburn et al. ) . this commentary explores what responses to can tell us about what may and may not be possible or desirable in a transition to a net-zero future. we have seen how quickly and effectively governments can intervene to completely reshape society and lifestyles; and that society in turn has largely been supportive of this intervention in service of a widely recognized urgent health threat. this level of intervention however has rarely been observed previously, except during war-time. but while it may be possible for governments to do this, is it possible or desirable for them to do so in order to address climate change? we argue that a rapid zero-carbon transition is possible, and that with the right policies some of the behaviour changes that the lockdown has imposed might be sustained. however, such a transformation needs to be underpinned by a clear social mandate and public support, and it needs to be well planned to avoid the disruptive effects of covid- . by a social mandate, we refer to a situation where society offers support to another actor (e.g. government) to take action to protect our collective well-being, with the processes and the outcomes of this action being broadly accepted as being legitimate. such a mandate is also sometimes referred to as a social contract or a social license for action. deliberative citizen engagement can support both these objectives. the two problems of covid- and climate change are strikingly similar and different at the same time. both have been described as global emergencies. both raise the prospect (indeed the reality) of unprecedented risks to human health and prosperity. both problems are global, need individuals to act for the common good, require government intervention in lifestyles, challenge societal resilience, and have some common solutions (e.g., travelling less). covid- has increased our awareness of how vulnerable we can be in the face of global risks, whether a pandemic or climate change, and how without foresight and planning we are left ill-prepared. the two problems share common drivers-including global travel, deforestation and land-use change-and are mutually reinforcing-climate change is known to affect the survival, reproduction, abundance, and distribution of pathogens, vectors, and hosts (wu et al. ) . however, the two emergencies are also different in important ways. while the threat of covid- is immediate and direct, the impacts of climate change are longer term and more diffuse. driven by necessity, the global response to covid- has been radical and swift. the global response to climate change, in contrast, has lacked a sense of urgency (despite a large number of climate emergencies being declared). perhaps this is because climate change is less tangible and emotive than covid- : the victims of climate change are less likely to be people we know (or ourselves), and even how we measure climate change (in e.g., greenhouse gas emissions or temperature increase) is more abstract and less visceral than how we measure covid- (in number of deaths). indeed, health concerns usually rank higher than environmental ones, because they are more psychologically proximal (maibach et al. ) . there is lower self-efficacy in mitigating climate change than covid- : the latter risks to the individual can be mitigated by hand-washing and social distancing, whereas no amount of individual energy saving will necessarily mitigate climate change risks to the individual. there is a lower social norm to act on climate change than on covid- : simply looking out of the window during lockdown, we can see others are (largely) complying; but low-carbon lifestyles are a long way off being 'normal' or even aspirational. this social norm is, of course, partly a product of stricter government regulation in the case of covid- than climate change. the timescale for interventions is also different: lockdown and other covid- responses are likely to be relatively short-term (months or perhaps years); whereas climate change policies may be in place indefinitely. we have learnt how quickly people can adapt their lives to a sudden disruption like a pandemic-induced lockdown. the social response to this exogenously enforced shock is likely to have varied in different countries reflecting the social norms and contracts in place in those different contexts. for example the use of draconian restrictions on movement such as fines and permits in france and italy (and the rapid behaviour change resulting from this) reflects the culture, role and acceptance of law enforcement within those countries; an approach less accepted in more libertarian countries such as the uk. typically, one of the biggest barriers to behaviour change are habits, and we know times of disruption are when we can most effectively change habits (verplanken et al. ; graham-rowe et al. ) . covid- is the biggest moment of disruption we have seen since world war and, while a tragedy, it has created some behavioural patterns that are intrinsically low-carbon (less travel, less consumption in some areas). they could have great value in shaping the response to climate change, but it is not yet clear how durable they will be. it takes on average days to create a new habit. so, after weeks, new patterns of behaviour may become routines that stick with us (lally et al. ) . in most countries, the lockdown has lasted at least weeks, suggesting new ways of living might endure postlockdown. however, this is highly contingent on these habits being locked in by appropriate infrastructure, incentives and norms (e.g., continuing to allow people to work from home at least some days; providing more cycling infrastructure) and ideally aligned with people's core values (e.g., spending time with family; environmental protection; verplanken et al. ). together, this ensures the motivation and ability to maintain low-carbon habits. the extent to which policies on covid- are driven by 'consent', as opposed to 'necessity' also needs consideration. covid- has shown that, under certain conditions, the relationship between citizen and state is far more elastic than previously thought. citizens have relied upon the state to keep them safe, imposing measures that would have previously been dismissed as unthinkable. for their part, citizens have accepted and encouraged these measures, with compliance levels beyond what was expected or modeled, as suggested by evidence from the uk. the modelling of non-pharmaceutical interventions by imperial college (ferguson et al. ) , used by the uk government to plan lockdown measures, assumed compliance rates of - %, whereas surveys in april showed over % compliance with social distancing (weinberg ) . however, the lockdown measures were-until days before being in place-unforeseen and implemented with lead times considerably shorter compared to normal democratic processes (which would include consultations, white papers, debates in parliament), considering the speed at which the virus was spreading (e.g. arriving in italy in february and the uk in march). there was therefore very little scope for undergoing a weighing up of values, trade-offs and competing priorities that any other far-reaching policy issue would require as standard democratic practice (hagendijk and irwin ) . the emergency measures brought in to freeze almost all 'in person' social and economic activity were driven by necessity, rather than based on considered societal choices. while unplanned transitions like the covid- response are thus workable in the short-term, they may be difficult to maintain in the long-term. while covid- has proven that radical behavioural change is possible, in the short-term, the economic, social and personal consequences of the lockdown have been devastating. the impact on greenhouse gas emissions has been large by historical standards, but modest relative to the deep emission cuts that are needed (le quéré et al. ) . the sudden and disruptive response to covid- is in stark contrast to the sustained, carefully calibrated but urgent and long-term response that climate change requires. the more deliberate planning of the zero-carbon transition is important for two reasons. first, a planned transition will allow people to be engaged and included. involving publics and stakeholders in climate change decision-making not only tends to improve the quality of decision-making (by incorporating a broader range of views and expertise, howarth et al. ) , it also builds trust in the process and buy-in to the outcomes (dietz and stern ) . second, planning a transition provides more time to minimise negative impacts and to maximise the co-benefits associated with tackling climate change. while the hurried nature of the covid- response was inevitably costly, the evidence is growing that a well-executed zero-carbon transition can be achieved without compromising on economic prosperity (kruse et al ; bowen and hepburn ) . a carefully planned response allows identification of those areas where the structural adjustment costs of decarbonization are real and putting in place safeguards for a just transition (bowen and fankhauser ; newell and mulvaney ) . planning will allow exploitation of the co-benefits associated with mitigating climate change, notably for health (e.g., reducing car use improves air quality and reduces obesity, limited red meat consumption cuts heart disease and cancer risks; jennings et al. ) . the complexity of these measures, together with genuine uncertainty and disagreement about how to address climate change in an effective and fair way (hulme ), means that climate change tends to be a less salient concern for individuals, organisations and policymakers than more tangible and immediate issues, not least covid- at the moment-but more generally, personal and family health, financial security, and social/community cohesion (e.g., bain et al. ; whitmarsh and corner ) . this means that measures that align emission reductions with those individual concerns are more likely to motivate behaviour and policy change (graham and white ; whitmarsh ) . but it also speaks to the imperative of securing a strong social mandate for continuous change beyond covid-style emergency measures. covid- has highlighted the role of experts in informing government responses to the pandemic crisis. scientists have been called upon to inform and influence on an issue fraught with uncertainties (lövbrand and Öberg ) . the prominence of government committees like the scientific advisory group for emergencies (sage) in the uk has prompted widespread debate about the role of science in policymaking. when faced with an issue like covid- , politicians often claim that their decisions are based on 'the science', or that 'science' itself is somehow making the decisions. this is a problematic presentation for two reasons. first, because the science itself is not value-free, and neither are the scientists who promote it. second, because scientific evidence cannot, in and of itself, determine political decisions-it can only inform them. political decisions are moral and ethical choices in conditions of uncertainty, not merely a product of machine-like evidenceprocessing (wilsdon and willis ) . thus the role of political decision-making should be seen as a negotiation between different actors, including scientists, policymakers, interest groups (such as businesses, ngos, professional associations and trade unions) publics and others, informed by scientific and social scientific evidence (davies and oreszczyn ) . involving diverse groups in decision-making processes-and crucially, including those who policies will impactleads to more robust outcomes (stirling ) . this is as true for covid- as it is for climate action. the rapid onset of the pandemic in the first half of meant that decisions were taken very quickly, which did not allow widespread consultation and engagement. people accepted lockdown measures because they understood the benefits, in terms of saving lives (including their own), and because the most restrictive measures were seen as relatively short-term. as countries begin to lift strict lockdowns, learn to live with the virus, and begin the process of economic and social recovery, it is both possible and necessary to widen the decision-making process (smith and hughes ) . crucially, this should include deliberative democratic processes such as citizens' juries, which we discuss below. for the reasons discussed above, support for government intervention to address climate change is less visible and more uncertain than for covid- . scientific understanding of climate change is very well established, has been built up over many years, and there is a very high degree of consensus; there is also good understanding of the measures required to bring emissions down to safe levels (ipcc ). however, there is limited debate, and no societal consensus on how we reach net zero, in terms of which measures governments should implement. there is public support for climate change action, including a low-carbon economic stimulus post-lockdown (figueres ) , but there is a need for a more comprehensive debate, bringing scientific evidence together with public views and values-a key characteristic of deliberative governance activities such as citizen juries and assemblies (kythreotis et al. ). in considering climate policy, politicians have been reluctant historically to contemplate imposing restrictions upon citizens. they did not feel under pressure from the electorate to act on climate (willis ) and prioritised policies which they perceived were 'neutral' in terms of lifestyle-such as switching to grid-based renewables or shifting from petrol and diesel to electric vehicles. the school strikes and climate protests which began following the publication of the report of the intergovernmental panel on climate change (ipcc ) have helped to persuade politicians of the importance of this agenda, but they are still reluctant to speak out about the fundamental changes that are required to shift economies and societies to net-zero emissions (willis ) . the pandemic has not necessarily changed the political calculus. the fact that drastic measures were accepted for covid- does not imply they would be accepted for climate. the lesson from covid- is more subtle: it shows that the challenge for climate strategy is not to assume, as politicians have done, a limited room for manoeuvre on climate, but rather to work with citizens to explore what is possible. using the language of political theory, this can be thought of as the negotiation of a contract between state and citizen to build a social mandate for change. such a mandate consists of contributions from both sides-state and citizen-each conditional upon the actions of the other side (boucher and kelly ) . this is where deliberation comes in. traditional polling measures and social research, can explore people's attitudes and values on particular issues, such as their views on renewable electricity technologies. but the fundamental question about the social mandate requires deliberation-an exchange between citizens and state (dryzek ) . citizen juries and assemblies are a tool out of several, which can help to create the social mandate to move forward on socially-inclusive climate action. by co-producing and including citizen's input into designing solutions they help increase public trust and ensure publics are on board and more receptive to any conditions (behavioural or other) that are implemented (warren and gastil ) . covid- interrupted an extraordinary wave of grassroots climate activism and experimentation with deliberative forms of climate governance. in the uk, for example, two thirds of local councils declared a climate emergency in , and many complemented their emergency declarations with the establishment of a citizens' assembly or citizens' jury. a national-level climate assembly sat between january and may in the uk (climate assembly uk ) and, similarly, the french citizen's convention met seven times from october to june (convention citoyenne pour le climat ), the impacts of which will need to be analysed. some of these citizen's assemblies and juries are already having an impact (e.g. in leeds, uk, the process led to substantial citizen engagement and recommendations on what the city should do to address the climate emergency), however it is too early to tell what the widespread impact of citizens assemblies (e.g. in france and the national uk assembly) on the climate debate has been. citizen assemblies have certainly been influential in other contexts, such as the irish debate on abortion (farrell et al. ) . to its supporters, they have demonstrated the power of randomly selected citizens that reflect the wider population, coming together to navigate their way through complexity to create a set of citizen-led visions and recommendations for the future. as such, these 'mini publics' can be an important instrument to obtain a social mandate for the zero-carbon transformation. they are a structured way of equipping citizens with a coherent and robust narrative on climate change, supporting citizens to imagine different ways of living and giving politicians the mandate to take action. they make society a co-designer of climate action rather than having solutions imposed on them (capstick et al. ) . deliberative processes including citizens' assemblies and citizens' juries have involved in-person meetings, which is problematic given the restrictions that covid- imposes. but innovation is happening quickly: the uk's climate assembly moved online in april , and initial evaluation by participants showed that it worked remarkably well (allan ). the first entirely-online citizens' jury on climate change is about to start, in the town of kendal. such processes can test to what extent people have emerged from the pandemic with different values, altered behaviours and perhaps an increased appetite to envision and shape their own future. citizen assemblies may not always generate the results that we expect, but by encouraging collective debate and decision-making they frequently propose a more positive future and different ways of getting there, which people are ready to support. covid- has increased our awareness of how vulnerable we can be in the face of global phenomena, and how without foresight and planning we are left ill-prepared. we have been made aware of our co-dependence and how we are citizens in a society as well as individuals in an economy. the pandemic has strengthened the case for an economic recovery that puts emissions reduction, and indeed climate resilience, at its heart. the global response to covid- has had environmental side effects that the climate community has aspired to achieve over a number of decades: reduced carbon emissions, cleaner air, less noise, more space for nature. however, these benefits have been achieved at a massive cost to welfare and the economy. the covid- response is therefore not a suitable model for climate action. climate change requires a more carefully planned and calibrated, inclusive, less disruptive and more sustained response. we argue that in order to accomplish such a response, there is a need for a clearer social mandate between citizens and the state. we suggest that deliberative engagement mechanisms, such as citizens' assemblies and juries, could be a powerful way to build a social mandate for climate action post-covid. this 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national politicians in global climate governance see-through science: why public engagement needs to move upstream impact of climate change on human infectious diseases: empirical evidence and human adaptation key: cord- -ssv arr authors: hodgkinson, tarah; andresen, martin a. title: show me a man or a woman alone and i'll show you a saint: changes in the frequency of criminal incidents during the covid- pandemic date: - - journal: j crim justice doi: . /j.jcrimjus. . sha: doc_id: cord_uid: ssv arr objectives: to investigate the effect of the covid- pandemic on the frequency of various crime types (property, violent, and mischief) in vancouver, canada. methods: crime data representing residential burglary, commercial burglary, theft of vehicle, theft from vehicle, theft, violence, and mischief are analysed at the city level using interrupted time series techniques. results: while covid- has not had an impact on all crime types, statistically significant change has been identified in a number of cases. depending on the crime type, the magnitude and direction of the change in frequency varies. it is argued that (mandated) social restrictions, shifted activity patterns and opportunity structures which are responsible for these findings. conclusions: we find support for changes in the frequency of particular crime types during the covid- pandemic. this is important for criminal justice and social service practitioners when operating within an extraordinary event. which can allow the federal government to quarantine canadians. this means that canada went into lockdown efficiently and, arguably, effectively in comparison to other nations. this creates an opportunity to explore the preliminary effects of this lockdown on crime trends in one of canada's major cities, with the intention of determining if the shift in opportunity structures have changed crime trends and to improve planning for safety and crime prevention for potential further outbreaks of the pandemic and future exceptional events. this research contributes to a growing literature on crime trends and exceptional events, suggesting that addressing the opportunity structures presented by particular exceptional events, the social costs of these events can be reduced. exceptional events, such as natural disasters, riots, blackouts, and pandemics are unusual incidents that often result in a significant shift in human behavior. these events create an opportunity to explore our understandings of the world. in particular, as the social order shifts and human behavior shifts with it, these events enable us to test the bounds of social theories and policies (andresen & tong, ; barton, ; drabek, ) . these incidents are acute, stressful, often unanticipated, and can disrupt the informal regulatory processes of social life (ritchie & gill, ) . while the effects can sometimes be transitory, some exceptional events can have long term impacts on both population composition and behavior in affected communities. exceptional events usually fall into three spheres, the lithosphere, atmosphere and biosphere (tipson, ) . lithosphere events include changes to the earth's crust which can cause volcanic eruptions, earthquakes, and tsunamis. atmosphere events involves temperature changes in the water or air that can result in droughts, wildfires, hurricanes, tornadoes, and floods (tipson, ) . pandemics, such as covid- , are biosphere disasters in which microorganisms can shift or evolve and potentially lead to a significant loss of life. biosphere disasters also emphasize the vulnerabilities of social and political systems, including access to health care, safety and economic security. these exceptional events can result in competition and hoarding, social instability and unrest, and competition for health care and vaccines (tipson, ) . covid- has exposed the impacts of a succession of neoliberal governments in the western world as political leaders fight for access to ventilators and masks (martin, ) , vulnerable citizens lack access to social safety nets and protections, and others are forced to work for inadequate compensation while putting their lives on the line (north, ) . these situations make it difficult to respond to needs in an efficient and effective way, devoid of corruption. indeed, the world has already witnessed the extent of corporate greed and disaster/monopoly capitalism (klein, ; robinson, ) as ceos capitalize on insider information to sell off stock and prey on small businesses unable to weather the economic impact of a global shut down (neate, ) . there are three main theoretical explanations of how crime emerges or shifts during an exceptional event. these theoretical explanations include social cohesion and altruism, social disorganization, and opportunity theories. interestingly, these explanations produce contradictory predictions about crime trends during an exceptional event. social cohesion, or altruism theories, predict that crime rates decline or remain stable during an exceptional event (zahran, shelly, peek, & brody, ). this argument has largely emerged from natural disaster research, in which proponents of the theory argue that during an emergency, people are more likely to help each other and act altruistically (barton, ) . for example, quarantelli ( ) argues that an increase in opportunistic crime, such as looting or theft, is rare after a disaster. after the earthquake in los angeles, violent crime did not increase (siegel, bourque, & shoaf, ) . furthermore sweet ( ) found a temporary increase in the levels of social support and cohesion after the ice storm in new york in january . a similar situation occurred the same year in quebec, canada, in which the ice storm there led to electrical blackouts. lemieux ( ) found that property crime dramatically decreased as instrumental altruism (government financial support) increased. however, this theoretical perspective has been heavily critiqued as many groups do not receive equal assistance during exceptional events such as earthquakes, ice storms, or blackouts. indeed, fothergill and peek ( ) found that exceptional events often exacerbate social inequality. this was evident in the lack of support provided to non-white americans in the aftermath of hurricane katrina (craemer, ) . furthermore, exceptional events can lead to changes in police reporting practices as front-line officers redirect their efforts to other, more pressing, issues (barsky, trainor, & torres, ) , and this can affect crime trends more broadly. this is particularly the case in natural disasters where the police role is expanded. another theoretical explanation of crime in exceptional events is social disorganization theory, which would predict an increase in crime rates as the social order is disrupted. as mentioned, exceptional events tend to exacerbate social inequality and emphasize the disadvantage of certain groups. advocates of this explanation argue that social cohesion and collective efficacy are not strengthened by an exceptional event, but rather weakened. in turn, these systems are unable to control antisocial behavior (curtis, miller, & berry, ; davila, marquart, & mullings, ; harper & frailing, ; prelog, ) . for example, in the new york city blackout in , genevie et al. ( ) found increases in looting in neighborhoods that already had higher levels of violent crime, illicit economy activity, and unemployment. however, in their study of the floods in brisbane in , zahnow, wickes, haynes, and corcoran ( ) found a displacement effect (in which crime incidents move from one location to another) for property crime to affluent, non-flood affected neighborhoods, suggesting that crime may shift as opportunity changes. this leads us to the final theoretical explanation, which accounts for the variations in findings from the other two theoretical perspectives. opportunity theories, such as routine activities theory, would predict that during an exceptional event, crime rates will both increase and decrease depending on the crime type and the shift in opportunity structure (leither et al., ) . routine activity theory argues that in order for a crime event to occur, a suitable target, a motivated offender, and the lack of a capable guardian need to come together in time and space (cohen & felson, ) . this theory emerged in response to the inability of other sociological and criminological theories to explain how a dramatic increase in financial prosperity had paralleled a dramatic increase in crime in the s and s in the united states. cohen and felson ( ) reasoned that the increase in crime was not a result of disadvantage or strain, but rather an increase in opportunities. peoples' routine activities had changed. entertainment options increased and this shifted people into the public sphere. women moved into the workforce in droves and were no longer at home to act as capable guardians. there was also an increase in the manufacturing and proliferation of lightweight durable goods that were cheaper to purchase and easier to steal (felson & cohen, ) . this research suggests that as the balance between suitable targets, motivated offenders, and capable guardians shift, this will have an indirect effect on crime trends (lebeau, ) . exceptional events can increase or decrease target availability by destroying, moving, or shifting targets (zahran et al., ) . for example, as capable guardianship decreases, as people leave their homes during a flood or hurricane, opportunities for burglary may increase (lebeau, ) . opportunity theories can also explain an increase in altruism as a protective factor against crime through the creation of informal guardianship (curtis & mills, ) . in the aftermath of hurricane andrew in florida, in , informal control mechanisms emerged to protect these properties (citizen-led patrols), which prevented an increase in opportunities and, in turn, property crime (cromwell, dunham, akers, & lanza-kaduce, ) . research on the long-term trends of crime after an exceptional event also suggest that when the opportunity structure returns to its pre-event status, crime trends in the affected areas also return to pre-event levels (zahnow et al., ) . exceptional events can have an important impact on crime. as the social composition of the community changes, so too can crime rates. the research on the impact of exceptional events on crime rates explores hurricanes, floods, earthquakes, blackouts, and more. however, as noted above, the findings of this research are mixed. for example, in , hurricane hugo resulted in a higher rate of property crime (quarantelli, ) . hurricane katrina, in , also led to a significant increase in burglary rates (harper & frailing, ) . however, varano, schafer, cancino, decker, and greene ( ) found that violent crime rates, such as robbery and homicide, did not change in the aftermath of hurricane katrina. in fact, they demonstrated that the increase in other crimes was a result of a shift in population, because a large number of survivors moved into one area. leitner, barnett, kent, and barnett ( ) and leitner and helbich ( ) found that crime rates after hurricane katrina remained relatively stable or even declined. hurricane rita, which hit later that same year, resulted in an increase in burglaries. interestingly, this increase did not occur after the hurricane hit, but after the evacuation notice was given (leitner & helbich, ) . changes in crime trends in exceptional events are not limited to hurricanes. the electricity blackouts in and in new york city led to dramatically different experiences of crime. the blackout had a negligible impact on crime rates (farrell, : montgomery, , while the blackout resulted in an increase in property crime and arson (corwin & miles, ) . the earthquakes in christchurch, new zealand in and resulted in a significant decline in crime in the central business district (cbd) after the earthquake, in particular where alcohol outlets had disappeared. however, a displacement effect occurred to some areas outside of the cbd that were not hit as hard by the earthquake and where alcohol was still available (breetzke & andresen, ) . during the flood in brisbane, australia in , zahnow et al. ( ) found that crime fluctuated across neighborhoods during the flood but returned to previous levels soon after the event was over. similarly, a flood in nigeria in , that killed ten times the number of people compared to brisbane and displaced over million others, led to an increase in violent crime rates. however, these rates also returned to pre-existing levels after the disaster (kwanga, shabu, & adaaku, ) . the research focusing on the effects of pandemics on crime rates is relatively scarce. importantly there is minimal criminological research about how pandemics affect other types of crime, such as corporate crime and corruption, cybercrime, and environmental crimes. at the time of writing, some research has emerged to say that the trends of certain crime types are changing. for example, eisner and nivette ( ) commented on the increase in hate crimes against east asian persons and care providers. they also reported dramatic increases in domestic violence and child abuse (eisner & nivette, ) . mcdonald and balkin ( ) have found some increases in rape, arson, and auto theft in five major cities in the united states. mohler et al. ( ) , investigated crime in los angeles and indianapolis, found that vehicle stops notably decreased and domestic violence was increasing. however, other crime types had minimal changes, and most were modestly decreasing. and ashby ( ), in a study of cities across the united states, found no increases in violent crime with mixed results for property crime. however, many of these preliminary findings require additional research. shifting crime trends alone should not guide policy or practitioners. it is possible that these early changes could be an anomaly. it is necessary to explore both how crime rates may be changing, but also how these changes occur within the context of other controls such as seasonal and yearly trends. in , outside of asian countries, canada was hit the hardest by the sars epidemic. while the death toll seems insignificant compared to that of covid- globally, the outbreak led to several changes in how the government of canada responded to pandemics. importantly, canada created the public health agency of canada to monitor outbreaks of disease, appointed a chief health officer to advise on health care precautions, prepared a set of guidelines to respond to a pandemic, enhanced research capacity, and improved their working relationship with the world health organization (who). as covid- started to spread in march of , canada moved quickly to bring canadians abroad home, shut down borders, and provide ongoing financial support to canadians who lost employment. these decisions supported canadians in staying home and isolating. additionally, they differ dramatically from the united states, in which leadership made inconsistent decisions and offered few protections to citizens who had lost employment or were unable to access health care. it is likely that these decisions will affect the nature of crime in canada during the pandemic. the current study examines crime trends across vancouver, british columbia during the initial twelve weeks of the covid- shutdown and compares these trends with projections of previous years. eight crime types are examined. this is conducted at the city level, in order to explore if the global pandemic is having a city-wide effect. additionally, most policing services operate at this level and a larger geographic area will be useful for helping to guide practitioners and policy in a rapidly changing environment. we examine eight different crime types as research suggests that different crime types will have different patterns during an exceptional event (prelog, ) . we believe these early studies are necessary to inform resource distribution and pandemic response strategies and better aid in a thoughtful and effective recovery. the city of vancouver is part of the vancouver census metropolitan area (cma), the third most populated metropolitan area in the country and the most populated in western canada, had a population of approximately . million in . over the past years, - , the vancouver cma's total crime rate decreased by %. however, from the previous year, , the total crime rate in the vancouver cma has only decreased by %, with an increase in violent crime ( %) and a decrease in property crime ( %). of the three largest metropolitan areas in canada, the vancouver cma had the highest total crime rate ( criminal code offenses per , people). this is slightly more than two times the rate of crime in the toronto cma ( per , people) and the montreal cma ( per , people). similar differences are present for property crime, but violent crime is at similar levels for all three cmas (moreau, ) . the city of vancouver had a population of approximately , in , making it the largest municipality within the vancouver cma. vancouver's total crime rate has only fallen % from to , currently at per , ), approximately double compared to toronto ( per , ) and montreal ( per , ). however, vancouver has experienced a notable drop in violent crime over this time period ( %) with a more conservative drop in property crime ( %) at the city level. vancouver's violent crime rate is similar to that of toronto and montreal, but twice the rates of both cities for property crime. fig. shows daily covid- infections in british columbia, most of which occurred in the vancouver area. evident from this bar graph is that covid- infections began to rise significantly late february and early march . daily infections reached their peak by the end of march and have been decreasing, albeit slowly, until the th of may . march is marked on fig. to indicate changes in routine activities and, therefore, opportunities for criminal activity as that is the day the bc public health emergency was declared, and restrictions began to be put in place. on march , grade school students began spring break and were instructed to not return to school once the break was over. on march large gatherings were banned and significant travel restrictions were put in place. on march the province declared a state of emergency, with restrictions on food and with the rise of covid- infection rates and public health warnings, followed by significant government policy changes and enactments, we expect any changes in temporal crime patterns to emerge at or around late february and early march when sharp increases in covid- infections and public health policy changes occurred, consistent with decreases in activity patterns outside of the home (google, ) . the current study is a time series analysis of crime at the city level, vancouver. these data are available from may to may . the crime incident data used for this study were obtained from the vancouver police department's geodash crime map website (https://geodash.vpd.ca/). in the analyses below we consider the following crime types: total crime, residential burglary, commercial burglary, theft of vehicle, theft from vehicle, theft, mischief, and violent crime. weekly counts of each criminal incident type are used for the analyses to identify any changes in the frequencies related to the timing of covid- . in order to identify short-term changes in the volume of criminal incidents in vancouver, we use weekly crime counts to maximize the number of observations while minimizing volatility. we analyse one year of data, weeks, and search for breaks in the trends of these time series, as outlined below. however, though the time before covid- may serve as a control, and trend-related control variables are used in the analyses, there may be unaccounted for phenomena that occur in march/april/may each year that may make it appear as though a change in trend has occurred when it is simply a recurring annual change in trend. to account for this, we undertake a similar analysis for the previous years to add another dimension of control into our evaluation. as noted above, weekly criminal incident counts are used in the analyses. however, as seen in the figures below, weekly crime counts are still rather volatile. as such, we use a data smoothing technique to abstract the trend of the data and, subsequently, identify deviations from trend, related to the timing of covid- . we use the hodrick and prescott ( ) filter to identify the trend component of the time series. extensively used in the macroeconomics literature, the hodrick and prescott ( ) filter separates the trend, cyclical, and error components of a time series: where y t is the time series of interest, τ t is the trend component, c t is the cyclical component (weekly pattern, for example, and ϵ t is the error ( ) the first term in eq. ( ) is the sum of squared deviations of the original times series and its trend; the second term is the sum of squares of the squared second differences, penalizing variations in the growth rate of τ t . the hodrick and prescott ( ) filter can be specified such that it can be used in all temporal frequencies: daily, weekly, monthly, seasonally, annually, and so on. all hodrick-prescott filter calculations are undertaken in r using the mfilter library, developed by balcilar ( ) . the benefit of using the hodrick and prescott ( ) filter is that it smooths/identifies the trend in the data without the loss of observations that occurs when using more traditional methods such as moving average calculations. however, the hodrick and prescott ( ) filter is not without its critiques, particularly around its identification and analysis of the cyclical component in a time series (hamilton, ) . but because we are not using the hodrick and prescott ( ) filter to identify and analyse the cyclical component of times series, but rather smooth volatile (weekly) time series data, is a lesser concern. and the ability to maintain all data points is of particular importance given the recency of potential changes in crime trends. interrupted times series is used to identify any changes in the trends of these time series, in conjunction with a chow ( ) testing methodology. interrupted time series tests are increasingly being used within the criminological literature to evaluate policy changes and crime reduction programs (hodgkinson, andresen, & saville, ; piehl, cooper, braga, & kennedy, ; reid & andresen, ) . moreover, these tests can be adapted for both known (exogenous) and unknown (endogenous) trend changes. in the current context there is a known policy intervention through the restriction of large gatherings, as discussed above. however, because we do not know how well these restrictions were enforced at their commencement date, march , we use a sequential chow ( ) test to endogenously search for a change in the trends of the criminal incident types: we account for the known seasonal component in crime data breetzke & cohn, ; cohn & rotton, ; farrell & pease, ; linning, andresen, ghaseminejad, & brantingham, ; mcdowall, loftin, & pate, ) through the inclusion of both week and week-squared variables that are represented by sequential values ( , , , …, ) representing the weeks may to may , for example; the week-squared variable is the square of week, allowing for any seasonality in the data to be identified. these two variables measure the underlying trend in the data. the interrupted times series (break in trend) variables (break, break trend, and break trend-squared) allow us to identify any changes in the frequency of criminal incidents, as both a sudden increase at the timing of the policy change (break) and changes in the trend at the timing of the policy change (trend and trend-squared). with weekly observations there are potential break points to identify; however, we exclude the first and last observations in order to facilitate the actual calculation of the break trend variable. as such each break-based variable has the value of zero before its representative break time and unity (break dummy variable) or sequential values (break trend variable) thereafter. for each of the crime variables (counts of total crime, residential burglaries, commercial burglaries, thefts of vehicle, thefts from vehicles, theft, mischief, and violence), we estimate possible break points. the actual structural breaks are determined at the time period that has the greatest value t-statistics for the break variables using robust standard errors (heteroskedastic and autocorrelation consistent). all estimation for the sequential chow tests is undertaken using r: a language and environment for statistical computing, version . . (r core team, ). the weekly time series for the various crime types in vancouver are shown in figs. - . all crime types have control years ending may and may , respectively, in order to identify changes in vancouver's temporal crime patterns that are not expected. table summarizes the results from the interrupted times series analyses. figs. to show that there is a clear seasonal pattern to crime in vancouver that is consistent with previous research (andresen & malleson, , . the seasonal pattern is not as strong as found in some research (e.g. mcdowell et al., ) but is present, nonetheless. the typical pattern, present for most crime types in most years, is an increase in event frequency during the summer and early fall, with subsequent declines in the winter months. as such, there is an expectation for most crime types to have (moderate) increases in their frequency at the time covid- restrictions were put into place. this is evident (in figs. , , , and ) for total crime, theft of vehicle, theft from vehicle, and theft, particularly in . the remaining crime types (violence, mischief, residential burglary, and commercial burglary) exhibit non-obvious or non-notable changes at this time of year given expected seasonal changes. this is not the case, however, for the temporal crime patterns from march to may . considering figs. to and table , there are a number of unexpected changes that occurred in march/april/may that did not occur in previous years. this is most easily seen in the hodrick and prescott ( ) trends, despite the volatility of weekly crime data. what can be seen from these results (graphed data and the breaks in trend identified by the interrupted time series analyses) is that total crime, theft from vehicle, and theft. all present decreasing trends in march/april/may when they are expected to be increasing or stable during these months. additionally, theft of vehicle has no obvious change in temporal trend march/april/may when it would be expected to increase, particularly based on the two most recent previous years. perhaps most dramatic is the significant increase, and of particular interest is the timing of the breaks in trend identified by the interrupted time series analyses. because of the volatility of weekly crime data, even those data smoothed by the hodrick and prescott ( ) filter, the timing of the breaks in trend are not always at or after week when a public health emergency was declared. for example, in the years ending in and , see table , breaks in trend have almost no consistency crime type to crime type. however, in the year ending in , there are notable changes in trend that occurred well within the covid- related restrictions in british columbia when health warnings were underway. collectively, the consistency in the timing of the breaks in trend and the changes in the expected directions of those trends (a lack of change in trend for the case of theft of vehicle) shows that covid- and its corresponding implications on opportunity structures have had significant impacts on crime in vancouver. consistent with opportunities theories, such as routine activities, the results demonstrate that as the nature of social life shifted into isolation, crime rates fluctuated accordingly. unlike social cohesion or altruism theories that predict crime would remain stable or decrease, or social disorganization theories that would predict crime would increase, routine activity and opportunity theories suggest that crimes may increase or decrease depending on the opportunity structure and the character of the exceptional event. in vancouver, we found that there was a significant decrease in total crime, when the previous seasonal patterns suggest we should have found an increase or stability. when we broke that pattern down by crime type, we found that there was a significant increase in commercial burglary, followed by a decrease, when in previous years it remained relatively stable during this time. theft and theft from vehicle decreased despite typically stable trajectories for these crime types in previous years. auto theft was stable when it would be expected to be increasing. violence, mischief, and residential burglary had no obvious change over this time period compared to previous control years. according to routine activity theory many of these changes in crime trends are unsurprising. as motivated offenders, suitable targets, and a lack of capable guardianship converge differently, crimes trends should fluctuate. for example, commercial burglary increased during the covid- shut down because businesses were forced to close, and owners and employees were no longer present to act as capable guardians. subsequent decreases would be expected if capable guardians reacted to this shift in crime. in vancouver, the police responded to the surge in calls for commercial burglary by arresting offenders and urging business owners to increase securitywhich many did by boarding up their shops (cbc, ) . this provides not only support for routine activity theory, but also the importance of natural surveillance (cozens, saville, & hillier, ) . theft and theft from vehicle likely declined, and auto theft failed to increase as expected, as suitable targets were t. hodgkinson and m.a. andresen journal of criminal justice ( ) relocated, and capable guardianship increased. for example, it might be more difficult to steal or steal from vehicles that are now parked in the garage of the owner, rather than an above ground parking lot during the workday (hodgkinson, andresen, & farrell, ) . the theory could predict that mischief would increase as young persons have more time outside of school to engage in troublemaking. however, because of the nature of the pandemic restrictions, capable guardianship actually increases as youth are expected by both parents and government officials to not leave the home. if they were witnessed out alone or in a group, this may increase their likelihood of being caught, because this behavior may appear more suspicious. thus, the findings are consistent with routine activity theory. counterintuitively, violence and residential burglary trends did not demonstrate a significant change over the study period, aside from expected seasonal patterns, as would be predicted by routine activity theory. residential burglary should have witnessed a significant decrease. as residents are home throughout the day, they can act as capable guardians making it very difficult to break in. however, vancouver had experienced significant declines in residential burglary across all neighborhoods prior to covid- (hodgkinson & andresen, ) and, thus, residential burglary counts may have already been too low to demonstrate any change. regarding violence, routine activity theory would predict that assault may decline as there are fewer opportunities for motivated offenders and suitable targets to converge in certain locations, such as alcohol outlets (bars, pubs, etc) or schools (fights). at the same time, the theory would predict that domestic assault would increase as offenders and victims are now consistently in the same space without capable guardianship. in our study, rates of violence do not change over the study period. however, violence is a combined measure of all crimes against person. thus, maintaining its expected seasonal trajectory is unsurprising as the decrease in assault may counter with the expected increase in domestic violence. it may also be useful to consider that women may be even less likely to report domestic violence if their access to social services in the pandemic is limited. as a result, they would be unlikely to find shelter if reporting leads to an escalation in violence. unfortunately, the data here do not provide us with the opportunity to explore this issue, but it is an area that desperately needs further attention. outreach services may be in a better position to understand what is happening in this area and how to address this gendered aspect of community safety during an exceptional event like a pandemic. overall, however, the changes in crime trends in vancouver are consistent with the change in routine activities. we believe this research may be useful in guiding policy and practice. for example, as opportunities for commercial burglary increase, so too must alternative forms of capable guardianship. this may include the police or citizenled patrols. in vancouver, many businesses boarded up their storefronts to add additional layers of security (singh & chan, ) . in a more creative response, a local printing shop has encouraged local artists to cover these boarded-up storefronts with art that celebrates front-line workers in order to reduce the "fortress" look of these business areas (crawford, ) . as the opportunity structure of crime changes during an exceptional event like a pandemic, it is important to address opportunities for crime early in the outbreak. as with any analysis, ours is not without its limitations. because of the source of our data, vancouver police department, we are limited to the impact covid- has on criminal incidents reported to the police. estimates for such reporting are not available at the city level, but in canada the average reporting rate to the police is % in (perreault, ) , a percentage that has been decreasing since the late- s (perreault, ; perreault & brennan, ) . moreover, reporting rates vary significantly from % (sexual assault) to % (burglary). as such, we cannot know the full impact of covid- on all criminal victimizations. additionally, the classification for violent crime is not available in a disaggregated format. because of this, we are not able to differentiate between different violent criminal incidents. in the current context, this could be quite important. as mentioned, with self-isolation and the closure of liquor establishments, we expect decreases in assaults, particularly those related to alcohol (grubesic & pridemore, ) ; similarly, with increased time together coupled with the stress of a pandemic, we expect increases in domestic violence (parkinson, ) . depending on their changes in frequency, one may cancel out the other in the aggregate such that there is no identifiable change overall. however, without disaggregated crime data we cannot assess this possibility. there are also unknowns at this point in the research. we are unable to say if calls for service and police activity have been affected by the virus. police may change their behavior to protect themselves from contracting the virus. furthermore, police may change how they respond to calls as it becomes increasingly irresponsible to arrest and incarcerate people who may then be at an increased risk of contracting the virus. we were unable to explore these changes to police behavior in this study. importantly, the research is limited by its focus on the city-level. this means any changes to opportunity structures at the local level, such as shifts in land use, are unable to be investigated here. additionally, in many countries, academics, legal practitioners, and social workers are demanding de-incarceration and improved housing policies for the homeless, in order to prevent the spread of the virus (bartels & anthony, ; kim, ; kirby, ) . the changes in land use, coupled with shifts in social policy will likely affect the nature of routine activities at both the local and city levels and require further examination. directions for future research follow from the limitations outlined above. first, qualitative research that can more thoroughly investigate victimization should be undertaken in order to establish a deeper understanding of the impacts of covid- on criminal victimization. second, research that disaggregates violent crime into its component parts is necessary to identify any changes that are more nuanced than total violent crime. third, researchers should attempt to obtain and analyse disaggregated data that account for shifts in land use and opportunity structures as a result of local shifts in covid- related policies. and fourth, though the impacts over time of covid- may not be known for some time, continued research in this area should be undertaken to identify medium-and long-term effects, if any, of the pandemic. we predict that as the pandemic ends, and routine activities return to their pre-pandemic state, so too will crime trends. this will include the return of informal social controls such as the capable guardians of commercial establishments, that have already resulted in decreases in commercial burglary. we expect that other crime types described above such as theft and auto theft will likely return to their previous seasonal trajectories as well. we may witness an increase in cybercrime, as many systems have moved online in order to cope with the crisis and some may remain there. however, as potential victims become more computer savvy, this may actually decrease (e.g potential victims may be become capable guardians). at the point of writing we do not know how long the pandemic will last. routine activity theory assumes motivated offenders as a constant. however, as the economy changes, and people are unable to work and become more desperate, it is possible that crime trends may increase to match changes in motivation. there are parts of the world that are suffering already, where staying indoors is not an option and corporations and politicians are beginning to meddle in covid- related decisions (wilson, ) . furthermore, there are certain groups who are already marginalized and struggling who will be affected more deeply by continued restrictions on work and the economy. indeed, it would be unfair to claim that the shift in routine activities alone may be driving crime rates, when other countries do not experience the same institutionalized altruism from which many canadians benefit (lemieux, ) . comparative research is necessary to determine if similar social support policies may benefit other countries who are experiencing greater rates of social inequality and, subsequently, increased motivation, during the pandemic. finally, early and continued methodologically rigorous research is necessary on crime and exceptional events to not only identify how crime is changing, but how to better prevent it. regardless of these limitations, and subsequent directions for future research, we contribute to the literature on exceptional events and crime through the analysis of a (ongoing) pandemic as a natural experiment. similar to previous research that has investigated the impact of natural disasters on crime, this topic is not to be taken lightly. rather, given its presence we can use it to better understand social phenomena, hopefully making cities safer during the crisis and in its aftermath. while we have yet to know the full extent of the economic impact globally, with small businesses closing, and people 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social cohesion: a longitudinal study natural disasters as threats to peace, special report a tale of three cities: crime and displacement after hurricane katrina canada and covid- : learning from sars. the lancet the rightwing groups behind wave of protests against covid- restrictions disasters and crime: the effect of flooding on property crime in brisbane neighborhoods natural disaster and social order: modelling crime outcomes and disasters in florida key: cord- - m iozj authors: yang, hyeonchae; jung, woo-sung title: structural efficiency to manipulate public research institution networks date: - - journal: technol forecast soc change doi: . /j.techfore. . . sha: doc_id: cord_uid: m iozj with the rising use of network analysis in the public sector, researchers have recently begun paying more attention to the management of entities from a network perspective. however, guiding elements in a network is difficult because of their complex and dynamic states. in a bid to address the issues involved in achieving network-wide outcomes, our work here sheds new light on quantifying structural efficiency to control inter-organizational networks maintained by public research institutions. in doing so, we draw attention to the set of subordinates suitable as change initiators to influence the entire research profiles of subordinates from three major public research institutions: the government-funded research institutes (gris) in korea, the max-planck-gesellschaft (mpg) in germany, and the national laboratories (nls) in the united states. building networks on research similarities in portfolios, we investigate these networks with respect to their structural efficiency and topological properties. according to our estimation, only less than % of nodes are sufficient to initiate a cascade of changes throughout the network across institutions. the subunits that drive the network exhibit an inclination neither toward retaining a large number of connections nor toward having a long academic history. our findings suggest that this structural efficiency indicator helps assess structural development or improvement plans for networks inside a multiunit public research institution. public research more inclines to distribute its findings than commercialize in contrast to industrial research (geffen and judd, ) . in general, institutes conducting public research are largely government funded and target the public domain (bozeman, ) . because of their national orientation and stable funding source, public research institutes do cutting-edge research at least one academic field through long-term plans (greater than three years) (bozeman, ) . a public research institution often develops as an association of research institutes rather than a single organization. research entities with a public research institution enjoy institutional autonomy in choice of subjects notwithstanding the fact that they are under the same umbrella of governance. naturally, research organizations have different characteristics depending on national circumstances. some public research institutions, such as the max planck gesellschaft (mpg) in germany, are faithful to pure research (philipps, ) , while others have significance within a particular national context: part of the national laboratories (nls) in the united states (us) addresses defense-related technologies (jaffe and lerner, ) , and the government-funded research institutes (gris) in korea attempt to assist in the country's economic development by promoting indigenous public research (mazzoleni and nelson, ; arnold, ; lee, ) . with recent advances in our understanding of network, it is possible to apply novel network knowledge to manage public research institutions in response to internal and external changes. for example, entities in national innovation systems (freeman, ) or the triple helix models (phillips, ; leydesdorff, ) can be external factors affecting research of public research institutions. the notion of national innovation systems provides a framework to explain underlying incentive structures for technological development at a national level and international differences in competence from a network perspective of public and private organizations (patel and pavitt, ) . the triple helix model considers coevolving academic, industry, and government which provokes techno-economic developments of a country (leydesdorff et al., ) . in these systems, public research institutes provide fiscal and technical assistance to other organizations. kondo (kondo, ) pointed out that public research institutes dedicated to transferring technologies to industry by means of consulting, licensing, and spinning off. by doing so, they contribute to promoting integration and coordination within the system (provan and milward, ) . in order to formulate policies and procedures to steer the entire system, system organizers are able to guide public research institutes properly. in this context, control of those key agencies is important to achieving desirable outcomes. moreover, there is a growing need for an efficient implementation throughout public research institutions composed of multiple sub-organizations in order to deal with internal controls (yang and jung, ) . for example, most public research institutions have undergone transformations in recent years due to modernization, imperatives for efficiency, and the promotion of collaboration with the industry (buenstorf, ; cohen et al., ; simpson, ; senker, ) . in unfavorable economic conditions, declining government funding causes the restructuring of research areas (malakoff, ; izsak et al., ) or the government demands more practical outputs from them, such as conducting applied research and setting standards (oecd, ) . in an attempt to harness technology for socio-economic development, governments often prioritize future research through foresight activities (priedhorsky and hill, ) and accordingly assign new academic missions to public research institutions. in particular, developing countries have lately been paying more attention to the technology-driven development model under government supervision (arnold, ) . at that time, controlling every entity enables the institution to fully guide those internal changes but entails great expense. from to , public research institutions engaged in national strategic areas, including exploration of mineral resources, industrial development, and military research and development (r&d) (oecd, ) . after the termination of world war ii, the establishment of public research institutions grew in an effort to advance military technology in many countries. moreover, at that time, public research institutions extended almost all areas with which governments were associated, such as economic and social issues. they continued growing until the s. in the s and s, many countries expressed doubts on their contributions to innovation. however, as deepening the understanding of national innovation systems or the triple helix models, public research institutions started to be seen in a new light. in these models, public research institutions have played an indispensable role in preventing systemic failures, which reduce the overall efficiency of r&d (lundvall, ; sharif, ) due to their relations with external collaborators (klijn and koppenjan, ; mcguire, ) . still, the importance of public research institutions are emphasized in particular for scientific innovation (cabanelas et al., ) . in this regard, a network approach is necessary to efficiently implement transformations throughout sub-organizations, and the academic interest also grows for the effective operation of the network (cabanelas et al., ; jiang, ) . there is, however, a lack of empirical research on managing public research institutions through a network system. hence, in this paper, we conceptualize three major public research institutionsthe mpg, nls in the us, and gris in koreaas networks, identify the sub-organizational network structure of each, and examine its structural efficiency. a collaborative research network is one of the most prevalent inter-organizational configurations (shapiro, ) . however, we deem that topical similarity between research institutes is suitable to represent a relation between them in research interests. most transformations involve changes in research areas, and changes in organizational research topics frequently occur when governments prioritize specific research fields or delegate new roles to institute (wang and hicks, ) . prior studies emphasized the importance of similarity in knowledge content among entities to effectively manage inter-organizational networks as well (tsai, ; hansen, ) . for these reasons, a network here is formed by pairs of subunits having the most similar research profiles. with the addition of temporal dynamics to inter-organizational relations, a chain of networks over time allows the description of the structural evolution of public research institutions. based on revealed networks, we determined the structural efficiency with which network-wide actions can influence entities for finite time periods. no matter the measure puts in place, all members of network need to adopt it to achieve collective actions. in the early stages of change implementation, network organizers select initiators to change among entities. as the change initiators propagate control actions to the remainder of entities, a public research network can be steered in the desired direction like a car. we can derive a minimum number of suitable initiators from a theory of "structural controllability" (yuan et al., ) . in the theory, change initiators refers to injection points of external energy used to steer the network, which are theoretically selected depending on network structure. in this process, structural efficiency is obtained by calculating the share of change initiators in the network: the lower the efficiency value, the smaller the number of entities the network manager is required to handle. therefore, by comparing efficiencies with structural properties over time, we can estimate network characteristics specific to institutions. in this study, we divided institutional research portfolios into six time periods based on scientific output over eighteen years ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) , and estimated structural efficiencies of research similarity networks. considering structural efficiency, we can observe that networks in all three research institutions can be managed with less than % of sub-organizations, and the values reflect the changes that have occurred in research institutions. each research institution has some suborganizations consistently selected as suitable change initiators over a period of time. our results primarily highlighted young subordinates as appropriate change initiators, which means that information blockades in network might occur unless the selected units are properly managed. moreover, the estimated changes initiators tend to have a lower connectivity in network than the rest of nodes. we expect that our work has implications for decision-making bodies and network managers seeking to an efficient way to influence their intention on a network of public research institutes. the remainder of this paper is structured as follows: in section , we briefly describe the impact of structure on network effectiveness associated with public research institutions based on past research. section is devoted to an explanation of data sources, network construction processes, and the calculation of structural controllability in a network. we discuss the results of our experiments in sections and , and offer our conclusions in section . methods for utilization and development of networks have grown in an attempt to address complex problems that require collective effort. when the purpose of the network is to deliver public services, independent organizations are generally involved in the process, and interdependency between participants facilitates the formation of links (kickert et al., ) . by exchanging knowledge through a network, public research organizations attain a higher level of performance, at the same time, create a greater ability to innovate (morillo et al., ) . goldsmith and eggers ( ) claimed that using a vehicle for networks is favorable to organizations that require flexibility, rapidly changing technology, and diverse skills because actors can exchange goals, information, and resources while interacting with each other. resources usually refer to units of transposable value, such as money, materials, and customers, and information signifies exchangeable units between agencies, such as reports, discussions, and meetings. with regard to exchanged goods between organizations, van de ven (van de ven, ) underlined the importance of information and resources as "the basic elements of activity in organized forms of behavior." in research systems, organizations can take advantage of network participation to have a greater possibility of funding, to broaden their research spectrum, or to reduce the risk of failure (beaver, ) . therefore, networks are beneficial because they can pool resources, permit the mutual exploration of opportunities, and create new knowledge (priedhorsky and hill, ) . however, strategies are needed to coordinate interactions while managing networks because different actors have different goals and preferences concerning a given problem (kickert et al., ; o'mahony and ferraro, ) . the capability of network management is also necessary to promote innovations (pittaway et al., ) , but there remain questions as to how to manage such organizational interactions as beaver (beaver, ) pointed out. orchestrating activities seems unnecessary because of interactions between autonomous organizations, but addressing conflicts keeps agencies cooperative in effort to achieve the goal of the network, thereby facilitating the effective allocation and efficient utilization of network resources. furthermore, a network sometimes needs to be intentionally formed to boost management by governing parties which would be either an external organization or network participant(s) (provan and kenis, ) . public research institutions can be said to be governed by external organizations, considering that different entities are in charge of their administration in general, such as ministries, research councils, and other steering bodies. both the mpg and korean gris are apparently steered by a single entity. the fundamental management policy of the nls in the us also originates in a federal agency, although several laboratories are operated by contract partners. by frequently repeating interactions among actors, networks produce certain outcomes. the performance of a network is evaluated according to whether the network effectively attains its goal. the outcome varies depending on governing strategies, and the course of attainment can be enhanced by taking advantage of structural properties of the network (kickert et al., ; goldsmith and eggers, ) . provan and milward ( ) argued that the assessment of network effectiveness should involve consideration not only of beneficiaries, but also of administrative entities and the participants of the network. nevertheless, the literature on networks has paid more attention to the evaluation of their effectiveness by treating networks as a whole, such that the common goal is primarily involved in network-level accomplishment (provan and milward, ; möller and rajala, ) . there remain difficulties in determining network effectiveness. the problem primarily resides in the impossibility of quantifying the exact network outcome (provan and lemaire, ) . as agranoff ( ) claimed, networks are not always directly related to policy adjustments because some interactions are forged by voluntary information exchange or educational service. in the public research institution, researchers engaged in specialized fields have the opportunity to share ideas across administrative boundaries given that they have the goal and intend to generate public knowledge. outcomes of research networks can be approximated by proxy variables, such as patent and paper citations, innovation counts, new product sales, and productivity growth (council, ) . furthermore, such networks also indirectly affect subsequent movements and policies. thus, network efficiency needs to be measured for various types of networks, by considering factors beyond collaborations. in order to increase network effectiveness, structural efficiency in networks is important: since all entities are connected, damage to one part can cause the collapse of the entire system through a cascade of failures. in this regard, considerable research on networks has focused on deliberately building efficiently manageable networks (cabanelas et al., ; kickert et al., ; van de ven, ; provan and kenis, ) . certain network structures can affect innovation performance by catalyzing knowledge exchange (valero, ) . enemark et al. ( ) argued the importance of the network structure to collective actions via experimental tests that demonstrated structural variations in a network can either improve or degrade network outcomes. however, there is ambiguity in appropriate network structures to achieve effective control. pittaway et al. ( ) suggested that longitudinal network dynamics need to be taken into account when designing network topologies. a network is required to change its members or structures in order to adapt to environmental changes. much of the literature on networks emphasized that instability is an opportunity for transformation (hicklin, ) . although the capability of flexible response is one of the strongest features within a network model, such network dynamics challenge for effectively managing networks. with regard to network size, it is widely known that the greater number of actors involved, the more difficult it becomes for the network to achieve collective cooperation (kickert et al., ) . increasing the number of participants results in more complex network governance because the number of potential interactions also exponentially escalates. however, prior research found that research networks evolved to be more centralized as growing the network (ferligoj et al., ; hanaki et al., ) . the growing patterns of research networks imply that adding an entity does not always increase complexity of network management. theorists rather claimed that the introduction of a new node would improve efficiency to control networks (klijn and koppenjan, ) . centralization captures the extent of inequality with which important nodes are distributed across the network, and is often measured in terms of freeman's centralities (freeman et al., ) . a degree (the number of connections) centralized network is known to readily coordinate across agencies and closely monitor services (provan and milward, ) . in complex networks, a minority of nodes, referred to as hubs, dominates connections while the majority is connected with a small number of points (barabási and albert, ) . research revealed that complex networks were robust against random attacks (albert et al., ) . hubs in research networks were not only empirically impressive in their performance (echols and tsai, ; dhanarag and parkhe, ) , but also easy to access new knowledge developed by other entities (tsai, ) . hanaki, nakajima and ogura (hanaki et al., ) also found r&d collaboration networks evolved toward more centralized structures because organizations prefer to collaborate with reliable partners based on referrals obtained from former partners. however, a high degree of integration is not always desirable. provan and lemaire ( ) proposed that connective intensity between organizations should be appropriately controlled for effective network structure. cabanelas et al. ( ) also found research networks producing high performance featured nodes with low degree centrality. no matter the types of networks that develop out of interactions, the goal achievement is possible only when the relevant information spreads throughout the network to encourage actors to conform. in recent years for public research institutions, the controllability of organizational portfolios has been seen as constitutive of dynamic capabilities, which means the "ability to integrate, build, and reconfigure internal and external competencies to address rapidly changing environments" (teece et al., ; floricel and ibanescu, ) . in this sense, estimating efforts to control entities of public research institutions is related to assessing the feasibility of research reorganization over networks. at the same time, the number of key points in information flow within a network affects burden on the network administration. although earlier work emphasized that selectively activating critical actors is more effective to integration than full activation, the system must secure the capability to exercise influence across agencies (kickert et al., ; provan and lemaire, ) . furthermore, the efficiency with which network structure can be manipulated would be a suitable criterion to evaluate the built structure. this section is devoted to describing methods of network construction based on collected bibliographies and analytical methods. we describe a quantification method for structural efficiency given structure to control the whole network, and explain structural properties to explore their relation with structural efficiency. in the process of efficiency calculation, we extract suitable organizations to initiate transformation. this investigation was conducted in the r ver. . . environment (r core team, ) , and used the following add-on packages for convenience: ggplot (wickham, ) and igraph (csardi and nepusz, ) . we identified research portfolios based on scientific output, and gathered bibliographic data regarding nls, mpg, and gris from the thomson reuters web of knowledge. academic output over eighteen years ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) was compiled according to institutional names and abbreviations of authors' affiliations. we only used affiliations in english for this study. subordinate research institutes listed in official websites were considered, and their portfolios were tracked using at least twenty papers for each. all disciplines, which are the constituent elements of a portfolio, need to be identified using the same classification system for ease of institutional comparison. we utilize the university of california-san diego's (ucsd) map of science (borner et al., ) as a journal-level classification system. the map classified documents into subdisciplines belonging to disciplines on the basis of journal titles. naturally, a research portfolio has two levels of classification: discipline and sub-discipline. particularly, a discipline refers to the aggregate level of sub-disciplines in the hierarchical structure in this study. fig. shows an example of disciplinary mapping using sci (sci team, ) in order to analyze the thematic evolution of network over time, we split the portfolios into time intervals. with regard to an adequate duration of assessment period to represent scientific output being measured, abramo et al. ( ) claimed that a three-year period is adequate to assess scientific outputs. by accepting their recommendations, we observed the development of institutional portfolios for six consecutive time slices. as a well-known analytical method, a complex network is suitable for exploring dynamic topology changes (strogatz, ) . here, an inter-organizational network is formed between subordinate institutes building similar research profiles. representing sub-organizations, nodes are connected by a link when two sub-organization have similar research portfolios. in order to measure similarities, we used "inverse frequency factors" for weighting system and "second-order cosine similarities" (garcía et al., ) . the inverse frequency factor borrows from a term discrimination method for text retrieval (salton and yang, ; salton and buckley, ) . the factors weight each subdiscipline in the research portfolio. the weight of sub-discipline m for research institute i is determined by w m;i ¼ f m;i  logð n nm Þ where f denotes the number of articles; and ð n nm Þ implies the inverse frequency factor to file out prevalent research (jones, ) . the logarithmic frequency factor is calculated inversely from the ratio number of subunits n m that publish their achievements in sub-discipline m to the total number n of research institutes. as a result, the set of weights generates a sub-disciplines-by-institute matrix. the similarities between two institutional research portfolios primarily take the cosine measure (salton and mcgill, ; baeza-yates and ribeiro-neto, ) . for the purpose of improving the accuracy of similarity, we applied second-order approaches to the sub-disciplineby-institute matrix. colliander and ahlgren ( ) explained that first-order approaches directly reflect the similarity between only two profiles, whereas second-order similarities determine those between two given portfolios and other institutional portfolios. a large number of studies have confirmed the superior performance of the secondorder approach as well (ahlgren and colliander, ; thijs et al., ) . moreover, to render easier structural analysis and network visualization, we strip weak similarities from the research similarity matrix. using the maximum spanning tree (mst) algorithm (kruskal, ) , we extracted tree like-structures. the mst algorithm ensures that all institutes are connected with maximal similarity, which implies that the institutes are connected through the most relevant links. therefore, a linked pair of institutes indicates greater potential for common intellectual foundations. among various well-known algorithms to detect mst, the backbone of thematic networks is derived by prim's algorithm (prim, ) . in order that the network structure can efficiently elicit the desired response from its elements, a certain amount of energy needs to be injected into the network to change the behavior of actors. thus, the selection of several agencies to initiate changes depending on network structure is inevitable. at the same time, it is important to minimize the number of injection points due to management cost. studies on complex networks consider that nodes can dynamically make decisions or can dynamically change their states by responding to information received through links between nodes. as individual actors, nodes on research networks can be researchers or research institutions, and the nodal states can be represented by individual research interests or disciplinary composition. here, we estimate the capability that controls the behavior of such nodes in complex networks with the minimum involvement of intervention adopting the notion of structural controllability. in recent years, a number of studies have focused on driving networks to a predefined state by combining control theory and network science (liu et al., ; wang et al., ; lombardi and hörnquist, ; gu et al., ) . according to network controllability, if a network system is controllable by imposing external signals on a subset of its nodes, called driver nodes, the system can be effectively driven from any initial state to the desired final state in finite time (kalman, ; lin, ) . thus, network controllability depends on the number and the placement of the control inputs. for this reason, structural efficiency refers to the share of the driver nodes. in this study, agencies found using structural controllability are key locations to steer the entire inter-organizational research network. we applied the structural controllability for undirected networks, introduced by yuan et al. ( ) , to matrix representation of our temporal msts. each temporal matrix g(a) was considered a linear timeinvariant model _ xðtÞ ¼ axðtÞ, where the vector x ∈ ℝ n represents the state of the nodes at time t, a∈ ℝ n×n denotes the research similarity matrix of mst, such that the value a i,j is the portfolio similarity between institutes i and j (a ij = a ji ). the controlled network g(a, b) corresponds to adding m controllers using ordinary differential equations _ xðtÞ ¼ axðtÞ þ buðtÞ, where vector u(t)∈ ℝ m is the controller and b ∈ ℝ n×m is a control matrix. the problem of finding the driver nodes of the system is solved by the exact controllability theory following the popov-belevitch-hautus (pbh) rank condition (hautus, ) . to ensure complete control, the control matrix b should satisfy rank[λ m i n − a, b]=n, where i n is the identity matrix of dimension n, and λ m denotes the maximum geometric multiplicity μ(λ l ) (=n-rank(λ l i n − a)) for the distinct eigenvalues λ l of a. therefore, from a theoretical perspective, changes initiated from the drivers are likely to affect the entire structure. hence, driver institutes are crucial to the functioning of networks for public research institutes. in this paper, we regard the share of drivers in all agencies as an efficiency indicator in that the number of drivers is important for efficient control. network properties have been utilized by a considerable amount of literature in the area to better understand structural features of networks (newman, ; albert and barabasi, ; woo-young and park, ) . in order to understand the relation between efficiency and the inter-organizational research network, we extracted major features across institutions based on some structural properties, such as network size and connectivity. the number of participants represents network size associated with network volume. centrality is one of the most studied indicators in network analysis, and measure the influence of a node in a network using degree centrality (freeman et al., ; borgatti et al., ; freeman, ) . we examine the degree feature of driver nodes. as a nodal attribute, we assign research experience in time periods to nodes to characterize driver nodes. this section contains the major results of our investigation of the structural features of the inter-organizational networks. to form our desired skeletal network, we extracted pairs of academically close institutes based on portfolio similarities among their participants using the construction algorithm of the maximum spanning tree (mst). the results obtained from the backbone networks are related to structural controllability. in order to address the evolution of inter-organizational research, we assessed the structural features of temporal msts. figs. - show tree-like structures of institutions over time. each node represents a sub-organization, and its size is proportional to the total number of documents published. the colors filling the nodes were determined by the discipline in which the institute was found to be most productive. the portfolio similarities between pairs of linked institutes represented the weight on the network, and these weights affected the width of links as well. the descriptive statistics of portfolio similarity summarize and describe the distribution of the skeletal relationships between subordinates, as listed in table . for all institutions, we found that the distributions were biased toward high similarities between research portfolios. for the nls networks in the us, the overall greater averages and smaller standard deviations of portfolio similarities than the other two institutions indicated that most research units were seen as connected, with the smallest difference in their research areas. on the other hand, in case of the gris, the lowest values of average similarity signified that each unit had a distinct research portfolio. the largest standard deviation and the low values of kurtosis for most time periods also showed that their research similarities were the most widely distributed. in order to represent the dynamic characteristics of msts, their structural properties are listed in table . the number of nodes n increased and, accordingly, the number of links increased to n − following the definition of an mst in the context of a connected network. in spite of sparsity of the network, nodes having a relatively large number of links could be found in some institutions, in particular the oak ridge national laboratory (ornl) within the nls, which was connected to approximately a quarter of the other organizations for four time periods, and the korea research institute of standards and science (kriss) and the korea institute of science and technology (kist), which appeared as a maximally connected node in each half of the dataset. however, there was stiff competition among institutes with the maximum number of connections in the mpg. we noted that network density ( /n) could be obtained from the number of nodes, and the transitivity always reduced to zero because mst rules out cycles. we calculated a periodical change of structural efficiency, and then examined the relations between network efficiency and structural properties. following this, we investigated the features of estimated driver nodes in terms of degree and period of appearance. note that although the number of driver nodes is theoretically fixed in a network, there can be multiple sets of drivers (jia and barabasi, ) . we randomly selected a set where multiple driver sets existed. as an indicator of network efficiency obtained from structural controllability, fig. denotes the share of driver nodes over time. according to the graph in the figure, the proportion of drivers varied, but institutions did not have to consider all their agencies for network-wide transformation. less than % of nodes were selected as suitable points at which to inject external information in all three institutions because the maximum value of structural efficiency in the entire datasets was about % at the second period ( ) ( ) ( ) in the gris. in particular, the nls could be influenced with a relatively small share of drivers among the institutions at all times, which the exception of the period - , whereas in the gris, the largest portion of nodes mostly needed to initiate changes. the efficiency fluctuation of the mpg was more stable than other two institutions over time periods. an understating of drivers enables administrators to take preemptive action to prevent information isolation, like the knowledge of the relation between the share of drivers and network efficiency can help plan structural development. the total number of driver appearances for the entire period corresponded to , , and for the nls, the gris, and mpg, respectively but , , and agencies were selected as drivers. this was an evidence for the existence of memory in the drivers. moreover, figs. and capture some features of the drivers. fig. compares the average number of links between drivers and the entire nodes over different periods. despite the common knowledge that nodes possessing large connectivity are influential, our results showed that drivers with low connectivity tended to determine collective agreement on the network. fig. shows the average durations of appearance by institutional drivers. we see that the driver nodes were the ones newly entered to the network based on the average durations. of the institutions, the research units of the nls showed a wide difference between drivers and non-drivers. public research has contributed to major innovations by improving competitiveness among existing industries and developing new ones. as prominent contributors to public research, governments have implemented a variety of support policies and programs for higher efficiency and excellence. among the actors involved in public research, public research institutions aim to disseminate their knowledge, by providing various functions: priority-driven research to address national and academic agendas or blue skies research engaging large-scale research facilities to complement university research (pot and reale, ) . to maintain such diversity, public research institutions seek to coordinate elements with varying specializations and missions in adapting to dynamic technological environments. as a part of the effort, institutions occasionally attempt to restructure research portfolios or modify organizational placements in relation with other research units. in order to assess the development of public research institutions, we examined structural evolution derived from research similarities in the context of networked organizations in this paper. more precisely, this study focused on public research institutions composed of several specialized research units, and extracted a network from similarities between sub-organizational research portfolios over eighteen years. a pair of connected agencies would be most influenced by the same type of exertion on a specific research area. in addition, suborganizations connected to each other can be potential partners to collaborate because they share similar academic backgrounds. for example, the similarity networks of the gris give implications for inter-disciplinary research groups operated by the research council. in the research group, researchers working at different gris seek a solution together to technological difficulties and research similarities can indicate proper gris to resolve the difficulties. moreover, offering the advantage of predictable network controllability, network modeling helps to understand the system's entire dynamics, which could be guided in finite time by controlling the initiators (liu et al., ) . as a result of the modeling, we can measure the efficiency of the network, where network efficiency implied the proportion of elements required as initiators to change the states of the entire agencies. the lower the proportion, the greater the network efficiency because the initiators are injection points for external information. we also revealed the structural properties of estimated initiators. our research here is different from other studies concerning network effectiveness in that it quantitatively estimated the effort required to control an entire inter-organizational network based on its structure. naturally, if we send control signals to every single node, the network is operated with high controllability but involves significant cost. thus, by employing the concept of structural controllability, we can theoretically detect the initial spreaders of information that need to be properly treated. otherwise, they would have produced barriers to the exertion of authority; in extreme cases, the information blockades could have caused network failure (klijn and koppenjan, ) . however, handling these elements incurs extra cost, because of which it is important to build networks with the minimum possible number of initiators to reduce enforcement costs incurred for complete control (egerstedt, ) . common structural features of estimated initiators can direct network management of public research institutions. we generated results to provide a clear idea of how structural efficiency of research network is related to structural properties, such as size and nodal degree. previous work on network governance structures has provided recommendations on how to build and design inter-organizational networks for innovation acceleration. for example, related to the number of participants, it is natural to expect that the share of drivers would also increase owing to a higher risk of insularity in information due to increasing structural complexity. however, our findings suggest not necessarily complying with the idea. each of the institutions considered by us was different in size from others: the mpg was the largest-scale organization, whereas the nls formed the smallest group in terms of number. however, according to our results, the size of the network did not seem to meaningfully affect table network properties of inter-organizational network. time span - - - - - - the proportion of drivers in public research institutions. despite being a medium-sized institution, the networks of the gris were more likely to be inefficient than those of the mpg and the nls. we think this was because an institution more experienced with managing such a union has built more effective structures. even we found that the gris took advantage of the structural reorganization of the network because additions improved their network efficiency. in this regard, kickert et al. ( ) claimed that the introduction of new actors can be a strategy to accomplish a mutual adjustment, since the new institute would cause structural changes within the network. proposition . a subset of nodes positioned in structurally important locations will have ability to steer a whole network of public research institution. our findings indicated that control actions applied to only less than half of the research units can lead to changes of an entire system, and the units repeatedly appear over time. we suspect the reason that public research institutions are designed to be a cost effective and resilient, as do their infrastructure networks. however, as national research structures can be affected by government policies (hossain et al., ) , the network efficiency also changes over time. the drastic fluctuation in the share of drivers would be related to changes in the relevant institution's strategy or operation. for example, the gris underwent a restructuring to remove redundancy, and began operating under the research councils after . we can capture drastic changes at the same time in our results because their structural efficiency significantly increased between the second and third periods between and . the results would imply that the organizational rearrangements in the gris worked well. besides, the research subjects of the nls were revamped in the - period due to several events, i.e. the september attacks and the outbreak of the severe acute respiratory syndrome (sars). since the terrorist attacks of september , , the nls have made greater efforts to reinforce national security by working on nuclear weapons or intelligent detection of potentially dangerous events. moreover, a sudden epidemic of sars accelerated multidisciplinary research in the nls on vaccines, therapeutics, bioinformatics, or bioterrorism. we also find that the structural efficiency of the nls were severely affected during the readjustment period. these changes in portfolio composition would cause temporary disarray in the structure of the networks. on the other hand, the property of stable fluctuations in the mpg would be attributable to internal transitions for scientific advances rather than external impact. the mpg makes an expansion of research topics by mostly spinning off units because each unit has its own research area. proposition . variations in structural efficiency of research networks will reflect structural changes in research composition. another difference between past research and our work here is that degree centralization is not invariably recommendable. policy makers and network scientists have hitherto paid attention to highly connected institutes because hubs are regarded as network facilitators. however, our findings indicated that most key elements were apt to have low degrees. our study focused on revealing the injection points to infuse their nearest neighbors with energy regardless of the amount of energy required, and the nodes impart directions to connected neighbors at a time rather than exuding control forces over their adjacencies simultaneously. obviously, the energy-entering hub can effectively reach agencies within its orbit, but there is a diffusion range. thus, our observations suggest that network-wide influence was dependent upon nodes with a low connectivity. in this context, a network with moderately distributed focal points can be more effective to influence all organizations than a thoroughly concentrated one. furthermore, emergent sub-organizations show a tendency to have greater effect on structural efficiency than sub-organizations with a long research experience. we suspect that this is why a new research institute is often derived from a larger unit in public research institutions, holds low research similarity with other units than its parent, and takes position at the border, beyond any energy ranges. another possible is that that a newly-established research institute has unstable research portfolios, as braam and van den besselaar ( ) pointed out. the instability that a new research institute has in its research areas can increase uncertainty about the consequences of network-wide changes. therefore, network managers may have the need to monitor the degree of acceptance of a network-wide action especially among emerging sub-organizations. this result is also consistent with recent observations, whereby driver nodes in real-world networks tend to be reluctant to link with high-degree nodes (liu et al., ) . proposition . other things being equal, a possibility to control the whole research network will increase when control actions work properly at nodes with a low connectivity and a short research history. we consider the differences in network effectiveness between existing studies and our findings originate from: whether the complete functioning of all elements was considered. previous studies regarding the maximization of network effectiveness implicitly presupposed the complete performance of all entities a priori despite conflicts between participants, but at least network managers need to ensure complete operation of their network. for the full functioning of a network, all elements are required to be within the sphere of influence of the network manager for network-wide control. we deal with a possibility to manage the network behavior in public research institutions by quantifying the effort required to implement maneuvers. in order to avoid control blockades, we showed the importance of the elements, inter alia, with low connectivity and brief experience in academia. this study provided theoretical results for structural controllability assuming some ideal situations, such as that measures were implemented on network skeleton without redundant connectivity, sufficient resources were provided to change the network, all institutes respected the administrator's intention, and there were no conflicts between a pair of connected institutes. the success or failure of such measures can be determined once the processes are completely implemented because a network's dynamic nature in inter-organizational network raises difficulties in coordination. nevertheless, estimating the completion of network-wide objectives is still critical to network planning and design. our theoretical calculations here can assist decision making for structural improvement plans. moreover, the common features of the selected initiators are sufficient to suggest elements significant to attaining a synchronized response across and institutional network to reorganize research portfolio. public research institutions continue to gain prominence in the development national agendas of science and technology. institutions have their own strategies according to their values and interests in research trends to a greater or lesser extent. governments and research councils significantly affect these institutes through polices, programs, funding, and financial support in an effort to better coordinate their research agencies (rammer, ) . therefore, guiding the subunits of these institutes in a network is important to efficiently deliver managerial control. in doing so, administrators should be concerned with improving network structure to enhance its outcomes. however, manipulating network structure is difficult because of complex and dynamic states of the sub-organizations. in this study, we quantified network structural efficiency to maneuver a set of spontaneous elements into network-wide goals by using the theory of structural controllability (yuan et al., ) , and tracked the efficiency of networks of these public research institutions: the gris in korea, the nls in the us, and the mpg. for the relevant calculations, we extracted a hidden network structure from each institution based on similarities between the profiles of their subordinate organizations. the results of structural efficiency enabled the assessment of the operational strategies of each institution for eighteen years. the elements selected by structural controllability implied suitable points to inject external energy for governing networks. revealing the injection points was important to prevent information blockages that hinder collective action. apparently, the greater number of injection points required, the lesser the efficiency of network due to the increased burden of management. our findings indicate that structural efficiencies reflect changes in research interests of an institution. in this sense, research institutions have the necessity to track the structural controllability to assess their structural changes, such as portfolio adjustments on all of sub-organizations. the structural controllability can also provide the suitable spots for an intervention by a network manager (ministries, research councils, or steering bodies) as driver nodes with regard to structural changes. according to our results, the proper intervention points tend to be with a low connectivity as well as young suborganizations. in spite of these implications for managing strategies of interorganizational networks, this study has shortcomings that limit the generalizability of our findings. scientific articles represent only part of an institute's capacity for research. major scientific outputs are classified into two types: scientific articles and patents. depending on the major research types, some institutes concentrate on patents instead of publications. as a result, research portfolios derived from richer data sources than were used would more precisely depict institutional research capacity. another limitation of this study is that network properties other than those considered here, such as network density, clustering coefficient, and betweenness centrality, might affect structural efficiency like. furthermore, our findings raised several questions that suggest directions for future research. these include exploring the range 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resources in rtd establishing 'green regionalism': environmental technology generation across east asia and beyond emergence and development of the national innovation systems concept after the reforms: how have public science research organisations changed? r&d manag exploring complex networks dynamic capabilities and strategic management do second-order similarities provide added-value in a hybrid approach knowledge transfer in intraorganizational networks: effects of network position and absorptive capacity on business unit innovation and performance effective leadership in public organizations: the impact of organizational structure in asian countries on the nature, formation, and maintenance of relations among organizations detecting structural change in university research systems: a case study of british research policy optimizing controllability of complex networks by minimum structural perturbations the network structure of the korean blogosphere a strategic management approach for korean public research institutes based on bibliometric investigation exact controllability of complex networks hyeonchae yang is a ph.d. candidate of the graduate program for technology and innovation management at pohang university of science and technology the authors are grateful to the editors of the journal and the reviewers for their support and work throughout the process. this work is supported by mid-career researcher program through the national research foundation of korea (nrf) grant funded by the ministry of science, ict and future planning ( r a a a ). key: cord- -rcft xfh authors: hulme, mike; lidskog, rolf; white, james m.; standring, adam title: social scientific knowledge in times of crisis: what climate change can learn from coronavirus (and vice versa) date: - - journal: wiley interdiscip rev clim change doi: . /wcc. sha: doc_id: cord_uid: rcft xfh nan of knowledge (e.g., certain disciplines within natural sciences) are seen as inherently superior, rather than as complimentary. such a diversification of knowledge would benefit both the effectiveness of decisions made, as well as the legitimacy of those decisions among publics. there is widespread misunderstanding on the part of publics, propagated by dominant political (and scientific) discourses, that scientific and technical knowledge can provide clear and unequivocal answers to policy problems. this obscures the reality that political decision-making is rooted in careful consideration of a variety of options and tradeoffs-in jasanoff's terms, "science will not come on a white horse with a solution". these discourses are already prominent in normal periods, but become even more so in times of crisis when uncertainty, the risk of policy failure and associated blame are at stake. by obscuring the "messy" nature of knowledge production and its relation to policy-making, the danger is that the expertise on which policies are based will become delegitimized whenever those policies are seen to fail. this is not a call to abandon or dilute expertise. rather it is a call to strengthen the public standing of expertise through pluralizing it. the most recent report from the intergovernmental panel on climate change (ipcc) claims that rising global temperature changes the likelihood and distribution of local, regional and global disease outbreaks. this is due to a number of factors, including the expanding geographical range of some disease vectors (ipcc, : ) and the changing seasonality and transmission intensity of some infectious diseases (semenza & menne, ). medical, public health and epidemiological knowledge needs to be complemented with rigorous and varied social scientific knowledge on the fundamental social causes behind mobilities and movements of populations and products that act as vectors of disease. social science has the analytical capacity to show why the risks associated with these movements are harder to govern, the multilevel and transboundary nature of the risks, and the strengths and weaknesses of global institutions of governance that deal with risk. national and local responses to pandemic disease similarly vary widely. so do the vulnerabilities of populations and their faculties for resilience, adaptation and mitigation of risk. social scientific knowledge adds to our understanding of these differences. it also grants decision-makers, as well as publics, critical capacity to evaluate the trade-offs, whether political, economic or social, that responses will entail. numbers should not be allowed to substitute for or obscure political judgment. the covid- pandemic has shone light on a scientific discipline that, in an age of improving sanitation and public health systems, we rarely see entering the public sphere, at least in the global north: epidemiology. in particular, epidemiological mapping and statistical modeling have ameliorated an unknown and unpredictable situation for governments across the world (rhodes, lancaster, & rosengarten, ) . governments always have to weigh up different forms of knowledge and expertise, from scientific and technical knowledge to policy and political considerations. in normal times, this is stage-managed to show a consolidated and consistent position. but in periods of crisis-when information is more uncertain, susceptible to rapid change and when more attention is given to decision-making processes-it becomes increasingly difficult to ensure the effectiveness of government interventions. this was witnessed recently in the cases of both the uk and sweden, where the respective governments' positions and advice have been subjected to increased scrutiny and criticism. climate change too has seen mathematical modeling take a prime position in the search for authoritative knowledge in the context of deep uncertainty (wynne, ) . in the epistemic battle fought in the early days of the ipcc's scientific assessments, it was mathematical models of the climate system that won out over more experimental or observational methods of generating relevant knowledge (coen, ) . it has been argued that such models provided the objectivity and authority that policymakers craved in order to legitimatize decisions (oppenheimer et al., ) . such mathematical models of reality-whether of the climate system or a pandemic-are always partial. not all physical processes are known or simulated. and many of the social systems which condition their predictions are either excluded or else the assumptions upon which human behavior is simulated remain hidden from view. apart from offering inevitably uncertain predictions, mathematical models also obscure the social nature of the climate risk being faced (wynne, ) . this has also been the case in the initial stages of covid- in which uncertain or incomplete data, habitual thinking and the desire to conform to political sensibilities has led to diverging estimates of the risk the virus posed (qi, du, liu, zhao, & dong, ) . parallels between the framing of scales for covid- and climate change have already been drawn. the claim of dr michael mcbride, the chief medical officer of northern ireland, that "viruses don't recognise borders" echoes a similar refrain about air pollution and greenhouse gases from those who wish to emphasize the global character of climate change. the spread of the pandemic, and the spatially differentiated responses, emphasizes the importance of local and contextual knowledge. this goes beyond comparative analysis of health systems-although this is of great importance. it embraces questions of state power, organizational uncertainty and trust in institutions in order to better understand the relative effectiveness of specific interventions in different circumstances. central to these questions is an appreciation of varying cultural and political values and how they interact with scientific or medical knowledge. equally, pleas to "listen to the experts" have emerged in response to the avalanche of misinformation regarding both the covid- pandemic and climate change. but which experts and what advice? when does valid dissent become obstructionist denial? (nemeth, ) . social science helps illuminate the problems inherent to knowledge legitimacy, epistemic authority and competing truth claims. as with climate change, the impact of covid- is likely to be as far-reaching in its secondary consequences as in its primary ones. with stock markets falling, transport links suspended, unemployment reaching record levels and the prospect of global recession looming, the economic and social ramifications appear to be as salient as the medical ones. the pandemic serves to highlight the interrelated nature of environmental, health, social and economic issues. there are multiple dimensions to the risk being faced; trade-offs are inevitable. the pandemic also focuses attention on the variable power available to different states, regions, groups and individuals to adapt and mitigate crises. a pertinent example is the different levels of social and economic capital available to people that are self-isolating. social scientific knowledge deepens our understanding of how perceptions of risk, fear and trust impact on crisis mitigation. it also illuminates the importance of structural factors, social positioning and cultural belongings-such as wealth, race and gender-in developing responses at the individual and institutional level, and how this provides different opportunities for and constraints on action. crises exacerbate existing vulnerabilities and inequalities, both among populations and between states (rhodes et al., ) . the covid- pandemic has already highlighted an uneven distribution of social and institutional support networks. social scientific knowledge will prove invaluable in understanding the long-term consequences of quarantine, social distancing and isolation on the mental and physical well-being of populations and vulnerable groups. this has proven to be the case in scholarship on previous epidemics, such as hiv/aids (emlet, ) . in the current pandemic we can already see that socio-economic class poses different levels of risk, both in terms of contracting the virus in the first place and then the subsequent prognosis for the infected. the search for a technical fix to reduce the numbers infected-typically through various forms of social distancing and quarantine-is rarely sensitive to the (in)capacity of many people to adhere to such requirements, dependent as they are on leaving their house to work. one of the most important lessons to be learned from covid- is a fuller appreciation of how framings such as "crisis" and "emergency" mobilize, legitimate and yet also constrain certain forms of action. both carry with them medical connotations. the origin of the word "crisis" can be traced back to classical greek, kρίσις, meaning a moment of decisive intervention, in the medical sense a choice between life and death (koselleck, ) . the covid- pandemic has seen unprecedented state intervention in society and the economy, from locking down and quarantining entire populations, tenacious surveillance of the individual, banning free assembly and travel, and placing a moratorium on debt repayments. on the one hand, this undermines arguments that have been made in relation to climate change that such decisive interventions are either impractical or impossible. on the other hand, it will be necessary for social scientists to analyze and understand the differing political, social and cultural conditions that made these types of intervention possible, not to mention whether the corporate and state-led incursions into our private lives are necessary or desirable. how did different population groups accede to or resist such impositions? what was the long-term political fall-out of the suspension of normal decision-making mechanisms? how was political accountability either maintained or eroded during the crisis? these questions will play out differently in different political cultures under different types of governance regimes. this again highlights why one-size-fits-all technical solutions are inappropriate and why a more plural knowledge paradigm, inclusive of social-scientific knowledge, is necessary. an important distinction to make between covid- and climate change concerns temporality. the impact of the pandemic appears clear and immediate, while the impact of climate change is diffuse, variable and uncertain. the underlying drivers of climate change are much more deeply rooted in global economic, technological, cultural and political structures than are those for covid- . deploying a crisis/emergency frame can help to attract attention, concentrate resources and provide public legitimacy for action. but it can also serve to obscure the hidden and inertial causes of climate change (asayama, bellamy, geden, pearce, & hulme, ) . the scientific expertise deployed in response to the pandemic seeks to return us to the status quo ante, whereas climate knowledge recognizes the inherent unsustainability of the underlying conditions, suggesting that things can never be the same again. crises highlight how important knowledge is to politicians and policymakers. but they also bring into focus some of the underlying tensions in this relationship between science and policy. technical and scientific knowledge is always partial, uncertain and often contradictory-as we see particularly well in the case of mathematical modeling. that is not to say that such knowledge is not valuable. it is rather to say that to effectively deal with crises, multiple forms of knowledge and expertise are required and political judgment is then necessary to sort, select and present it to publics. resolving a crisis is never about just getting the numbers right. not for climate-it is not just about net-zero -and nor for covid- -it's not just about minimizing some arbitrary mortality statistic. it is about providing effective and ultimately trustworthy transformative change that is grounded on the broadest base of knowledge and to which normative judgments are applied. the effectiveness and legitimacy of interventions in a crisis is reliant on well-informed, transparent but subjective processes of decision-making. it is not sufficient to present the interpretation of knowledge as automatic or to restrict the expertise that presents, interprets and discusses relevant knowledge. decision-makers must be attuned to the insights that social sciences can reveal about the reasons for different individual and collective human behaviors in the face of a threat-their beliefs, values, cultures, norms, expectations and trust. it is vital for the public to retain its trust in the expertise which informs decisions, but also to understand the normative judgments which then guide future policy. this is true for dealing with covid- and it is true for dealing with climate change. the guardian, march , . https://www.theguardian.com/world/ /mar/ /catastrophe-sweden-coronavirusstoicism-lockdown-europe [accessed on may , ]. https://www.standard.co.uk/news/uk/northern-ireland-confirms-first-case-of-coronavirus-a .html [accessed on may , ]. https://news.un.org/en/story/ / / [accessed on may , ]. brookings institute march , . https://www.brookings.edu/blog/up-front/ / / /class-and-covid-how-theless-affluent-face-double-risks/ [accessed on may , ]. why setting a climate deadline is dangerous changing the intellectual climate climate change and the quest for understanding an examination of the social networks and social isolation in older and younger adults living with hiv/aids meet the humanities special report: global warming of . c who speaks for the future of earth? how critical social science can extend the conversation on the anthropocene in defense of troublemakers: the power of dissent in life and business discerning experts: the practices of scientific assessment for environmental policy the variability of critical care bed numbers in europe a model society: maths, models and expertise in viral outbreaks climate change and infectious diseases in europe strange weather, again. theory, culture & society experts' conservative judgment and containment of covid- in early outbreak the authors have declared no conflicts of interest for this article. adam standring: initiator of the idea for this commentary, intellectual lead and corresponding author; rolf lidskog: contribution to drafting; james white: contribution to drafting; mike hulme: contribution to drafting and final editing. key: cord- -a jc g i authors: shrivastava, paul; stafford smith, mark; o’brien, karen; zsolnai, laszlo title: transforming sustainability science to generate positive social and environmental change globally date: - - journal: one earth doi: . /j.oneear. . . sha: doc_id: cord_uid: a jc g i despite the decades-long efforts of sustainability science and related policy and action programs, humanity has not gotten closer to global sustainability. with its focus on the natural sciences, sustainability science is not able to contribute sufficiently to the global transition to sustainability. this perspective argues for transforming sustainability science into a transdisciplinary enterprise that can generate positive social and environmental change globally. in such transformation, the social sciences, humanities, and the arts can play an important role to address the complex problems of culture, institutions, and human behavior. to realize a truly integrated sustainability science, we need renewed research and public policies that reshape the research ecosystem of universities, funding agencies, science communications, policymaking, and decision making. sustainability science must also engage with society and creatively employ all available sources of knowledge in favor of creating a sustainable earth. global environmental challenges are now manifesting at a planetary scale in the form of climate change, ocean acidification, biodiversity loss, environmental pollution, deforestation, and land-use change, among many others. these characteristics of the anthropocene era have been driven by accelerating socioeconomic trends, including population growth, various measures of consumption and economic growth, urbanization, telecommunications, and other aspects of globalization. the so-called great acceleration, which started in the s, has been compounded by the rapid development and spread of information technologies and telecommunications. decades of international conferences and reports-beginning with the report the limits to growth by the club of rome and the united nations (un) conference on the human development in that same year and evolving to the creation of the sustainable development goals (sdgs) by the un general assembly in -have warned of the challenges and risks associated with current development pathways and have fostered the evolution of sustainability science at the global scale ( figure ). science has played a pivotal role in helping society to identify and understand the scope and scale of planetary environmental changes. it has also helped to guide policies and practices that address sustainability challenges from local to global scales. in recent years, global-scale challenges have become a growing concern. the s saw the development of a number of global-change research programs and various environmental science initiatives, such as the world climate research program, the international geosphere biosphere program, diversitas, the international human dimensions program, and some attempted unification in the earth systems science program. sustainability science emerged from the recognition of a need to extend global-change research to seek solutions, which in turn requires multidisciplinary and interdisciplinary research. this is slowly moving further from the perceived hegemony of the disciplinary expert toward the transdisciplinary co-design and co-production of research. international science coordination and funding approaches have also evolved over the past decades, as promoted by the international science council, the belmont forum, and others. many of the global-change programs merged into future earth in . , in , a unified coordination of the natural and social sciences was advanced by the formation of the international science council through the merger of the international science union and the international social sciences council. this year, the transdisciplinary sustainability research & innovation congress was planned to unite global researchers, industry practitioners, and world leaders to inspire action and promote sustainability transformation. although the event is now postponed because of the coronavirus disease pandemic, its aim of cultivating a space for sustainability scholarship, innovation, collaboration, and action won't be delayed. global-change research reflects a growing demand for research to become more impactful and solution oriented, as spelled out in the future earth vision, which is aimed at a multiscale (from local to planetary) understanding of environmental changes that transcend national boundaries. over the past decade, global-change research has made significant efforts to be more inclusive of science communities from china, russia, africa, and the global south. global-change research has first and foremost promoted a systems approach to environmental problems. this approach has focused on relationships and interactions among the atmosphere, biosphere, hydrosphere, cryosphere, and soils and explores how they are influenced by (and influence) human activities. an understanding of human impacts on earth system processes gave rise to the notion of planetary boundaries, the idea of tipping points, , and the concept of the anthropocene. these scientific metaphors have been useful in articulating the risks of continuing with business as usual and development as usual. it is fair to say that global sustainability science has contributed significantly to a number of global agreements to address the challenges of the anthropocene, most notably the paris agreement and the un agenda , which were signed in . these universal agreements reflect a global consensus to address climate change and strive for sustainable and balanced social and economic development that promotes the well-being of socio-ecological systems. they also provide a powerful statement of intended direction for humanity, as captured in the un's sdgs. they create an imperative for transformative change in response to global challenges. however, such changes have not been forthcoming at the rate and scale required by global agreements. as a consequence, we continue to see increasing concentrations of co in the atmosphere, biodiversity loss, land degradation, and growing inequalities among people and nations. , the great acceleration is generated and sustained by powerful economic mechanisms, including globalization, marketization, and financialization, which are based on the mainstream model of doing business and promoting economic growth. to foster growth and maximize profit, companies and other organizations aggressively appropriate value as much as possible from nature and society and externalize the costs to natural ecosystems and to human communities, including future generations. , the mainstream business model drives competition and consumption and guarantees that human and social activities continue to drive the overuse of ecosystems beyond their carrying capacity toward breaching several planetary boundaries. the current economic paradigm has led us to the point where the functioning of our global life-support system is no longer assured. the global economy and functioning earth system are on a collision course that will ultimately have dire consequences for humans and the biosphere; we need an urgent course correction, and current research paradigms are not meeting this challenge. in this perspective, we argue that in order to generate positive social and environmental changes globally, sustainability science must transform into a transdisciplinary enterprise. our modest goal here is to lay out the special research challenges posed by the emerging anthropocene era and exhort sustainability science to address these challenges impactfully. we focus our suggestions primarily on research policy measures that shape the conduct of science and agency of scientists and only secondarily on institutional policy measures. this is not intended to be a broad public-policy discussion on sustainability research. sustainability science needs deep integration of the social sciences, humanities, and the arts with its dominant natural sciences. the focus should be on complex problems of nature, culture, institutions, and human behavior and the co-creation of knowledge with stakeholders. to do this, sustainability science and its stakeholders will need to better understand the funding dynamics and higher levels of research funding. to make sustainability science impactful, we need to modify the current research ecosystems. sustainability science must engage with society (economic, social, and cultural spheres) to extend many successful small-scale seeds for change. the goal of sustainability science is to create a sustainable earth by creatively deploying all available knowledge of humanity. the evolution of what is now known as sustainability science has been important, but clearly it is not yet enough to play a pivotal role in social transformations needed for human preservation in the face of accelerating changes of the anthropocene. we argue that we need to transform our approach to research itself. a number of known attributes of global change currently create not only are these changes fast, but in most instances they are also increasing non-linearly, as described by the great acceleration. the human capacity to respond to these changes has not kept pace. some of the historical drivers of social change, such as generational turnover, have slowed as a result of increasing longevity. consequently, cultural and value changes that influence how humans relate to the environment have to occur within a generation rather than over generations. researchers themselves are challenged to think differently and engage in interand transdisciplinary research processes. however, it takes time to develop common understanding between disciplines and engage across different stakeholder knowledge epistemologies; therefore, integrative research risks lagging behind the changes it is addressing (table ) . spatial connectivity sustainability policies and practices must be local and context specific, yet they are embedded in larger systems that are increasingly and more rapidly connected. although developments in inter-and transdisciplinary research over the past few decades have delivered much success at local levels in agriculture, development, and other domains, replicating this success across scales and globally is genuinely challenging. transformations of social, economic, and institutional systems must also be negotiated across scales. sustainability science research has not reconciled the gap between global-scale problems and locally based solutions. we still do not know how to scale up solutions in an interconnected world (table ) . social-ecological systems have always been affected by social and cultural norms, vested interests, and power dynamics. however, in today's globalized system, many social norms have been shaped by market-based capitalism and consumption, and the rates and distributed extent of change mean that managing the dynamics of winners and losers is increasingly challenging. for example, oil profits in one part of the world could finance investments that undermine sustainability interventions in another. at a time when there is a need to build engagement across all levels of society, identifying and transforming the nature of power relationships can be difficult. sustainability science has not fully acknowledged or integrated global power dynamics into understandings of change (table ) . selective embeddedness of science in society science is extremely specialized and operates within universities and corporations, increasingly fulfilling the mandates of government and corporate funders. science does not reach wide swathes of global society, particularly the communities where challenges of the anthropocene are concentrated. many sectors, especially the informal sector of the economy, are not benefiting from scientific knowledge. the informal sector accounts for up to % of global output in developed countries and over % in developing countries. according to the international labor organization, two billion women and men make their living in the informal economy, which provides over % of non-agricultural employment in africa, latin america, and asia. in africa, the informal economy could account for %- % of the gross domestic product (gdp) and % of new jobs. even in sectors where science does influence economic and social decisions, it does not always effectively serve broad public interests. science funding for the public good and commons protection is insufficient to address the massive and expanding problems of the anthropocene (table ) . above all, the challenges involve culture, society, behavior, and institutions. yet, the current approach to sustainability science still pays insufficient attention to the social and human dimensions, and holistic integrated research remains the exception rather than the norm. sustainability science is still dominated by the natural sciences, and there is grossly less investment in the social sciences, the humanities, and arts than in the ''hard sciences,'' as well as a general underinvestment in research overall. the uptake of research insights from the social sciences and the arts and humanities has been limited to the findings that fit within the existing paradigm. particularly debilitating is the failure to open up to new perspectives and different types of knowledge in the name of keeping science ''objective'' and apolitical. even where efforts have been made to integrate social, human, and natural sciences in transdisciplinary research, these successes are challenged by scale. in a recent review, norströ m et al. identified four key principles for good transdisciplinary co-production of knowledge: the engagement by research should be context based, pluralistic, goal oriented, and interactive. these principles, which draw on a great body of understanding from past transdisciplinary successes, are all challenged by the attributes of the anthropocene. engagement must be context specific, yet it seems to be required at all levels, including globally. it must be pluralistic, yet the diversity of stakeholders even at the local level is immense, and more so for global processes. goal-oriented science is challenging with diverse actors across scales, and making engagement interactive, with feedback loops that promote reflection and learning, requires new thinking at the global level. systems thinking emphasizes that the most powerful leverage points for system change come through modifying the design and intent of the system. paradigms-or the shared thought patterns that influence the way that systems are perceived and designed-are also recognized as a powerful driver of systems change. understanding how to achieve paradigm shifts that influence the goals, values, and behaviors of systems is not the normal purview of the natural sciences. in fact, alternative paradigms are usually dismissed rather than encouraged within research systems. although global assessment initiatives such as the intergovernmental science-policy platform on biodiversity and ecosystem services (ipbes) recognize and include cultural beliefs, values, worldviews, and indigenous and local knowledge, many perspectives from the social sciences and humanities have not been taken seriously in sustainability science. for example, research within feminist political ecology, decolonization studies, and ''new materialism'' offers new thinking on ethical and political challenges. , to capture the breadth and depth of global challenges, sustainability science needs to support efforts to recognize multiple perspectives and ways of knowing. this includes developing profoundly holistic views on how natural and cultural systems coevolve. interdisciplinary and transdisciplinary research activities in academic institutions are seeking such integration. there have been numerous attempts to encourage transdisciplinary collaboration and team science. in higher-income countries, there is a tradition of collaborative cross-sectoral research, such as the agricultural extension research in the us and that conducted by the cooperative research centres (crcs) in australia. recently, the us national science foundation has encouraged research collaborations across science fields, which it calls ''convergence science,'' by awarding grants for themes such as ''navigating the new arctic.'' global programs such as future earth are developing ''knowledge-action networks'' to promote transdisciplinary research. however, the societal ecosystems in which such initiatives operate do not embrace their logic or provide resources to scale them globally as necessary. current efforts to support integrated and transdisciplinary research do not match the scale of the problem. maintaining the power of conventional, objective science has come at a cost for sustainability science. not only has failure to integrate important insights from the social sciences and environmental humanities limited the perceived ''solution space'' for responding to global challenges, but sustainability science has also failed to engage with the ''how'' of transformative change. it has had limited success in connecting with key stakeholders (policymakers, corporate decision makers, political leaders, and civil society) through a language that they understand. it has also failed to convey not only the sense of urgency underscored by global changes but also the possibilities for responding. currently, research on global environmental change largely speaks to the converted-to those who are influenced by rational arguments and scientific evidence. very little attention has been paid to alternatives that challenge the tenets of conventional science and business as usual. the normative dimension of sustainability science is one area that is often challenging to researchers. the question of which changes are ethical and the ''right ones'' to pursue introduces a normative dimension to science that can also be seen as highly political. however, this is an area where global agreements have provided direction for sustainability in recent years-the sdgs of agenda clearly articulate normative visions for global sustainability in alignment with providing food, water, and energy to the approximately ten billion people expected to live on earth by , just as the paris agreement clearly states an intent to stay below c warming, and . c if possible. although they provide a strong statement of goals and intents, both still contain ambiguities around more detailed interpretations relating to different places, levels of governance, and interests, which together can limit their impact for sustainability. to integrate the diversity of interpretations of particular goals, the research system has to open itself up to a wide range of disciplines and epistemologies. many examples of existing research can contribute to a more integrative discourse within sustainability science. however, it is important to consider these different perspectives together rather than as separate boxes or silos. drawing on an integral framework, figure depicts how sustainability science has been largely confined to the domain of ''systems'' science and how less attention has been paid to behavior, culture, and experience. an integral approach to sustainability science includes a more balanced representation of these other three perspectives. below, we highlight some specific examples of research in these other ''quadrants'' to illustrate the potential to broaden the systems-oriented perspective of sustainability science and deepen our understanding of how to respond to environmental change. behavioral change there is still a tendency within sustainability science to draw on the information-deficit model and assume that once individuals ll open access and organizations realize the risks associated with environmental change, they will take action or at least can be manipulated or nudged in that direction. yet, plenty of research in psychology-including research on the relationship between the intrinsic and extrinsic motivation of human actors, i.e., between motivation that is inherent in the task itself and motivation that relies on external rewards or sanctions -shows that human behavior is more complex than this. the latter can result in the crowding-out effect, where external rewards actually reduce the intrinsic motivation of the actors. a crucial result of this mechanism is that people's environmental commitment declines and more environmentally harmful behavior emerges. , a key message here is that intrinsic motivation is critical to sustainable behavior within rapidly changing systems. research also describes how mechanisms of moral disengagement enable otherwise considerate people to commit transgressive acts without experiencing personal distress or guilt. in other words, moral control can be selectively disengaged from detrimental conduct through psycho-social mechanisms. selectively activated disengagement strategies (such as moral justification, euphemistic labeling, advantageous comparison, displacement of responsibility, diffusion of responsibility, disregard for or distortion of the consequences, dehumanization, and attribution of blame) are at work in harmful environmental practices of corporations and other economic entities. this points to the need for a more critical engagement with the social structures and systems in which sustainability measures are enacted, as emphasized in social practice theory. cultural change embedding sustainability sciences and practices at a global scale will require a new and evolutionary narrative about hu- an integral framework recognizes four perspectives or domains of reality that present different types of knowledge about a subject, in this case sustainability science. global-change research has tended to prioritize the systems domain and put less emphasis on research from the meaning-making, cultural, and behavioral domains. integration does not diminish the systems perspective but rather adds valuable knowledge that can help society meet the sdgs. adapted from wilber. man-environment relationships. research on the power of narratives (language, metaphors, and storytelling) points to the need for new cultural narratives on the journey of humans in the universe across geological time. , the current narrative that humans are a special, privileged species destined to dominate over nature is deeply flawed, and it has contributed to a fascination with techno-fixes, such as geoengineering. cultural changes need to embrace research on the power of the arts, aesthetic inquiry, and the humanities to heal the emotional disconnection between humans and nature, which is at the root of our current environmental crisis. the role of artists and art-based practices in achieving sustainability is increasingly being recognized. dutton refers to the art instinct of humans as having survival value across cultures and time. art is ubiquitous in all human communities globally over most of history. communities able to tell better stories and draw better pictures of natural and predatory hazards were able to avoid annihilation more effectively. the rich human legacy of the arts also allows us to connect cognitive understanding to emotional empathy and embodied learning about sustainability. the experiential dimensions of environmental issues are becoming increasingly pronounced through emotions such as hope, grief, anger, and despair. whether and how we identify and respond to environmental problems are also closely tied to the ways that we perceive the world and our place in it. issues such as climate change will be interpreted differently depending on the beliefs, values, and worldviews held by individual or groups. within psychology, meaning making is defined as ''the process of how people construe, understand, or make sense of life events and experiences.'' research in developmental psychology shows that multiple worldviews coexist today and that these can change over generations and within individual lifetimes. acknowledging both differences and developments in perspective-taking capacities and meaning making can provide insights into why people relate to sustainability issues differently and how to connect with a diversity of worldviews. for example, in a study of meaning making among farmers in el salvador as it related to climate-change adaptation, hochachka found differences in the object of awareness, the number of perspectives taken, the complexity of thought, and the scope of time considered in one's understanding of climate change and adaptation. such findings suggest that it could be time for sustainability science to engage with the diversity and dynamics of meaning making rather than to assume that sense making of global challenges is universal and static. in the three research areas described above, one can already see the ways in which researchers are synthesizing knowledge within and across the ''quadrants'' to study the dynamics of sustainability at a deeper level. for example, understanding behavioral changes involves grasping the processes of motivational crowding, understanding socio-cultural changes involves studying the narratives that underpin social values, and understanding experiential responses to environmental change involves indepth research into meaning making. yet, questions remain on how to forge this integrative research more broadly within sustainability science. the integration of different perspectives and domains of knowledge and methodological approaches to research can be enhanced through awareness and education. yet, disciplinary science has privileged cognitive modes of learning, and as disciplines have fragmented, so have the content domains of learning. in highly scientized fields such as medicine, expertise is being isolated and narrowed into increasingly restricted specializations. education for global-change problems needs to be more holistic and contextually anchored through the incorporation of systems and their interconnectedness with culture and experience. education that embraces an integrative discourse recognizes multiple perspectives and the need for critical thinking, reflection, and experience-based learning that leads to action and a sense of agency. one way of integrating different perspectives is through ''action research,'' which focuses on driving change and doing rather than just reaching understanding. otto scharmer and colleagues at the presencing institute created an actionresearch platform at the intersection of science, consciousness, and social change. their ''theory u'' framework and methodology for leading profound innovation proposes that the quality of the results that people create is a function of the quality of awareness, or consciousness, from which the participants in the system operate. the inner shift, from fighting the old to sensing and presencing an emerging future, is at the core of deep leadership work. people are encouraged to expand their thinking from the head to the heart. it is a shift from an ego-system awareness to an eco-system awareness that cares about the well-being of all, including oneself. the presencing institute has reached over , people in countries to help leaders at any level find new solutions to the disruption that humanity faces. there are a variety of approaches to linking research epistemologies with experiential knowledge systems. the crcs in australia have been a generally successful approach to making public funding contingent on a close working relationship between researchers and stakeholders, the latter of which provide funding input, in a vast array of different topics (https://www. business.gov.au/grants-and-programs/cooperative-research-centres-crc-grants). crc structures increasingly require a formal, even dominant, role for stakeholders in governance; even so, the growing experience suggests that it takes up to years for the diverse partners to really work out a common mission and deliver on the promise of transdisciplinarity to get novel research into action. another promising approach is through greater investment in coordinated, applied, local innovation hubs or sustainable development labs. at community colleges and universities in the us, ''living labs'' are using university operational facilities to create engaged action learning opportunities for students and engaged scholarship opportunities for researchers. examples of integrated and holistic knowledge-creation and action initiatives that embrace more diverse epistemologies are also emerging from social and cultural institutions, as exemplified by the encyclical letter laudato si' by pope francis. the encyclical presents an integral ecology that underscores the human origins of the ecological crisis and proposes fundamental changes in organizing economic life. among the important suggestions put forward by the pope is increased frugality in consumption, and he also acknowledges the intrinsic value of nature. to realize his vision, pope francis initiated the economy of francesco project. this international cooperative platform brings together , young economists, entrepreneurs, and change makers to co-create and put in place a new economic model in the spirit of integral human development. notwithstanding the examples of integrative approaches described above, sustainability science has not been transformed enough to successfully address the mounting grand challenges faced by society. the challenge of evolving sustainability science cannot be just endogenous and isolated. science is an instrument of society, and it operates within a broader socio-economic, cultural, and technological milieu that sets the operating context for research. at present, the external context is characterized by neoliberal capitalism that sets up competition rather than collaboration; devalues or overlooks social, human, and environmental capitals; fails to see systemic effects; and encourages inequality. science needs to rethink its social relevance and social connections to effectively contribute to the grand challenges of the anthropocene. it must play an active role in the concurrent co-evolution of economies and their underlying cultural assumptions in sustainable directions. within that broader context, scientific institutions and business organizations supporting science would need transformation of their core purpose, administrative processes, and reward systems. sciences would need deep unification across epistemic and disciplinary lines and be willing to play a more active, participatory role in the transformations required for global sustainability in the world at large. at a more synoptic level (see figure ), waddell et al. have argued that research needs to support the establishment of a ''transformation system'' in parallel with the system being transformed (see https://transformationsforum.net/). they outline four strategies that can contribute to transformation: doing change, forcing change, directing change, and co-creating change. according to these authors, most transformations involve a mix of all four types, and research can play a different role in each of these strategies. there is research to be done on actual transformations, as well as on transformation processes, practices, and pathways. although systems thinking emphasizes that addressing design and intent are the strongest leverage points, these ll open access are also the hardest to achieve, particularly when the personal sphere of transformation is considered to be ''external'' or irrelevant to systems change. an integration of experience, culture, behavior, and systems within sustainability science recognizes that the system is not ''out there,'' and it highlights the importance of ''being change,'' i.e., embodying the changes that are considered necessary at all levels, right here and now, and critical to the process of creating an equitable and sustainable future. recognizing that people engage with the future differently is also essential for activating transformations to sustainability. the three horizons framework helps to generate agency and future consciousness by recognizing different ways of addressing uncertainty. this approach distinguishes between incremental and transformative change and emphasizes the need for participatory, reflective, and action-based research. research on how to pre-condition the system for transformation through incremental changes also provides valuable insights on how to create transformative change , by drawing, for example, on parts of the australian wine industry that were adapting incrementally as opposed to transformationally. when change is seen as a process, incremental changes offer an opportunity to shape the pathways for transformative change, as depicted on the righthand side of figure , and thereby activate easier systems interventions (left side) in order to pre-condition the system for more transformative change in the harder systems characteristics of structure and intent. in practice, this requires a diversity of the intervention strategies (top of cone), as can be seen in examples such as the german energy transition or marriage equality in the us. then arises the question, how can similar insights be applied to sustainability science itself? the cone of increasingly transformative change: systems interventions with increasing leverage (from bottom to top on left side , ) as the cone opens out have greater potential to cause transformational change and require a larger application of the four strategies for systems change (top plane ); easier but directed incremental changes can contribute to the transformability of the system (right side). , enabling sustainability science action sustainability science is a part of the global system that needs to transform to achieve the sdgs. this means that researchers have to reflect on how science should evolve from the research that is already available on transformations to research that can activate sustainability transformations in the wider world. we note four lessons in this regard, examine incremental changes in research organizations (where a great deal of science is operationalized), and then share some thoughts on how science can drive transformation across society. first, most environmental problems, especially the global ones, are complex, messy, and wicked. too often the problem formulation by researchers is partial and inadequate because they reduce the real-world, multifaceted problems into a single scientific-technical dimension and seek to solve this. instead of providing a precise answer to the wrong question (i.e., committing an ''error of the third kind''), approaches should address the full scope of the problem in question (the scientific-technical, the interpersonal-social, the systemic-ecological, and the existential-spiritual) and then develop some satisficing balance among them. the job of sustainability science is to identify problems and then to produce response options that are substantively adequate and ethically acceptable in broad socio-economic contexts. the same logic can be applied to thinking reflexively about sustainability science itself. second, the growing literature on transitions and transformations in society covers the many dimensions and scales needed for responding to global environmental challenges. [ ] [ ] [ ] much of this research emphasizes small-scale (pilot) experiments as important to demonstrate proof of concepts. it also explores how local success stories can be scaled. we need rapid cross-system learning about the successful ones in a context of changing the institutions and rules (scaling ''up'') and the cultural milieu (scaling ''out'') in which these can be scaled. a growing set of examples and opportunities for scaling can influence how we do sustainability science. we are at a point in time when sustainability science needs to think about how to selectively scale these exemplars massively, including the transformation of science itself. third, as already noted (figure ) , systems thinking highlights how the strongest leverage points for changing a system are in modifying its design and intent. it is usually much easier to fiddle with parameters and perhaps feedbacks. the resilience literature addresses what makes a system more or less ''transformable.'' it highlights how important well-chosen incremental changes can be to pre-conditioning a system to be more likely to transform in the ''right'' direction when a suitable impetus comes along. therefore, a good strategy for transforming sustainability science (as for transforming the world) should integrate well-directed incremental changes with preparedness to transform at the appropriate opportunity. fourth, enabling sustainability science to be impactful could also involve modifying the identity and role of scientists. historically, the role of scientists has been to be ''objective'' observers of phenomenon, scientific data collectors, unbiased analysts, and accurate recorders and reporters of findings. in a world beset with grand challenges, scientists must also become translators of knowledge, communicators to the public, policymakers, implementors of action, advocates of solutions, and co-designers of the future. they must also listen deeply and maintain reflective, open minds as they engage not just with theory but also with actions that generate a sustainable world. noting these points, we turn to asking how sustainability science could become more pre-adapted to transform (bottom of figure ), as well as actually transform (top of figure ), in order to play a more effective participatory role in global transformations. this must be in the context of the challenges of the anthropocene, i.e., that change is rapid, spatially interconnected, and more than ever influenced by global power dynamics as it is increasingly embedded into all societal systems. acknowledging universities as a key operational venue of sustainability sciences, and knowing how difficult it is to change them, we suggest some modest yet potentially transformative incremental changes. progressing incremental changes in research and public policy scientific research is guided by research policies and public policies, both of which progress through incremental changes. research institutions, especially universities, are being challenged to produce impactful research. it is widely held that good transdisciplinarity (convergence science) is a means to greater and more enduring impact, especially in more complex and value-laden problems, such as many of the sustainability challenges discussed here. such transdisciplinary challenges almost inevitably end up also requiring interdisciplinary approaches. , the incremental moves noted above from universities and funders toward promoting more inter-and transdisciplinarity must now be accelerated and empowered in both intent and design. however, there are a number of well-recognized barriers to this-traditional mainstream pressures and incentives (e.g., university tenure based on citation counts and grant dollars or corporate instrumentality of research-delivering products); systemic problems in publication, granting, and training processes (e.g., difficulties in publishing integrated research, funding review processes that favor narrow disciplines, and underresourced student training); and disciplinary research cultures of universities that undermine the commitment to inter-and transdisciplinarity and solving real-world public problems. these barriers need to be systemically addressed. change can be initiated if funders of science articulate more demand for impactful science. funders should also modify the institutional constrains to addressing challenges in the global south, where research funding is woefully inadequate and sustainability problems are also pervasive. universities where researchers are employed should modify tenure and promotion rules and reward systems to actively encourage transdisciplinary, solution-oriented sustainability science. even where inter-and transdisciplinarity are encouraged, we have noted that their co-design and co-production processes can be too slow to meet the rates of change we are experiencing. this could require additional resources to speed up these social processes. many current funding mechanisms and processes do not recognize the need for these additional resources to be effective. however, we must also work harder to recognize where transdisciplinarity is really needed and where it is not. we must obtain better evidence for what works and what doesn't in multistakeholder processes so the resources can be transparently focused where they will have the best effect. a critical impact of science-related public policies occurs via funding priorities. inter-and transdisciplinarity need to be supported by a much greater investment in social science and the humanities if we are to promote an equal partnership across the disciplines. as noted earlier, this investment is miniscule in comparison with that in the natural sciences, even if these also are under-resourced. even if we don't wait for more deep interdisciplinarity, many research areas require greater attention: understanding the deeply social nature of desire and consumption as it expands globally, mapping power dynamics across world consumer demand, exploring how culture change can support global transformations, rethinking the idea of ''progress'' in western cultures, and understanding global social and cultural tipping points and other aspects of mobilizing leverage points for systems change. , in addition to this improved investment, there should be a significant effort to improve holistic conceptual models and research practices that draw on multiple traditions. there is potential for increasing resources via government university and private-sector partnering. , these could help to resolve epistemological conflicts among the natural sciences, social sciences, humanities, and arts. this is important both interdisciplinarily (e.g., through conceptual models that deeply support the indivisibility and universality of sustainable development as framed by the sdgs) and also transdisciplinarily (e.g., through extended engagement and experimentation with traditional knowledge systems and faith-based systems). another key incremental and ''no-regrets'' step is increased training of researchers. this encompasses building the cohort of early-career researchers comfortable with interdisciplinary and transdisciplinary processes. we should also provide researchers with the skills to engage with stakeholders, convene and facilitate multistakeholder meetings, identify community problems that need to be addressed, and communicate results to the public and to policymakers. effective co-evolution of science and society will also need to address joint training of the next generation of researchers and stakeholders so they will be able work together to meet sustainability challenges. last, we can begin to recognize reflexively that sustainability is itself an open-ended social learning process in which we all need to play a part. the notion of ''being change'' implies the need to ''walk the talk.'' sustainability scientists should be models of sustainable behavior yet also draw attention to the norms and systems that are impediments to sustainability. this means engaging in public debates and contributing to critical and reflective conversations about sustainability. drawing on the experiences described in this paper, we should engage our own organizations to practice sustainability, drive toward net-zero carbon emissions and zero waste, and deliver gender equity and better social outcomes to meet the sdgs in our institutions and communities. driving global transformations to sustainability moving toward a transformational agenda for sustainability science implies not only driving the incremental change in the right direction but also being sufficiently active so that transformational interventions can take place rapidly when the opportunity arises. , for this we suggest a number of interlinked approaches. first, building on the incremental efforts already taking place, sustainability science should be pursuing funding and other support for a large network of co-learning innovation hubs in all sorts of contexts. these can be modeled in various ways (e.g., sdgs and cities ). they share the attributes of obtaining close engagement between research and society at a local level in many projects in combination with a strong monitoring, learning, and knowledge-exchange infrastructure that enables success stories to be learned from and scaled rapidly in ways that are sensitive to understanding the contexts in which they will or will not work. this transformation of the ivory-tower model of research not only provides an innovation scaffolding for transformation in society when the opportunity arises but also creates good outcomes at the local level in the meantime. , it also engages a much wider portion of society in discussions and learning about sustainability. second, sustainability science needs to engage in an action research mode with the high societal leverage points in such a way that an opportunity for transformation will lead these levers to be pulled in the ''right'' direction. ethically, these should be in areas where the direction is both normatively justified by the framing of goals such as the sdgs and factually justified by research and analysis. examples already noted include dramatic changes to our economic system away from narrow valuation of gdp to broader human well-being, the removal of structural barriers related to the skewed distribution of wealth and consequent inequalities, and a fundamental rejection of simplistic marketbased capitalism, all of which are underpinned by a deep realignment of values to a more collaborative, reflexive, and kinder view of humanity's stewardship of our planetary home. needless to say, such changes would require supporting changes in all areas of institutional and governance design, but these are, of course, past human inventions themselves. the role of sustainability science (and scientists) here would be as activists, as envisaged in waddell et al.'s ''forcing change'' quadrant. third, in addition to playing this activist role, sustainability science needs to have a greater voice in societal processes, such as international un governance discussions or discourse with global religions and traditional societies. the past attitude of scientists has tended to emphasize an autocratic expert identity. the transformed role must be as a partner among equals in seeking the normatively defined outcomes. global-change research has sought this collaborative role, for example, at the un conference on sustainable development (rio+ ) and through the intergovernmental panel on climate change and ipbes assessment processes, but in the sdg development process it was still relegated to being one among many lobby groups. the un secretary general's science advisory panel is still seen as a set of experts on the outside of the political process, as are most government scientific advisory boards. this is a failing on both sides of the relationship, and it must be worked on without hubris in policy and other domains. fourth and finally, sustainability science needs to be goal oriented to pursue transformation and accept the challenging roles implied by this. in the absence of clear intentionality in the design of these systems, science is dragged along by the dominant socio-economic forces that themselves should be the subject of change. sustainability science should examine and prepare for its role in all the complementary transformation strategies noted by waddell et al. -understanding when to be an activist and when to partner and being ready to distribute these tasks in a credible way. the community of researchers and their co-design partners also need to accept the goal of transforming themselves to help enable societal transformation in order to meet the sdgs and thereby set the direction for all the above points. maintaining transparency and accepting open debate while seeking sufficient consensus to move forward rapidly to achieve global sustainability and human well-being will be the hardest part of the balancing act. it is difficult to reach final conclusions to the broad critique and transformational vision for sustainability science as explored in this paper. we acknowledge that the realization of this vision could take time, yet the current global situation is challenging assumptions about incremental and transformational change. now is the opportunity for sustainability science to engage and coevolve with the underlying socio-economic roots of environmental changes. can the various branches of science afford to remain at epistemological odds with each other? can research that is driven by funding needs and competition continue to serve private corporate interests rather than the public good? research has not sufficiently emphasized how the most pressing environmental problems are caused and conditioned by social, cultural, economic, and business activities based on individualistic, competitive, profit-seeking models of economics and finance. if there has ever been a time to explore paradigm shifts that support an equitable and sustainable world, this is it. in this article, we have offered some suggestions for moving sustainability science forward on its evolutionary path. profoundly, on this path, the interlinked nature of science and society means that an overall societal commitment to sustainability (as articulated in the un agenda ) is a prerequisite for transforming science and society toward sustainability. for this to happen, macroeconomies would need to be designed for human and biosphere well-being rather than for financial economic growth. , cultural values to support this economic transition would be based on a more eco-centric vision of life and organizations. [ ] [ ] [ ] [ ] [ ] [ ] transformations like these require a different, more impactful version of sustainability science. the research system of sustainability science itself needs to evolve rather than just seek to change everything else. this means that sustainability scientists and policymakers have to be more reflexive and challenge their own limiting assumptions related to global change. global sustainability research and practices strive to provide adequate food, water, energy, income, education, resilience, voice, jobs, health, and gender and social equity for all in an ecologically safe operating space for humanity. this will require weaving together models of economic growth with models of ecosystem changes and managing winners and losers and the related changes in power dynamics. it will also require addressing the structural economic barriers that have emerged over the past century. the most important of these is wealth and income inequality. currently, % of wealth is owned by less than % of the world population. two billion people still consume under $ per day and aspire to increase it. growing inequality makes ''economic growth'' ineffective at addressing the needs of vulnerable sections of society by concentrating wealth with the rich. the co-evolution of sustainability science and sustainable societies implies increasing consumption of some and decreasing consumption of others without degrading ecosystems. the research ecosystem of universities, funding agencies, policymakers, and decision makers needs to be modified in other ways. science operations are different in each country, and there is no one-size-fits-all solution. some general actions for consideration include the following: d universities explicitly prioritize impactful research on environmental grand challenges. d universities adjust their research productivity, measurement methods, and reward systems to beyond academic measures (publications, citations, and grant dollars) to include the impact on social and sustainable development metrics. d funding agencies make more stringent and concrete demands that research should have an impact on sustainability (via the sdgs) in communities. d science communication with policymakers and with the public should be made a priority. many global environmental changes are being addressed outside academia through citizen activism, non-governmental organizations, and social movements. sustainability science could be leading some of these activities instead of merely studying or observing them. however, in order to take such leadership, researchers will need to acknowledge that science is not just a neutral objective pursuit of knowledge when it comes to sustainability. by bringing in reflexivity, values, and ethics, they can justify participation in structural and systemic changes that would produce an equitable and thriving world. scientific pursuit of knowledge involves much more than constructing accurate and analytically powerful 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determination of dynamic macromolecular complexes by single particle cryo-em holger stark max-planck-institute for biophysical chemistry, goettingen, germany macromolecular complexes are at the heart of central regulatory processes of the cell including translation, transcription, splicing, rna processing, silencing, cell cycle regulation and repair of genes. detailed understanding of such processes at a molecular level requires structural insights into large macromolecular assemblies consisting of many components such as proteins, rna and dna. single-particle electron cryomicroscopy is a powerful method for threedimensional structure determination of macromolecular assemblies involved in these essential cellular processes. it is very often the only available technique to determine the d structure because of the challenges in purification of complexes in the amounts and quality required for x-ray crystallographic studies. in recent years it was shown in a number of publications that it is possible to obtain near-atomic resolution structures of large and rigid macromolecules such as icosahedral viruses. due to a number of methodological advances there are now also great perspectives for high-resolution single particle cryo-em studies of large and dynamic macromolecules. successful high-resolution structure determination of dynamic complexes requires new biochemical purification strategies and protocols as well as state of the art electron microscopes and high-performance computing. in the future cryo-em will thus be able to provide structures at near-atomic resolution and information about the dynamic behavior of macromolecules simultaneously. detection and rapid manipulation of phosphoinositides with engineered molecular tools tamas balla section on molecular signal transduction, program for developmental neuroscience, nichd, nih, bethesda, md , usa polyphosphoinositides (ppis) are ubiquitous lipid regulators of a variety of cellular processes serving as docking sites and conformational switches for a large number of signaling proteins. the localization and dynamic changes in ppis in live cells have been followed with the use of protein domain gfp chimeras. in this presentation we will show experimental systems that allow rapid manipulation of the levels of ppis in specific membrane compartments. we are also actively pursuing strategies that will allow us to map the distribution and possible functional diversity of the phosphatidylinositol (ptdins) pools within intact cells since they are the precursors of ppis. we will show our most recent progress in addressing this question: the use of a ptdins specific plc enzyme isolated from listeria monocytogenes together with a highly sensitive diacylglycerol sensor to determine the distribution and also to alter the level of ptdins in living cells. these studies reveal that a significant metabolically highly active ptdins pool exists associated with tiny mobile structures within the cytoplasm in addition to the known er and pm ptdins pools. we will show our most recent data on the consequences of ptdins depletion within the various ptdins pools on ppi production and on the morphology and functions of various organelles. the functionality of proteins is known to be intimately related to the motion of their constituents on the atomic/molecular level. the study of microscopic motion in complex matter is often reduced to the observation of some average mean square atomic displacement, a first, very partial characterization of the dynamics. the marked crossover in the temperature dependence of such quantities in hydrated proteins around k, the so called ''dynamic transition'' has been originally observed a quarter of century ago. the origin, nature and the key characteristics of the atomic motions behind this remarkable evolution of the mean square displacement in proteins remained controversial over the past decades. recent analysis of mö ssbauer, dielectric relaxation and neutron scattering spectroscopic data provide unambiguous evidence that this phenomenon is caused by the temperature dependence of a relaxation process spread over several orders of magnitude in the time domain, similarly to the b relaxation process observed in glasses. the review and critical analysis of the available data highlights the inherent ambiguities of commonly used data fitting approaches. emerging evidence from model independent observations tend to exclude some of the proposed mechanisms. microbial rhodopsins: light-gated ion channels and pumps as optogenetic tools in neuro-and cell biology e. bamberg, c. bamann, r.e. dempski, k. feldbauer, s. kleinlogel, u. terpitz, p. wood department of biophysical chemistry, max-planck-institute of biophysics, frankfurt, germany microbial rhodopsins are widely used in these days as optogenetic tools in neuro and cell biology. we were able to show that rhodopsins from the unicellar alga chlamydomonas reinhardtii with the transmembrane helix motif act as light-gated ion channels, which we named channelrhodopsins(chr , ). together with the light driven clpump halorhodopsin chr is used for the non-invasive manipulation of excitable cells and living animals by light with high temporal resolution and more important with extremely high spatial resolution the functional and structural description of this new class of ion channels is given (electrophysiology, noise analysis,flash photolysis and d crystallography). new tools with increased spatial resolution and extremely enhanced light sensitivity in neurons are presented. a perspective for basic neurobiology and for medical applications is given. extracellular signals consists of the induction of specific gene expression patterns and the re-organization in space and time of stereo-specific macromolecular interactions that endow the cell with its specific morphology. we develop quantitative experimental and computational approaches to derive and conceptualize physical principles that underlie these dynamics of signal processing and cellular organization. we have an experimental emphasis on functional microscopic imaging approaches at multiple resolutions to study the localization and dynamics of protein reactions/ interactions, maintaining the inherent spatial organization of the cell. we have a strong recursion between computation of molecular dynamics in realistic cell geometries as sampled by microscopy, and experiments that reveal the dynamic properties of networks in living cells. we investigate the cellular topography of activities that transmit signals from receptors at the cell surface. here we ask, how spatial partitioning of intracellular signalling activities is achieved by the causality structure of the signalling network, and how this partitioning affects signal response. this entails the experimental elucidation of connections between reactions and the determination of enzyme kinetic parameters in living cells. o- molecular photovoltaics mimic photosynthesis michael grä tzel laboratory of photonics and interfaces, institute of chemical science and engineering, station , ecole polytechnique fé dé rale, ch- lausanne, switzerland e-mail: michael.graetzel@epfl.ch the field of photovoltaic cells has been dominated so far by solid state p-n junction devices made e.g. of crystalline or amorphous silicon, profiting from the experience and material availability of the semiconductor industry. however, there is an increasing awareness of the possible advantages of devices referred to as ''bulk'' junctions due to their interconnected three-dimensional structure. their embodiment departs completely from the conventional flat p-n junction solid-state cells, replacing them by interpenetrating networks. this lecture focuses on dye sensitized mesoscopic solar cells (dscs), which have been developed in our laboratory. imitating natural photosynthesis, this cell is the only photovoltaic device that uses a molecular chromophore to generate electric charges from sunlight and that accomplishes the separation of the optical absorption from the charge separation and carrier transport processes. it does so by associating the molecular dye with a film constituted of tiny particles of the white pigment titanium dioxide. the dsc has made phenomenal progress, present conversion efficiencies being over percent for single junction and percent for tandem cells, rendering the dsc a credible alternative to conventional p-n junction devices. molecularly single-molecule imaging and tracking techniques that are applicable to living cells are revolutionizing our understanding of the plasma membrane dynamics, structure, and signal transduction functions. the plasma membrane is considered the quasi- d non-ideal fluid that is associated with the actinbased membrane-skeleton meshwork, and its functions are likely made possible by the mechanisms based on such a unique dynamic structure, which i call membrane mechanisms. my group is largely responsible for advancing highspeed single molecule tracking, and based on the observations made by this approach, i propose a hierarchical architecture of three-tiered meso-scale ( - nm) domains as fundamental organizing principles of the plasma membrane. the three tiers i propose are the following. [tier ] - nm compartments made by partitioning the entire plasma membrane by the membrane-associated actinbased meshwork (membrane skeleton: fences) and its associated transmembrane proteins (pickets). since the entire plasma membrane is partitioned by these structures, and the membrane skeleton provides important platforms for the molecular interactions and pools, membrane compartments are the most basic tier for the plasma membrane organization. [tier ] meta-stable - nm raft domains that can be turned into stable * - -nm domains (receptor-cluster rafts), based on ligand-induced homo-dimers of glycosylphosphatidylinositol (gpi)-anchored receptors (coupling with [tier ]) and facilitated by raft-lipid interactions. [tier ] protein complexes of various sizes ( - nm) and lifetimes. i will also talk about how domains of tiers and are coupled to the membrane partitioning (tier ). the concept of the three-tiered domain architecture of the plasma membrane and the cooperative interactions of different tiers provides a good perspective for understanding the mechanisms for signal transduction and many other functions of the plasma membrane. introduction: in the present study we investigate the effects of electromagnetic fields (emf) on the binding of norfloxacin (nrf) to human serum albumin (hsa) by fluorescence, three-dimensional fluorescence and uv-visible spectroscopic approaches. hsa is the most abundant protein in human blood plasma which works as a carrier that transports different materials in the body. nrf is used to treat variety of bacterial infections. it works by stopping the bacterial growth. methods: hsa, nrf and potassium phosphate buffer were purchased from sigma. fluorescence spectrofluorometer, uv-vis spectrophotometer, three-dimensional fluorescence and a home-built emf generator apparatuses were used. results: results obtained from this study indicated that nrf has a strong ability to quench hsa in nm. in addition, there was a slight blue shift, which suggested that the microenvironment of protein became more hydrophobic after addition of nrf. moreover, synchronous fluorescence demonstrated that the microenvironment around tyrosine (tyr) had a trivial increase. these, and the results of hsa-nrf in the presence of emf with khz, illustrates the same results inferred from quenching and blue shift. however, there was a significant decrease in k sv of nrf with hsa in presence of emf exposure. moreover, the binding parameters including the number of binding sites and the binding constant were calculated form hill equation. conclusion: it was shown that nrf could induce conformational changes in hsa both in the absence and presence of emf with no significant difference. yet, the affinity is decrease significantly in the presence of emf. the clinical implications are discussed in detail. characterization of the biochemical properties and biological function of the formin homology domains of drosophila we characterised the properties of drosophila melanogaster daam-fh and daam-fh -fh fragments and their interactions with actin and profilin by using various biophysical methods and in vivo experiments. the results show that while the daam-fh fragment does not have any conspicuous effect on actin assembly in vivo, in cells expressing the daam-fh -fh fragment a profilindependent increase in the formation of actin structures is observed. the trachea specific expression of daam-fh -fh also induces phenotypic effects leading to the collapse of the tracheal tube and lethality in the larval stages. in vitro, both daam fragments catalyze actin nucleation but severely decrease both the elongation and depolymerisation rate of the filaments. profilin acts as a molecular switch in daam function. daam-fh -fh , remaining bound to barbed ends drives processive assembly of profilin-actin, while daam-fh forms an abortive complex with barbed ends that does not support profilinactin assembly. both daam fragments also bind to the sides of the actin filaments and induce actin bundling. these observations show that the drosophila melanogaster daam formin represents an extreme class of barbed end regulators gated by profilin. electron spin echo studies of free chain-labelled stearic acids interacting with b-lactoglobulin rita guzzi, luigi sportelli, rosa bartucci dipartimento di fisica, università della calabria, rende (cs), italy b-lactoglobulin (blg) binds non-covalently fatty acids within its central calyx, a cavity in the barrel formed by the strands ba-bh. we present results of pulsed electron paramagnetic resonance (epr) spectroscopy on the interaction of blg with stearic acids spin-labelled at selected positions, n, along the acyl chain (n-sasl, n = , , , , ). d o-electron spin echo envelope modulation (eseem) fourier transform spectra indicate that all segments of the bound chains in the protein binding site are accessible to the solvent. the extent of water penetration decreases progressively on moving from the first segments toward the terminal methyl end of the chain. about % of the nitroxides in the upper part of the chain (n = , ) are h-bonded by a single water molecule and this fraction reduces to % at the chain terminus (n = , ). a lower fraction of the nitroxides are h-bonded by two water molecules, and it decreases from about % to a vanishingly small value on going down the chain. echo-detected ed-epr spectra reveal subnanosecond librational motion of small amplitude for both -and -sasl in the protein cavity. the temperature dependence of the librations is more marked for -sasl and it arises mainly from an increase in librational amplitude with increasing temperature. fusion peptides (fp) pertaining to the spike glycoprotein from severe acute respiratory syndrome (sars) coronavirus are essential for the fusion between viral and host cellular membranes. here we report a biophysical characterization of the interaction of two putative fps with model membranes. fluorescence and dsc experiments showed that both peptides bind stronger to anionic than to zwitterionic lipid membranes. esr spectra showed that toac-sars ifp rotational dynamics is modulated by lipid composition and ph as compared to the spectrum of this peptide in solution. however, stearic acid spin labels reported no changes on the dynamic structure of zwitterionic micelles, whereas the whole chain of anionic surfactants was perturbed by the peptides. finally, cd data revealed a predominant b-strand structure for sars fp and an a-helix for sars ifp in the presence of micelles, in contrast to their disordered structures in buffer. overall the results point out that electrostatic and hydrophobic interactions are both important to the energetic behavior of peptide membrane interaction. these findings might provide a useful rationale for the elucidation of one of the steps involved in the fusion process, and thus help understanding the more general way of action of fps at a molecular level. interaction of filamentous actin and ezrin within surface modified cylindrical nanopores daniela behn , and claudia steinem institute for organic and biomolecular chemistry, university of gö ttingen, tammannstraße , gö ttingen, germany, ezrin is a member of the ezrin-radixin-moesin (erm) protein family that acts as a dynamic linker between the plasma membrane and the actin cytoskeleton and is hence involved in membrane organization, determination of shape and surface structures and other cellular processes. the protein is highly enriched in microvilli of polarized epithelial cells, where it binds filamentous actin (f-actin) with its c-terminal domain, while the n-terminal domain is connected to the plasma membrane via specific binding to l-a-phosphatidylinositol- , -bisphosphate (pip ). nanoporous anodic aluminum oxide (aao) films provide similar dimensions as microvilli and are thus a versatile template to investigate the interaction of ezrin with f-actin within spationally confined areas. owing to their optical transparency, functionalized aaos can be used to measure the binding process of ezrin to a pip containing solid supported membrane by means of time resolved optical waveguide spectroscopy (ows). confocal laser scanning microscopy (clsm) will elucidate, whether f-actin binding to ezrin takes place within or atop the nanopores. furthermore, elasticity mapping of f-actin filaments by means of atomic force microscopy will allow determining binding forces and the lateral tension of the actin cytoskeleton. in vitro application of porphyrin photosensitisers on mcf , hela and g tumour cell lines binder s., kolarova h., bajgar r., tomankova k., daskova a. deparment of medical biophysics, faculty of medicine of palacky university, olomouc, czech republic tumour treatment presents a challenge to all scientists and clinicians. contemporary methods like radiotherapy, chemotherapy or surgery have many undesirable side effects. photodynamic therapy (pdt) seems to be one of alternatives which can be helpful in malignant cell therapy. pdt is not only limited to cancer treatment but is also used as an alternative for cardiovascular, skin and eye disease treatment. pdt employs photosensitive agents which need to be activated by light which is not harmful to a patient. the activated photosensitive agent provokes a formation of reactive oxygen species leading to cell damage or death. the phototoxicity of the two porphyrin photosensitizer (tmpyp, zntpps . h o) on the malignant cell lines (g , hela, mcf ) irradiated with the jcm - doses was evaluated by ros production assay, mtt assay and comet assay. our results indicate higher efficiency of tmpyp over zntpps . h o. as for the photodynamic effectiveness of the used photosensitizers on chosen cell lines we found that hela cell line is the most sensitive to phototoxic damage induced by tmpyp. p- nmr analysis of the respiratory syncytial virus m - protein structure and of its interaction with some of its targets c. sizun the respiratory syncytial virus (rsv) is a major cause of acute respiratory tract infections (bronchiolitis, pneumonia) in human and a leading cause of viral death in infants and immunocompromised patients. rsv genome is formed of a single non-segmented negative strand rna which transcription and replication is ensured by a specific rna-dependent rna polymerase complex formed of the large (l) polymerase subunit and of several cofactors. this complex has no cellular counterpart and represents an ideal target for antiviral drugs. among the cofactors, m - acts as an antitermination factor and increases the polymerase processivity. its central domains has been shown, in vitro, to bind the phosphoprotein p and genomic rna in a competitive manner. here we report the nmr structure of this central domain and its interaction with p and rna fragments. m - shares structural similarity with vp , a transcription factor of ebola virus. the binding surfaces for rna and p are distinct but overlapping. rna binds to a basic cluster located next to residues found to be critical for transcription both in vitro and in vivo by mutational analysis. we speculate that m - might be recruited by p to the transcription complex, where interaction with rna takes place, stabilized by additional elements. force spectroscopy at the membrane-cytoskeleton interface: interactions between ezrin and filamentous actin julia a. braunger , , ingo mey and claudia steinem institute for organic and biomolecular chemistry, georg-august-university of gö ttingen, tammannstraße , gö ttingen, germany, ggnb doctoral program: imprs - ezrin, a member of the erm (ezrin/radixin/moesin) protein family, provides a regulated linkage between the plasma membrane and the actin cytoskeleton. it contributes to the organization of structurally and functionally distinct cortical domains participating in adhesion, motility and fundamental developmental processes. ezrin is negatively regulated by an intramolecular interaction of the terminal domains that masks the f-actin binding site. a known pathway for activation involves the interaction of ezrin with phosphatidylinositol , bisphosphate (pip ) in the membrane, followed by phosphorylation of the threonine residue in the c-terminal domain. to date, it is unclear to what extent both regulatory inputs contribute to the activation. we developed an in vitro system that facilitates the specific analysis of the interaction forces between ezrin and f-actin by means of atomic force spectroscopy (afm). applying ezrin wild type and the pseudophosphorylated mutant protein ezrin t d, respectively, permits to monitor the individual influence of phosphorylation on the f-actin-ezrin interaction. thus, a thorough characterization of the acting forces at the ezrin-actin interface will elucidate the activation mechanism of ezrin. delivery system even more efficient, we have constructed nano-carrier by coating of ldl by polyethylene glycol (peg) . the hydrophilicity of peg should reduce the interaction of ldl with other serum proteins and consequently decrease the redistribution of loaded drug from ldl to the (lipo)proteins. dynamic light scattering was used for determination of hydrodynamic radius of ldl-peg particles. cd spectroscopy measurements didn't reveal structural changes of apoliprotein b- (ligand for ldl receptors on cell surface), after conjugation of peg with ldl. interaction of ldl-peg complexes with hypericin (hyp) a natural photosensitizer was studied by fluorescence spectroscopy. we have demonstrated accumulation of higher number of hyp in ldl-peg than ldl particles. however, the kinetics of hyp redistribution from hyp/ldl-peg complex to free ldl have similar parameters as those for the kinetics of hyp transfer between non-modified ldl molecules. we suggest that hyp molecules are mostly localized in the vicinity of the surface of the ldl-peg particles and they are prone to redistribution to other serum proteins. grant support: lpp- - , vega- - . modification of the head-group of aminophospholipids by glycation and subsequent lipid oxidation affect membrane's structure causing cell death. these processes are involved in the pathogenesis of aging and diabetes. non-enzymatic glycation forms in the first step a schiff base (sb), which rearranges to a more stable ketoamine, amadori product, which leads to the formation of a heteregenous group of compounds (ages). although several studies have been focused on identification of aminophospholipid glycation products, less attention has been paid to kinetic mechanism of the reaction. for that reason, in the present work, we compare the kinetic reactivity of polar head-group of phosphatidylethanolamine (pe) and phosphatidylserine (ps), the two target phospholipids components of mammalian cell membranes. the reaction of pe and ps's head-group with glycating compounds (glucose and arabinose) was studied in physiological conditions by using nmr spectroscopy. the obtained formation rate constants for sb are lower than those determined for the sb of the peptide ac-phe-lys with the same carbohydrates. it suggests that the phosphate group may delay the glycation process. moreover, the ps's head-group has a carboxilic group in the structure, which affects the stability of the sb. we developed ultrasensitive, elisa-like nanoimmuno assays suitable for proteomics/interactomics studies in low sample volumes. we exploit the approach of dna microarray technologies applied to proteomics [ ] , in combination with atomic force microscopy (afm) to generate functional protein nanoarrays: semisynthetic dna-protein conjugates are immobilized by bioaffinity within a nanoarray of complementary ssdna oligomers produced by afm nanografting (ng). a nanoarray of different antibodies or synthetic molecular binders can be generated in a single operation, once the dna nanoarray is produced. moreover, ng allows adjusting the packing density of immobilized biomolecules to achieve optimum bio-recognition. afm-based immunoassays with these nanoarrays were shown to achieve detection limit of hundreds of femto molar, in few nanoliters volumes, with very high selectivity and specificity [ ] . to detect the hybridization efficiency of our devices, we run a combined experimental-computational study that provides quantitative relations for recovering the surface probe density from the mechanical response (afmcompressibility measurements) of the sample. nucleoside analogues used as anticancerous drugs can be rapidly degraded within treated cells, constituting a major obstacle of their therapeutic efficiency. among the enzymes responsible for this degradation, the cytosolic 'nucleotidase ii (cn-ii) catalyses the hydrolysis of some nucleoside monophosphates. in order to improve the efficacy of anticancerous drugs and to define the precise role of cn-ii, new original inhibitors have been developed against cn-ii. virtual screening of chemical libraries on the crystal structure has allowed us to identify very promising candidates that turned to be competitive inhibitors of cn-ii. one molecule was included in the anticancerous treatment of tumoral cell lines in order to evaluate the potential benefit and could induce in fine a sensitization of certain anticancerous drugs. we also explore other inhibitors targeting the allosteric sites of this enzyme using a strategy that takes into account the dynamics of cn-ii. the chemical structures of the newly identified allosteric inhibitors as well as the atomic interactions with enzyme residues will be presented. the final goal of this study is to find molecules that can freeze the enzyme in a conformation for which its dynamics is severely limited and therefore its function. native mass spectrometry to decipher interactions between biomolecules sarah cianferani laboratoire de spectromé trie de masse bio-organique, université de strasbourg, iphc, rue becquerel strasbourg, france. cnrs, umr , strasbourg, france mass spectrometry is generally understood as ''molecular mass spectrometry'' with multiple applications in biology (protein identification using proteomic approaches, recombinant protein and monoclonal antibody characterization). an original and unexpected application of mass spectrometry emerged some twenty years ago: the detection and the characterization of intact biological noncovalent complexes. with recent instrumental improvements, this approach, called native ms, is now fully integrated in structural biology programs as a complementary technique to more classical biophysical approaches (nmr, crystallography, calorimetry, spr, fluorescence, etc.). native ms provides high content information for multiprotein complexes characterization, including the determination of the binding stoichiometries or oligomerization states, sitespecificities and relative affinities. recent developments of ion mobility / mass spectrometry instruments (im-ms) provide a new additional level for ms-based structural characterization of biomolecular assemblies allowing size and shape information to be obtained through collisional cross section measurements. these different aspects of native ms for structural characterization of biomolecular assemblies will be illustrated through several examples, ranging from multiprotein-complexes to protein/nucleic acid assemblies. complex coacervation is a process which may result by electrostatic interaction between charged polysaccharides. it depends essential on ph, ionic strength and biopolymers properties like ratio, concentration and charge density. in this case, the main work was to study the structural properties of a colloidal system of opposite charge -chitosan and gum-arabic by atomic force microscopy (afm). according to some of complexes show tendency to agglomerate. this depends on the molar ration of the macromolecules and their relative molecular weights. afm micrographs show, too, that some formation of irregular aggregates by both polymers were due to presence of noncharged polar monomers in chitosan molecule. at higher gum-arabic/chitosan ratios biopolymer concentrations, coacervates appear like a core-shell miccelar structure composed of hydrophobic core (charge neutralized segments) stabilized by the excess component (positive zeta potential) and non-charged segments of gum arabic. interaction of human serum albumin with rutin theoretical and experimental approaches Ícaro p caruso human serum albumin (hsa) is the principal extracellular protein with a high concentration in blood plasma and carrier for many drugs to different molecular targets. flavonoids are a large class of naturally occurring polyphenols widely distributed in plants. rutin (quercetin- -rutinoside) is the glycoside between flavonoids quercetin and disaccharide rutinose. like other flavonoids, rutin displays anti-inflammatory and anti-oxidant properties. the interaction between hsa and rutin was investigated by fluorescence spectroscopy, ab initio and molecular modeling calculations. fluorescence titration was performed by keeping the hsa concentration ( lm) constant and stoichiometrically varying the rutin concentration ( - lm) . the emission spectra were obtained in the range of to nm, with the excitation wavelength at nm. the obtained fluorescence data were corrected for background fluorescence and for inner filter effects. the stern-volmer quenching constant values were . and . m - at and k, respectively. from the modified stern-volmer association constants . (at k) and . m - (at k) were calculated the thermodynamic parameters dh = . kj mol - , dg k = - . kj mol - and dg k = - . kj mol - , and ds = . kj mol - k - . fluorescence quenching method was used also to study the binding equilibria thus determining the number of binding sites . and . , and binding constant . m - and . m - at and k, respectively. the efficient quenching of the trp fluorescence by rutin indicates that the binding site for the flavonoid is situated within subdomain iia of hsa. the distance r = . nm between the donor (hsa) and the acceptor (rutin) was obtained according to fluorescence resonance energy transference (fret). wavelength shifts in synchronous fluorescence spectra showed the conformation of hsa molecules is changed in the presence of rutin. the structure of rutin utilized in molecular modeling calculation was obtained by gaussian program. the optimization geometry of rutin was performed in its ground states by using ab initio dft/b lyp functional with - g(d,p) basis set used in calculations. the molecular electrostatic potential (mep) was calculated to provide the molecular charge distribution of rutin. the gap energy value between the homo and lumo of the rutin molecule was about . ev which indicates that rutin is classified as a reactive molecule. from molecular modeling calculation the interaction between hsa and rutin was investigated using the autodock program package. the three-dimensional coordinates of human serum albumin were obtained from the protein data bank (entry pdb code ao ) and of rutin were obtained from output optimization geometry of dft. the best energy ranked result shows that rutin is localized in the proximity of single tryptophan residue (trp ) of hsa that is in agreement with the fluorescence quenching data analysis. the effect of toxofilin on the structure of monomeric actin lívia czimbalek, veronika kollá r, roland kardos, gá bor hild university of pé cs, faculty of medicine, department of biophysics, pé cs, hungary actin is one of the main components of the intracellular cytoskeleton. it plays an essential role in the cell motility, intracellular transport processes and cytokinesis as well. toxoplasma gondii is an intracellular parasite, which can utilise the actin cytoskeleton of the host cells for their own purposes. one of the expressed proteins of t. gondii is the kda-sized toxofilin. the long protein is a monomeric actinbinding protein involved in the host invasion. in our work we studied the effect of the actin-binding site of toxofilin - on the g-actin. we determined the affinity of toxofilin to the actin monomer. the flourescence of the actin bound e-atp was quenched with acrylamide in the presence or absence of toxofilin. in the presence of toxofilin the accessibility of the bound e-atp decreased, which indicates that the nucleotide binding cleft is shifted to a more closed conformational state. the results of the completed experiments can help us to understand in more details what kind of cytoskeletal changes can be caused in the host cell during the invasion of the host cells by intracellular parasites. t bacteriophage, as a surrogate on non-enveloped viruses was selected as a test system. both tmpcp and bmpcp and their peptide conjugates proved to be efficient photosensitizers of virus inactivation. the binding of porphyrin to phage dna was not a prerequisite of phage photosensitization, moreover, photoinactivation was more efficiently induced by free than by dna bound porphyrin. mechanism of photoreaction (type i. versus type ii) and the correlation between dna binding, singlet oxygen production and virus inactivation capacity was also analyzed. dna binding reduced the virus inactivation due to the reduced absorbance and singlet oxygen production of bound photosensitizer, and altered mechanism of photoinactivation. as optical melting studies of t nucleoprotein revealed, photoreactions of porphyrin derivatives affected the structural integrity of dna and also of viral proteins, even if the porphyrin did not bind to np or was selectively bound to dna. anesthesia is a medical milestone (friedman & friedland, medicine's greatest discoveries, ) and local anesthetics (la) are the most important compounds used to control pain in surgical procedures. however, systemic toxicity is still a limitation for la agents as well as low solubility, as for tetracaine (ttc). approaches to improve la effects include macrocyclic systems formation, such as in cyclodextrins (cd). we have studied complexes formed between ttc and b-cd or hydroxylpropyl (hp)-b-cd through nmr and other (uv-vis, fluorescence, dsc and x-ray diffraction) techniques. at ph . a : stoichiometry of complexation was detected for both complexes, with association constants of m - and m - for ttc:b-cd and ttc:hp-b-cd, respectively. the nuclear overhauser nmr data disclosed trough the space proximities between hydrogens h h and h iat the aromatic ring of ttc -and hydrogens from the inner cavity of the cyclodextrins, allowing us to propose the topology of ttc:cd interaction. complex formation did not curb ttc association with model (liposomes) and biological membranes since the total analgesic effect (infraorbital nerve blockade in rats) induced by mm ttc increased % upon complexation. supported by (fapesp # / - , - ) brazil. p- itc as a general thermodynamic and kinetic tool to study biomolecule interactions philippe dumas , dominique burnouf , eric ennifar , sondes guedich , guillaume bec , guillaume hoffmann isothermal titration calorimetry (itc) is a powerful technique for thermodynamic investigations that is little used to obtain kinetic information. we have shown that, in fact, the shape of the titration curves obtained after each ligand injection is strictly governed by the kinetics of interaction of the two partners. a simple analysis allowed us to explain several facts (e.g. the variation of time needed to return to equilibrium during a titration experiment). all simplifications were further released to obtain a very realistic simulation of an itc experiment. the method was first validated with the binding of the nevirapine inhibitor onto the hiv- reverse transcriptase by comparison with results obtained by biacore tm . importantly, for more complex systems, the new method yields results that cannot be obtained in another way. for example, with the e. coli transcription-regulator thiamine pyrophosphate riboswitch, we could resolve kinetically and thermodynamically the two important successive steps: ( ) the binding of the tpp ligand and ( ) the subsequent rna folding. our results show that initial tpp binding is controlled thermodynamically by tpp concentration, whereas the overall transcription regulation resulting from rna folding is kinetically controlled. gfps, due to their tendency to dimerize at high concentration. we have characterized for the first time the selfassociation properties of cfp (cyan fluorescent protein), the fluorescent protein mostly used as fret donor. we found that the fluorescence quenching observed at high expression level in the cell cytoplasm and the fluorescence depolarization measured at high concentration in vitro are insensitive to the a k mutation, shown to dissociate other gfp dimers. both phenomena are satisfactorily accounted for by a model of non-specific homo-fret between cfp monomers due to molecular proximity. modeling the expected contributions to fluorescence depolarization of rotational diffusion, homo-fret within a hypothetical dimer and proximity homo-fret shows that cfp has a homo-affinity at least times lower than gfp. this difference is due to an intrinsic mutation of cfp (n i), originally introduced to increase its brightness and that by chance also disrupts the dimers. biomolecular recognition typically proceeds in an aqueous environment, where hydration shells are a constitutive part of the interacting species. the coupling of hydration shell structure to conformation is particularly pronounced for dna with its large surface to volume ratio. conformational substates of the phosphodiester backbone in b-dna contribute to dna flexibility and are strongly dependent on hydration. we have studied by rapid scan ftir spectroscopy the isothermal b i -b ii transition on its intrinsic time scale of seconds. correlation analysis of ir absorption changes induced by an incremental growth of the dna hydration shell identifies water populations w (po --bound) and w (non-po --bound) exhibiting weaker and stronger h-bonds, respectively, than those in bulk water. the b ii substate is stabilized by w . the water h-bond imbalance of - kj mol - is equalized at little enthalpic cost upon formation of a contiguous water network (at - h o molecules per dna phosphate) of reduced !(oh) band width. in this state, hydration water cooperatively stabilizes the b i conformer via the entropically favored replacement of w -dna interactions by additional w -water contacts, rather than binding to b i -specific hydration sites. such water rearrangements contribute to the recognition of dna by indolicidin, an antimicrobial -mer peptide from bovine neutrophils which, despite little intrinsic structure, preferentially binds to the b i conformer in a water-mediated induced fit. in combination with cd-spectral titrations, the data indicate that in the absence of a bulk aqueous phase, as in molecular crowded environments, water relocation within the dna hydration shell allows for entropic contributions similar to those assigned to water upon dna ligand recognition in solution. segmental-labeling expression of sh domains of cd ap protein to study interaction with their ligand i.f. herranz-trillo , j.l. ortega-roldan , n.a.j. van transient and low affinity interactions within the cell can be enhanced by the combination of more than one domain. up to now most of the effort has been put on the study of the regulation in the affinity and specificity of the binding to isolated single domains but little is known about the effect of the presence of a second or third domain. multiple examples of proteins containing tandem domains exist in the genome like the cin /cms family of adaptor proteins. in this family all three n-terminal sh domains are involved in a wide variety of different interactions, they share higher similarity among themselves than to any other sh domains, suggesting an overlapping specificities in binding. cd associated protein (cd ap) is an adaptor protein and belongs to this family, its n-terminus consists of three sh domains and the interaction of each one of them with its target(-s) might be ultimately modulated by the presence of its next-door-neighbor. in this work we present the expression and purification of the tandem cd ap-sh a/ sh b produced by segmental labeling techniques that allow us to express the domains with different isotopic label, improving the nmr signal and facilitating to study the interaction of the natural ligand in the presence of nextdoor-neighbor domain. there are plenty of molecules that exert their effects at the cell membrane. the evaluation of these interactions, frequently quantified by the nernst lipid/water partition constant (kp), helps to elucidate the molecular basis of these processes. we present here a recently derived and tested method to determine kp for single solute partitions using fpotential measurements. the concept was then extended to the interaction of supramolecular complexes with model membranes. a simultaneous double partition with an aqueous equilibrium is considered in this partition model. the results were validated by dynamic light scattering -dls, f-potential, fluorescence spectroscopy and laser confocal microscopy experiments. we evaluated the interaction of supramolecular complexes (peptides derived from dengue virus proteins with oligonucleotides) with luv to study our biophysical models. dengue virus (dv) infects over - million people every year and may cause viral hemorrhagic fever. no effective treatment is available and several aspects of its cellular infection mechanism remain unclear. the extension of the interactions of these complexes with biomembranes helps to elucidate some steps of dv life cycle. the aggregation of amphotericin b in the lipid membranes induced by k + and na + ions: langmuir monolayers study marta arczewska, mariusz gagoś department of biophysics, university of life sciences in lublin, poland the polyene antibiotic amphotericin b (amb) is currently the drug of choice in the treatment of fungal infections despite its undesirable side effects. according to the general conviction, the biological action of the drug is based on the formation of transmembrane channels which affect physiological ion transport, especially k + ions. this work reports the results of langmuir monolayers study of the effect of k + and na + ions on the molecular organizations of amb in the model lipid membrane. the two-component monolayers containing amb and phospholipid (dppc) have been investigated by recording surface pressure-area isotherms spread on aqueous buffers containing physiological concentration of k + and na + ions. the strength of the amb-dppc interactions and the stability of the mixed monolayers were examined on the basis of surface pressure measurements, the compressional modulus and the excess free energy of mixing. the obtained results proved a high affinity of amb towards lipids in the presence of k + than na + ions. the most stable mixed monolayers were formed with the : and : stoichiometry in the presence of k + and na + ions, respectively. this research was financed by ministry of education and science of poland within the research project n n . microcalorimetric study of antibiotic amphotericin b complexes with na + , k + and cu + ions arkadiusz matwijczuk, grzegorz czernel, mariusz gagoś department of biophysics, university of life sciences in lublin, poland amphotericin b (amb) as a metabolite of streptomyces nodosus is one of the main polyene antibiotics applied in the treatment of deep-seated mycotic infections. we presented microcalorimetric (dsc) study of molecular organization of amphotericin b in lipid membranes induced by na + , k + and cu + ions. the analysis of dsc curves indicates the influence of na + and k + ions on the main phase transition of pure dppc lipid. for the molar fractions of , , , mol% amb in dppc we observed the thermal shift towards higher temperatures in respect to pure lipid, both in the presence of na + and k + ions. this result may be connected with the changes in dynamic properties of the model membrane system. in case of amb-cu + complexes in aqueous solution at two ph values, . and . , the dsc measurements reported endothermic heat effect. this phase transition was related to the dissociation process of amb-cu + complexes. the formation of amb-cu + complexes are accompanied by changes of the molecular organization of amb especially disaggregation. these all observed effects might be significant from a medical point of view. this research was financed by ministry of education and science of poland within the research project n n . membrane proteins and peptides are acting in an environment rich in other proteins or peptides. aim of our study was to understand how such molecular crowding and resulting intermolecular interactions can influence the behavior of membrane proteins, using various antimicrobial peptides and membrane proteins as examples. in the case of antimicrobial peptides we have previously described a change in their alignment in the membrane at a characteristic threshold concentration. to understand whether this change is due to unspecific crowding or specific peptidepeptide interactions, we tested if this re-alignment depends on the presence of additional peptides. in most cases we found a similar re-orientation behavior irrespective of the added peptide type, indicating unspecific crowding. when pairing pgla and magainin- , however, we observed a distinctly different sequence of pgla re-orientation in the membrane, indicating a specific interaction between these two peptides, which correlates well with their known synergistic activity. a rather different effect of crowding was observed for the larger channel protein mscl, which was found to form clusters of functionally active proteins in the membrane. we propose that this clustering is caused by lipid-mediated protein-protein interactions. water, hydrophobic interaction, and protein stability j. raul grigera and c. gaston ferrara instituto de física de líquidos y sistemas bioló gicos (iflysib), conicet-unlp, la plata, argentina although there are several forces maintaining protein structure, it is well know that hydrophobic interaction is the dominant force of protein folding. then, we can infer that any factor that alters hydrophobic interaction will affect the protein stability. we have study by computer simulation a model system consisting in solution of lenard-jones particles in water (spc/e model) at different pressures and temperatures and analyzed the solubility i.e. the aggregation properties, of such a system. from the obtained data we are able to build up the phase surface determining the critical point. the computing results where compared with experimental data of binary mix of non polar substance in water and of protein denaturation, finding high coincidence on the critical point. since the behavior of our model system can only be due to hydrophobic effects, the coincidences with the denaturation of proteins allow us to conclude that the dominant factor that determine temperature and pressure denaturation of proteins is the hydrophobic interaction. the temperature and pressures at which the denaturation, as well the disaggregation of simple non-polar particles, starts agree with what we could expect based on the cross over line of the low to high density structure water transition. the functional reconstitution of a mitochondrial membrane protein into a lipid bilayer was studied using a quartz crystal microbalance. the xhis-tagged protein was immobilised via specific binding to a cu + terminated sensor surface, with a change in frequency indicating approximately % coverage of the sensor surface by the protein. a lipid bilayer was reconstituted around the protein layer, with a final change in frequency that is consistent with the remaining area being filled by lipid. incubation with a specific ligand for the protein resulted in a significant change in frequency compared to the interaction with the surface or lipid alone. the change is greater than expected for the mass of the ligand, indicating a possible conformational change of the protein, such as the opening of a channel and increased water content of the layer. electrical impedance measurements on the same system have provided additional evidence of protein-lipid bilayer formation, and it is intended that this system will be studied with neutron reflectometry to characterise potential ligand induced channel formation. valuable functional and structural information about this membrane protein was obtained by using surface sensitive techniques to study the protein in a biomimetic lipid bilayer. visualizing and quantifying hiv-host interactions with fluorescence microscopy jelle hendrix , *, zeger debyser , johan hofkens and yves engelborghs laboratory for biomolecular dynamics, university of leuven, belgium, laboratory for molecular virology and gene therapy, university of leuven, belgium, laboratory for photochemistry and spectroscopy (*present address), university of leuven, belgium protein-chromatin interactions are classically studied with in vitro assays that only provide a static picture of chromatin binding. fluorescence correlation spectroscopy (fcs) is a non-invasive technique that can be used for the same purpose. being applicable inside living cells it provides dynamic real-time information on chromatin interactions. transcriptional co-activator ledgf/p has well characterized protein and chromatin interacting regions. we studied ledgf/p in vitro and inside living cells with fcs and other techniques (luminescent proximity assay, spot/half-nucleus fluorescence recovery after photobleaching, continuous photobleaching). protein-protein interactions in living cells can be monitored with fluorescence cross-correlation spectroscopy (fccs) using fluorescent proteins as genetic labels. advantages over using fö rster resonance energy transfer (fret) are the independence from intermolecular distance and knowledge of absolute protein concentrations. we characterized fccs with fluorescent proteins in vitro and then studied the intracellular complex of ledgf/p and the hiv- integrase (in) enzyme both with fret and fccs. nucleus and its compartment nucleolus are a seat of enormous biosynthetic activity in human cancer cells. nucleolar proteins, e.g. b or c , play an important role in regulation of cell division and proliferation. one of the strategies how to intermit malignant cell proliferation is affecting, e.g. by drug treatment, a net of intracellular protein interactions to bring the cell on a way of apoptosis. a cytostatic agent actinomycin d initiates apoptosis in human cancer cells, as well as in normal peripheral blood lymphocytes. at the same time, translocation of b and c into nucleoplasm is observed in the treated cells. therefore interaction between nucleolar and apoptotic proteins comes into a question. co-immunoprecipitation, fluorescence microscopy and yeast two hybrid analysis are used to answer it. in co-immunoprecipitation experiments, tumor suppressor p showed up to be a promising candidate for the interaction. fluorescence deposits mostly constituted by variants of transthyretin (ttr), a homotetrameric plasma protein implicated in the transport of thyroxine and retinol [ ] . nowadays, the only effective therapy for ttr amyloidosis is liver transplantation. new therapeutic strategies are being developed taking advantage of our current understanding of the molecular mechanisms of amyloid formation by ttr [ ] . a significant effort has been devoted to the search and rational design of compounds that might decrease ttr tetramer dissociation, for example, through ligand binding at the thyroxine binding sites of ttr [ , ] . here, we use isothermal titration calorimetry (itc) to characterize the thermodynamic binding signature of potential ttr tetramer stabilizers, previously predicted by computerassisted methods [ ] . itc allows the measurement of the magnitude of the binding affinity, but also affords the characterization of the thermodynamic binding profile of a protein-ligand interaction. high affinity/specificity ttr ligands, enthalpically and entropically optimized, may provide effective leads for the development of new and more effective drug candidates against ttr amyloidosis. we have established a set of vectors to promote easy cloning of ecfp and eyfp fusions with any protein of interest. we exploit these fluorescent fusion proteins to study protein-protein interactions by fluorescence lifetime of ecfp. the decrease of ecfp lifetime reveals fret between ecfp and eyfp and hence the interaction between proteins in question. groel-groes chaperonin complex is required for the proper folding of eschericia coli proteins. bacteriophage t and its distant relative coliphage rb encode co-chaperon proteins (respectively gp and coco) that can replace groes in the chaperonin complex. gp is also required in the folding of the major capsid protein of the phage. prd is a large membrane-containing bacteriophage infecting gram-negative bacteria such as e. coli and salmonella enterica. it has kb long linear dsdna genome and the capsid has an icosahedral symmetry. the groel-groes chaperonin complex is needed in the assembly of prd . we have found evidence that prd protein p can work similar way as other viral co-chaperones and substitute groes in chaperonin complex. fluorescence lifetime studies between proteins groel and p reveals an interaction that backs up the theory. structural modification of model membranes by fibrillar lysozyme as reaveled by fluorescence study a.p. kastorna v.n. karazin kharkiv national university, svobody sq., kharkiv, , ukraine recent experimental findings suggest that protein aggregation, leading to the formation and depositions of amyloids play a central role in the neurodegenerative diseases, type ii diabetes, systemic amyloidosis, etc. in the present study we focused our efforts on investigation of the influence of fibrillar lysozyme on the structural state of model lipid membranes composed of phosphatidylcholine and its mixtures with cardiolipin ( mol %) and cholesterol ( mol %). to achieve this purpose, two fluorescent probes with different bilayer location, , -diphenyl- , , -hexatriene (dph) distributing in membrane hydrocarbon core and -lauroyl- -dimethylaminonaphthalene (laurdan) locating at lipid-water interface, have been employed. the changes in membrane viscosity under the influence of amyloid lysozyme were characterized by fluorescence anisotropy of dph. this fluorescence parameter was not markedly affected by fibrillar protein in all types of model membranes. the changes in emission spectra of laurdan were analysed by the generalized polarization value (gp). it was found that adding of amyloid lysozyme resulted in the increment of gp value. our data suggest that lysozyme fibrils cause reduction of bilayer polarity and increase of lipid packing density. isothermal titration calorimetry (itc) is the gold standard for the quantitative characterisation of protein-ligand and protein-protein interactions. however, reliable determination of the dissociation constant (k d ) is typically limited to the range lm [ k d [ nm. nevertheless, interactions characterised by a higher or lower k d can be assessed indirectly, provided that a suitable competitive ligand is available whose k d falls within the directly accessible window. unfortunately, the established competitive itc assay requires that the high-affinity ligand be soluble at high concentrations in aqueous buffer containing only minimal amounts of organic solvent. this poses serious problems when studying protein binding of small-molecule ligands taken from compound libraries dissolved in organic solvents, as is usually the case during screening or drug development. here we introduce a new itc competition assay that overcomes this limitation, thus allowing for a precise thermodynamic description of highand low-affinity protein-ligand interactions involving poorly water-soluble compounds. we discuss the theoretical background of the approach and demonstrate some practical applications using examples of both high-and low-affinity protein-ligand interactions. interaction of myoglobin with oxidized polystyrene surfaces studied using rotating particles probe m. kemper , , d. spridon , l.j. van ijzendoorn , m.w.j. prins , eindhoven university of technology, department of applied physics, eindhoven, the netherlands, dutch polymer institute, eindhoven, the netherlands, philips research, eindhoven, the netherlands the interaction of proteins with polymer surfaces is of profound importance for the sensitivity of biosensors. polymer surfaces are often treated in order to tune their chemical and physical properties, for example by oxidation processes. to get a better understanding of the association of proteins to treated polymer surfaces, we use the rotating particles probe (x.j.a. janssen et al., colloids and surfaces a, vol. , p. , ). in this novel technique, protein coated magnetic particles are in contact with a substrate and the binding is recorded for all individual particles using a rotating magnetic field. we investigate the interaction of myoglobin coated magnetic particles to spincoated polystyrene surfaces that have been oxidized with a uv/ozone treatment. the surfaces have been characterized by xps, afm and water contact angle measurements. we will demonstrate a clear influence of polystyrene oxidation on the binding fractions of the myoglobin coated particles. we interpret the results in terms of dlvo-theory: electrostatic as well as electrodynamic properties of the surfaces will be influenced by the oxidation. interact with monomeric and/or filamentous actins. twinfilin is a - kda protein composed of two adf-homologue domains connected by a short linker. in our work we studied the effects of the mouse twinfilin- (twf ) on the monomeric actin. we determined the affinity of twf to the atp-actin monomer with fluorescence anisotropy measurement (k d = . lm). the fluorescence of the actin bound e-atp was quenched with acrylamide in the presence or absence of twf . in the presence of twinfilin the accessibility of the bound e-atp decreased, which indicates that the nucleotide binding cleft is shifted to a more closed conformational state. it was confirmed with stopped-flow experiments that the kinetics of nucleotide-exchange of actin decreased in the presence of twf . we determined the thermodynamic properties of twf and investigated the effect of twinfilin on the stability of actin monomer with differential scanning calorimetry. the twf stabilized the stucture of the g-actin. our results can help us to understand the regulation of actin cytoskeleton in more details. magnetic np have attracted attention due to their potential of contrast enhancement of magnetic resonance imaging and targeted drug delivery, e.g. tumor magnetic hyperthermia therapy. potential nephrotoxicity of single i.v. administration of fe o np was studied in female wistar rats i.v. administered either placebo ( % v/v rat serum in . % nacl), suspension of tio np (positive control, bimodal / nm distribution), or fe o np (bimodal / nm distribution) in doses of . , . or . mg/kg. rats were sacrificed h, -, -and -days after np injection (n= - /each group). administration of np did not alter kidney size significantly; renal function of np administered rats as monitored by plasma creatinine and urea concentrations, creatinine clearance and protein excretion rate did not differ significantly in either interval from rats administered placebo. one week after administration significant rise in plasma ca, its urinary and fractional excretion was observed in rats administered mg fe o /kg. plasma mg levels rose in this group and weeks after administration. no significant changes in the expression of tnf-a, tgf-b, and collagen iv genes in renal cortex were revealed. no obvious nephrotoxic effects were observed in rats after a single i.v. dose of fe o np. study was supported by fp ec eu: nanotest (development of methodology for alternative testing strategies for the assessment of the toxicological profile of nanoparticles used in medical diagnostics.), grant no.: . biomimetic supramolecular assemblies for studying membrane interactions in vitro and in vivo s. kolusheva, r. jelinek ben-gurion university, beer-sheva, israel we designed a novel biomimetic sensor, composed of conjugated polydiacetylene (pda) matrix embedded within lipid vesicles. the system is capable of detecting various compounds occurring within lipid membranes through rapid colorimetric as well as fluorescent transitions. the colorimetric response of the sensor is correlated to the extent of compound-membrane binding and permeation and quantified binding sensitivity to lipid composition. we describe a new disease diagnostic approach, denoted ''reactomics'', based upon reactions between blood sera and an array of vesicles comprising different lipids and polydiacetylene (pda), a chromatic polymer. we show that reactions between sera and such a lipid/pda vesicle array produce chromatic patterns which depend both upon the sera composition as well as the specific lipid constituents within the vesicles. through attachment of chromatic polydiacetylene (pda) nanopatches onto the plasma membrane, real-time visualization of surface processes in living cells is possible. the ras protein is mutated in % of human tumors. ras acts as a switch, transmitting a growth signal in an active gtp-bound form and turning the signal off in an inactive gdp-bound form. the switch off is accomplished by gtp hydrolysis, which is catalyzed by ras and can be further accelerated by gtpase activating proteins (gaps). mutations which prevent hydrolysis cause severe diseases including cancer. we investigate the reaction of the ras gap protein-protein complex by time-resolved ftir spectroscopy. detailed information on the mechanism and the thermodynamics of the reaction was revealed: first, the catalytic arginine-finger of gap has to move into the gtp binding pocket, then cleavage of gtp is fast and h po hydrogen-bonded in an eclipsed conformation to the b-phosphate of gdp is formed. further, we performed for the first time atr-ftir spectroscopy of ras in its native environment, a lipid membrane. in this setup we are able to do difference spectroscopy of the immobilized protein. interactions with other proteins can be determined in a similar way as in spr experiments but with the additional information from the infrared spectra. galectins are a family of animal lectins that specifically bind b-galactosides and have gained much attention due to their involvement in several biologic processes such as inflammation, cell adhesion and metastasis. in such processes, several issues are still not clear including the mechanisms of interaction with different carbohydrates. galectin- (gal- ) is a tandem-repeat type galectin that contains two carbohydrate recognition domains (crd-i and crd-ii) connected by a linking peptide. in this study, we performed spectroscopic studies of the carbohydrate-recognition domains from human gal- . our goals are two-fold: ( ) to monitor conformational changes in each domain upon its binding to specific ligands and then to correlate the observed changes with structural differences between the crds and ( ) to investigate the interaction between the crds and lipid model membranes. to achieve such objectives we used a combined approach of spectroscopic techniques involving circular dichroism and electron spin resonance. overall the results obtained so far show that crd-i and crd-ii have distinct behaviors in terms of carbohydrate recognition and membrane binding. this may be due to specific differences in their structures and certainly suggests a non-equivalent role in protein function. hemoglobin influence on lipid bilayer structure as revealed by fluorescence probe study o.k. kutsenko, g.p. gorbenko, v.m. trusova v.n. karazin kharkov national university, kharkov, ukraine hemoglobin (hb) is a red blood cell protein responsible for the oxygen transport. its affinity for lipid bilayers represents interest for gaining insight into protein biological function as well as for some applied aspects such as development of blood substitutes or biosensors. hb influence on lipid bilayer structure was investigated using fluorescent probes pyrene and prodan. model membranes were prepared of phosphatidylcholine (pc) and its mixtures with phosphatidylglycerol (pg) and cholesterol (chol). hb penetration into membrane interior is followed by the increase of relative intensity of pyrene vibronic bands and decrease of prodan general polarization value suggesting an enhancement of bilayer polarity. this implies that hb incorporation into membrane interior decreases packing density of lipid molecules, promoting water penetration into membrane core. chol condensing effect on lipid bilayer prevents protein embedment into bilayer, thus decreasing membrane hydration changes as compared to pc bilayers. in the presence of anionic lipid pg hb-induced increase of bilayer polarity was found to be most pronounced, pointing to the modulatory role of membrane composition in hb bilayer-modifying propensity. we present optimized sialic acid-based mimics binding in the low nanomolar range. molecular interactions were determined with surface plasmon resonance (spr), characterizing the affinity and the kinetics of binding. furthermore, isothermal titration calorimetry (itc) was applied to dissect the standard free energy of binding (dg°) into the standard enthalpy of binding (dh°) and the standard entropy of binding (ds°). in order to pass the cell membranes, most of these medicines has to be administrated to patients as nucleoside pro-drugs and not directly as triphosphorylated forms. because of the poor phosphorylation of the nucleoside analogues used in therapy, it is important to understand and to optimize their metabolism. our aim is to understand how compounds of chirality l turn away -phosphoglycerate kinase (pgk) from its normal glycolytic function to be converted into the triphosphate forms. in order to elucidate pgk mechanism and substrate specificity, we have measured the kinetics of the different steps of the enzymatic pathways by rapid mixing techniques and studied the influence of the nature of the nucleotide substrate thereon. we first performed an extensive study with d-and l-adp (see poster by p. lallemand). we are now extending the studies to other nucleotide diphosphates (some of them used in therapies). changes in the nature of the nucleobase or deletion of hydroxyl group of the sugar affect the efficiency of phosphorylation by pgk, either by decreasing dramatically their affinity or by altering the phospho-transfer step itself. structural explanations are given based on docking data. probing drug/lipid interactions at the molecular level represents an important challenge in pharmaceutical research, drug discovery and membrane biophysics. previous studies showed that enrofloxacin metalloantibiotic has potential as an antimicrobial agent candidate, since it exhibits antimicrobial effect comparable to that of free enrofloxacin but a different translocation route. these differences in uptake mechanism can be paramount in counteracting bacterial resistance. in view of lipids role in bacterial drug uptake, the interaction of these compounds with different e. coli model membranes were studied by fluorescence spectroscopy. partition coefficients determined showed that lipid/antibiotic interactions were sensitive to liposomes composition and that the metalloantibiotic had a higher partition than free enrofloxacin. these results corroborate the different mechanism of entry proposed and can be rationalized on the basis that an electrostatic interaction between the metalloantibiotic positively charged species, present at physiological ph, and the lipids negatively charged head groups clearly promotes the lipid/antimicrobial association. oligomerization and fibril assembly of amyloid b peptide amyloid b peptide (ab) forms a large amount of extracellular deposits in the brain of alzheimer's disease (ad) patients and it is believed that this peptide is related to the pathogenesis of that disease. the most abundant monomeric form of physiological ab (* %) is constituted by amino acids and is benign, but by an unknown mechanism this endogenous material becomes aggregated and neurotoxic. increasing evidence suggests that membrane interaction plays an important role in ab neurotoxicity. in this work it will be studied the interactions of ab( - ) with ctac (cationic), sds (anionic), pfoa (anionic with fluorine atoms) and og (nonionic) amphiphiles in monomeric and micellar forms. the results demonstrated that ab( - ) forms fibrils with different morphologies in the presence of micelles. in addition, the presence of micelles accelerates the formation of fibrils and decreases the lifetime of oligomers. we present here the exploitation of the powerful approach of surface plasmon resonance imaging and mass spectrometry coupling for protein fishing in biological fluids such as human plasma at the same sensitivity. on one hand, multiplex format spri analysis allows direct visualization and thermodynamic analysis of molecular avidity, and is advantageously used for ligand-fishing of captured bio-molecules on multiple immobilized receptors on a spri-biochip surface. on the other hand, maldi mass spectrometry is a powerful tool for identification and characterization of molecules captured on specific surface. therefore, the combination of spri and ms into one concerted procedure, using a unique dedicated surface, is of a great interest for functional and structural analysis at low femtomole level of bound molecules. to reach these goals, particular surface engineering has been engaged to maintain a high level of antibody grafting and reduce non-specific adsorption. thus, various chemistries have been tested and validated towards biological fluids such plasma, keeping in mind the capacity of the in situ investigation by ms. finally, signal to noise ratio was magnified leading to the characterization of protein lag , a potential marker of breast cancer, in human plasma. atenolol incorporation into pnipa nanoparticles investigated by isothermal titration calorimetry mihaela mic, ioan turcu, izabell craciunescu, rodica turcu, national institute for r&d of isotopic and molecular technologies, cluj-napoca, romania e-mail: mihaela.mic@itim-cj.ro poly(n-isopropylacrylamide) (pnipa) is a thermo-sensitive hydrogel undergoing a volume phase transition at about of °c close to the body temperature. this volume phase transition is envisaged as a key property for drug binding and release. the purpose of our research is the thermodynamic characterization of the binding of atenolol by pnipa polymeric nanoparticles. the thermodynamic parameters which characterize the binding process are obtained using the isothermal titration calorimetry (itc) as the main investigation technique. when polymeric nanoparticles bind drug molecules, heat is either generated or absorbed depending on the amount of bond molecules and also on the exothermic or endothermic character of the binding process. the heat measurement allows the determination of binding constants, reaction stoichiometry and the thermodynamic profile of the interaction. itc technique has been used to investigate the binding properties of nanoparticles which shrink from the swollen to the collapsed state. the capacity of such nanogels to bind atenolol molecules is directly related to relevant differences between the binding properties in the swollen and in the collapsed state respectively. aggregation study of x-(alkyldimethylamonium)alkylaldonamide bromides p. misiak , b. ró _ zycka-roszak , e. woźniak , r. skrzela , k.a. wilk department physics and biophysics, wrocław university of environmental and life sciences, wrocław, poland, department of chemistry, wrocław university of technology, wrocław, poland sugar-based surfactants are of considerable research interest because they have improved surface and performance properties, reduced environmental impact, and have potential pharmaceutical and biomedical applications. x-(alkyldimethylamonium)alkylaldonamide bromides (c n gab) with different chain lenghs (n = , , , ) belonging to cationic sugar-based surfactants were newly synthesised. the aggregation processes of c n gabs were studied by means of isothermal titration calorimetry (itc), electric conductance method and molecular modelling methods. the critical micelle concentrations (cmc), the degree of micelle ionization (b), the enthalpies (dh m ) and the entropies (ds m ) of micellization as well as the contributions of the headgroups to the gibbs free energies (dg m (hy)) were calculated. the obtained values were compared with those for dodecyldimethylethylammonium bromide and literature data for analogical glucocationic surfactants. the latest compounds differ from c n gab surfactants by substitution of sugar chain by gluco ring. molecular modelling methods were used to relate the molecular properties of the compounds with their experimentally studied properties in solution. this work was supported by grant n n . every year over million people are infected with dengue virus (denv), transmitted by a mosquito (aedes aegypti). this enveloped virus, member of the flaviviridae family, has four distinct serotypes. it has a single stranded positive rna molecule with a single open reading frame that encodes a single poliprotein, which, after appropriate processing by viral and host proteases, gives rise to three structural proteins (c, prm and e) and seven non-structural proteins (ns , ns a, ns b, ns , ns a, ns b and ns ) [ ] . the surface of the immature virion is composed of e and prm heterodimers that are arranged as trimers protruding from the membrane [ ] . the virus is thought to enter the host cell via a receptor-mediated endocytosis, although, if any, the specific dengue receptors have not been described. once inside the cell, the acidified environment inside the endocytic vesicle triggers an irreversible trimerization of the envelope (e) protein, inducing the release of the nucleocapsid (composed of viral rna and multiple copies of c protein) to the cytoplasm, thus starting the infection process, where the poliprotein is translated and processed, originating all viral proteins. considering the structural proteins c and e, these are essential for the viral infection, specifically, protein c is thought to be involved in the viral assembly and specific encapsidation of the genome and protein e (a class ii fusion protein) plays a major role in the fusion process. as recently described by some studies [ ] , protein c is composed of four a helices connected by four short loops and has a highly hydrophobic region forming a concave groove that could interact with lipid membranes and a region with an increased concentration of positive charges, possibly interacting with the viral rna. as for protein e, it is composed of three b stranded domains. it is proposed that the fusion loop is located in domain ii of this protein and the putative receptor binding sites, considered essential for the viral entry, are supposedly located in domain iii. in this work, we describe the identification of the membrane active regions of both these proteins, considering both theoretical studies, hydrophobic moments, hydrophobicity and interfaciality values as well as experimental ones, namely fluorescence spectroscopy, where a fluorescent probe is encapsulated in model membrane systems, and differential scanning calorimetry [ ] . we have found one region in protein c and four regions in protein e with membranotropic activity. this is the first work describing experimentally the putative membrane interacting zones of both these proteins. this work was funded by grant bfu - -bm from ministerio de ciencia y tecnologia, spain, granted to jose villalaín. investigation of membrane-membrane interaction mediated by coiled coil lipopeptides gesa pä hler, andreas janshoff georg-august-university, tammannstrasse , gö ttingen, germany e-mail: gpaehle@gwdg.de specific cellular membrane interaction and fusion are crucial points in vivo which are in eukaryotic cells mediated by snare proteins. the definite mechanism behind these processes is still poorly understood, but the coiled coil formation of a snare core complex consisting of four a-helices seems to generate a fusogenic driving force. this offers the possibility to design a straightforward experimental setup to mimic the complex protein-mediated membrane-membrane interaction by using mere protein fragments or peptides attached to artificial lipid bilayers which self-assemble to a coiled coil structure. in our approach, two artificial three heptad repeat coiled coil forming peptides were synthesized and attached to maleimide functionalized membranes via an in situ-coupling reaction. thus, secondary structure changes, kinetic characteristics and binding energetics were monitored during coiled coil formation with real time ellipsometry, ir and cd spectroscopy. the lipopeptide mediated membrane-membrane interaction itself is investigated by colloidal probe spectroscopy and tirfm. these techniques and the setup of our model system allow screening the energetic and structural properties of variable coiled coil forming peptides, i.e. linker-modified or biologically inspired sequences. enzymatic reactions in nanostructured surfaces: unzipping and cutting the double helix pietro parisse , matteo castronovo , bojana lucic , alessandro vindigni , giacinto scoles and loredana casalis sincrotrone trieste s.c.p.a., trieste, italy, temple university, philadelphia, usa, protein-dna interactions are vital for living organisms. from viruses to plants and humans, the interactions of these two different classes of biopolymers control processes as important and diverse as the expression of genes and the replication, rearrangement, and repair of dna itself. to understand these processes at the molecular level, and to follow changes in cellular pathways due to different kinds of perturbations and/or diseases it is necessary the identification and quantification of proteins and their complex network of interactions. we have exploited the high spatial resolution given by atomic force microscopy to generate dna arrays of variable density by means of nanografting. on such nanostructures, we investigate the mechanism of different enzymatic reactions (from restriction enzymes to helicases). registering with high precision the height variation due to the action of the enzyme onto the engineered dna sequences (in the case of restriction enzymes) or taking advantage of the different mechanical properties of single and double stranded dna (in the case of helicases, where for the first time kinetic data were obtained on recq human helicase), we were able to monitor either the activity and/or the action mechanisms of these two important classes of enzymes. in this study an attempt has been made to investigate the structure, dynamics and stability of cyclic peptide nanotubes (cpnts) formed by the self-assembly of cyclic peptides (cps) using classical molecular dynamics (md) simulation and semiempirical quantum chemistry calculation employing pm . the structure and energetics of monomer and various oligomeric cpnts have been investigated by considering the (cyclo-[(d-ala-l-ala) ]) peptide as the model for cp. various geometrical parameters extracted from the md simulation reveal that the terminal residues are loosely hydrogen bonded to the inner subunits regardless of degree of oligomerization. the hydrogen bonds present in the inner core regions are stronger than the terminal residues. as the degree of oligomerization increases, the stability of the tube increases due to the hydrogen bonding and stacking interactions between the subunits. the results show that the binding free energy increases with the extent of oligomerization and reaches saturation beyond cpnt . in addition, hydrophobic and electrostatic interactions play crucial roles in the formation of cpnts. analysis of both structural and energetics of formation of cpnts unveils that the selfassembly of dimer, trimer and tetramer cpnts are the essential steps in the growth of cnpts. monolayers on a langmuir trough constitute a great biomimetic model to characterize protein-protein or protein-lipid interaction, where the physical state of the interfacial layer is completely controlled. we present here three studies performed on monolayers, with a wide panel of experimental (optical, spectroscopical, rheological) techniques. i) surface properties and conformation of nephila clavipes spider recombinant silk proteins (maspi and masp ) was studied at the air-water interface: we show that the mechanism of assembly of both proteins is different, although both proteins share the same sequence pattern and a close hydrophobicity. they both exhibit a certain propensity to form b-sheets that may be important for the efficiency of the natural spinning process. ii) the dystrophin molecular organization and its anchoring in a lipidic environment depend on the rod fragment used and on the lipid nature. moreover the interaction is guided by the lateral surface pressure. this lipid packing variation is essential to understand the role of the dystrophin during compression-extension cycle of the muscle membrane. iii) we evidence that non additive behavior of mixtures of food globular proteins leads to enhanced foaming properties or to self assembled objects. nucleolar-targeting peptides (nrtps) were designed by structural dissection of crotamine, a toxin from the venom of a south-american rattlesnake. at lm concentration, nrtps penetrate different cell types and exhibit exquisite nucleolar localization. the aim of this work was to pursue with the study of nrtps molecular mechanism for translocation, as well as to determine the ability of nrtp to delivery large molecules into cells. for the translocation experiments, rhodamine b-labeled nrtps were used and tested with giant multilamellar vesicles. confocal microscopy results show that there is an efficient translocation across model membranes. high levels of intracellular peptide were also seen in different cell lines and pbmc, soon after incubation with nrtp. furthermore, a conjugate of nrtp (nrtp c) bound to b-galactosidase was prepared by chemical synthesis and tested in hela cells. this conjugate maintains enzymatic activity and is stable at °c for several days. the work done so far with this new family of cell-penetrating peptides revealed strong interaction and translocation with lipid model systems. moreover, successfully cellular delivery of bgalactosidase was observed and quantified. interaction of zinc phthalocyanine with ionic and non ionic surfactants: uv-vis absorption and fluorescence spectroscopy for application in photodynamic therapy m. p. romero, s. r. w. louro physic department, pontifícia universidade cató lica de rio de janeiro puc-rio, brazil among the second-generation photosensitizer (ps) developed for the treatment of neoplastic diseases by photodynamic therapy (pdt), metallo-phthalocyanines (mpc) have been proposed as an alternative to the currently used ps in clinical application. unsubstituted mpc are not soluble in physiological solvents and their in vivo administration relies upon their incorporation into carriers or their chemical conversion into water-soluble dyes by the attachment of selected substituents. in this work, uv-vis absorption and fluorescence spectroscopy were used to study the ability of different micelles for dispersing zinc phthalocyanine (znpc). the following surfactants were tested: sds, ctab, hps, tween , tween , and pluronic f . znpc has low solubility in virtually all solvents, but dmf and dmso are observed to dissolve znpc in concentrations of the order of . and . mmol/l, respectively. stock solutions of znpc in dmf and dmso were prepared. micelles of the different surfactants containing znpc were prepared by dissolving in aqueous medium (milli-q water or phosphate buffer ph . ) small amounts of the stock solutions previously mixed with each surfactant. the amounts of each surfactant were calculated to give an average ratio of one znpc molecule per micelle in the final solution. the absorption and fluorescence spectra of znpc in the micellar systems were obtained, and were observed to change in time. immediately after dissolution the spectra are characteristic of monomeric znpc, suggesting formation znpccontaining nanoemulsions with the mixture of znpc-organic solvent in the hydrophobic region of the micelle. since dmso and dmf are miscible with water, the solvent diffuses out of the micelle and znpc stays inside the micelle in a monomeric or aggregated form. the different surfactants lead to different time evolution of znpc aggregation. aggregation lifetimes vary from one hour, in the case of pluronic f , to more than twelve hours, in the case of ctab and hps. it was observed that the ionic surfactants were more efficient than non ionic ones for monomeric delivery of znpc . work partially supported by cnpq, inami and faperj. nucleobase-containing peptides are an important class of molecules comprising both artificial (synthetic nucleopeptides) and natural (peptidyl nucleosides and willardiine-containing peptides) compounds characterized in many cases by interesting biological properties. , in this work, we report a spectroscopic study on the properties of a chiral nucleobase-bearing peptide obtained by chemical synthesis starting from commercial sources. the findings of this research strongly encourage further efforts in the field of the use of nucleopeptides as supramolecular assembling systems and open the way to novel drug delivery approaches based on nucleobase recognition. conformational plasticity. their structure depends tremendously on their local environment and confinment, and may accommodate several unrelated conformations, that are a strong challenge for the traditional characterizations of structure, supramolecular assembly and biorecognition phenomena. atomic force microscopy (afm) has been successfully exploited for both highly controllable nanolithography of biomolecules and for biorecognition studies, such as oriented prion protein -antibody interaction (sanavio et al., acs nano ( ) ( ): , bano et al. nano lett ( ) ( ): - ). here, we report different strategies for selective, oriented confinement of alphasynuclein at the nanoscale for sensitive and accurate direct detection, via precise topographic measurements on ultraflat surfaces, of biomolecular interactions in confined assemblies. a new class of cell penetrating peptides (cpps) was generated by splicing the ( - ) and ( - ) segments of crotamine, a toxin from crotalus durissus terrificus venom [ ] . as they localize preferably on the nucleolus, these novel cpps were named nucleolar-targeting peptides (nrtps). the extent of nrtp partition to zwitterionic (popc; popc:cholesterol : ) and anionic (popg; popc:popg : ) lipid vesicles was studied following the intrinsic tyr or trp fluorescence of the peptides. the partition curves into popc zwitterionic vesicles were characterized by downward slopes and higher partition coefficients (k p * - ). for pure popg, an upward curve and smaller partition coefficient point out for a different type of membrane-peptide interaction. popc:popg membranes present characteristics of both types of interaction. from red edge excitation shift and quenching experiments similar conclusions were attained. leakage assays ruled out lipid vesicle disruption by crotamine or nrtps. further studies on nrtp cellular translocation mechanism and large molecule delivery are currently in progress. dystrophin is essential to skeletal muscle function and confers resistance to the sarcolemma by interacting with cytoskeleton and membrane. we characterized the behaviour of dys r - , a five spectrin-like repeats from the central domain of human dystrophin, in the presence of liposomes and monolayers as membrane models. interaction of dys r - depends on the lipid nature, anionic or zwitterionic, with suvs, and on the lipid packing when comparing luvs to suvs. lateral pressure of lipid monolayers modifies the protein organization and leads dys r - to form a regular network as revealed by afm. trypsin proteolysis assays show that the protein conformation is modified following its binding to monolayer and suvs. label free quantification by nano-lc/ms-ms allowed identifying the helical amino acid sequences in repeats and that are involved in the interaction with anionic suvs. results indicate that dys r - constitutes a part of dystrophin that interacts with anionic as well as zwitterionic lipids and adapts its interaction and organization depending on lipid-packing and lipid nature. we provide here strong experimental evidence for a physiological role of the central domain of dystrophin on sarcolemma scaffolding through modulation of lipid-protein interactions. extracellular matrix proteins. overexpression of the mmps has been associated with a variety of diseases ranging from periodontal disease and arthritis to tumor invasion and metastasis. the majority of the more powerful synthetic inhibitors of mmps incorporate a hydroxamate group, but exhibit low selectivity and are toxic. in a recent modeling study, astaxanthin (ast), a carotenoid with potent antioxidant property, has been shown to be a potential inhibitor of mmp- function by occupying a binding site near the active center of the enzyme (bika di et al. ). in our ongoing project, we investigate the binding of ast to the catalytic domain of mmps using biochemical and ultimately crystallization to validate the proposed action of ast. along these lines, the catalytic domain of mmp- (cdmmp- ) was expressed in e.coli bl (de ) codon-plus and refolded using a novel effective refolding method. our results reveal that ast has a potent inhibitory effect on cdmmp- activity, however, determination of ic % or k i is difficult due to fast oxidation and structural instability of ast. ongoing work aims at optimizing the inhibition conditions and improving the refolding yield to allow analyzing structure and function of the ast-bound mmp- in more detail. hyaluronic acid (hyaluronan, ha) is a linear polysaccharide with a molar mass in the range of to da and is built from alternating units of glucuronic acid and n-acetylglucosamine. synthesized in the cellular plasma membrane, it is a network-forming and space-filling component in the extracellular matrix of animal tissues. here, we create hyaluronic acid films atop a porous alumina substrate, where they act as a barrier for macromolecular transport depending on their length and geometry. the geometry of the hyaluronic acid switches between a fully stretched and a mushroomlike state and is dependent on the concentration of hyaluronic acid. to bind hyaluronic acid selectively atop the nanoporous anodic aluminum oxide (aao), the aao is orthogonally functionalized by silane chemistry. by means of time resolved optical waveguide spectroscopy (ows) the transport of macromolecules, e.g. avidin, across the hyaluronic acid barriers can be recorded as a function of the pore diameter and hyaluronic acid concentration in a time resolved and label free manner. confocal laser scanning microscopy (clsm) provides an alternative method to investigate the orthogonal functionalization of the pores and to elucidate whether a molecule can cross the barrier at the pore entrance. we functionalized gold surfaces with a hydroxy-terminated self-assembled thiol monolayer exposing an adjustable fraction of biotin moieties. [ ] by in-situ acetylation or fluorination, surface properties could be fine-tuned to different protein immobilization scenarios. using streptavidin as a linker protein, immobilization of human abcc [ ] in liposomal and planar bilayer systems was possible. abcc -containing proteoliposomes doped with a biotinylated anchor lipid were successfully tethered to our streptavidin-coated surfaces. biotinylation of the extracellular glycosylation of abcc allowed direct immobilization with inside-up orientation and subsequent assembly of a lipid bilayer. outside-up orientation was achieved by exploiting the c-terminal histidin tag of recombinant abcc for immobilization via ni + and biotinnitrilotriacetate. all systems were thoroughly characterized by quartz crystal microbalance, atomic force microscopy and surface plasmon resonance techniques with respect to monitoring abcc mediated substrate transport in real time. because of its role in the apoptotic pathway, conformational transitions of cytochrome c (cyt c) have gain on interest. in native cyt c, met and his residues serve as heme axial ligands. cyt c interaction with the membrane causes the disruption of the iron-met bond. this allows the binding of others endogenous ligands forming alternative low spin species , or induces peroxidase activity through the formation of a five coordinated high spin iron specie. acquisition of this peroxydase activity by cyt c has been shown to be a key stage before its release from the mitochondria . in order to study these non native low spin species by checking the possible amino acids able to bind the human cyt c heme, different mutants have been designed and produced: h q, h n, and the double one h q/h n. sds in countries where seafood is an integrate part of the diet, fish are among the most common food allergen sources. the major fish allergen parvalbumins are abundant in the white muscle of many fish species. parvalbumin belongs to the family of ef-hand proteins and has a globular shape containing six helical parts. high pressure is known to unfold proteins. we performed high pressure ftir experiments, to explore the p-t phase diagram of cod parvalbumin, gad m , to test the possibility of its inactivation by high pressure treatment. the infrared spectrum of parvalbumin is characteristic for the helical conformation, in agreement with the crystal structure. a marked transition in the structure of the parvalbumin was observed with the central point of . gpa (at room temperature). the amide i position shifts to a wavenumber which is between the helical and the unfolded position. we assign this change to a native-molten globule transition. it was reversible as seen from the infrared spectra. it has been proven in the past that reflectometric interference spectroscopy (rifs) is a powerful measurement system for the detection of protein-protein interactions . we present here the development of a reflectometric sensor which allows for the detection of membrane-protein interactions in the micromolar regime. in this study we employ two different instrumental assemblies. the first installation enables direct detection and quantification of the interaction of membrane proteins with solid supported lipid bilayers. in the second assembly the original instrument is combined with an upright fluorescence microscope. the advantages of this installation are the direct optical control of the experiment as well as a smaller sensing area. the set-up allows for the detection of interactions on lipid-patches of just several micrometers in diameter. the aim of this work is an experimental system that enables the measurement of transport processes through lipid membranes. we attempt to achieve this by covering a closed porous substrate with a lipid membrane. the first steps to reach this goal were done by spanning membranes over anodized aluminum oxide substrates. initiation of actin polimerization in cells requires nucleation factors. a pointed-end-binding protein of f-actin -the lei-omodin -acts as a strong filament nucleator in muscle cells. the dynamical, structural and kinetic properties of a protein can provide important information to understand the intramolecular events underlying its function. we are interested in how does the leiomodin regulate the actin polimerization. our aim is to determine the dissociation constant of the actin-leiomodin complex, and study a possible side-binding effect of the leiomodin . the cardiac leiomodin of rattus norvegicus is a kda molecular weight protein, which contains a kda n-terminal, a kda leucin reach repeat (lrr) and a kda c-terminal region. the n-term and the lrr regions are together tropomodulin homologues. we expressed the wild type the n-term+lrr the lrr+c-term and the c-term protein fragments by using a ptyb vector that contains an amplicillin resistance gene. the expression of the proteins was carried out with the twin-cn (ne bio-labs) kit, which is a chitin-intein self-cleavage and purification system. the nucleation activity of leiomodin and the polimerization speed of actin in the presence of tropomyosin and leiomodin were studied by using pyrene-actin polimerization assay. we measured the stoichiometric, conformational and kinetic properties of the leiomodin-actin complexes with co-sedimentation assay, fluorescence spectroscopic and rapid-kinetic methods. the results showed that the rate of actin polimerization depended on the leiomodin concentration. the nucleator activity of leiomo-din was ionic strength dependent. the data also confirmed that leiomodin is a side-binding and pointed-end binding protein of f-actin. the binding of leiomodin to the sides of the actin filaments was slower than to the pointed-end of the f-actin. the structure of f-actin was changed by the sidebound leiomodin . these observations will contribute to the better understanding of the development and function of thin filaments in cardiac and other muscle tissues. leukemias are one of the most common malignancy worldwide. there is a substantial need for new chemotherapeutic drugs effective against these diseases. doxorubicin (dox), used for treatment of leukemias and solid tumors is poorly efficacious when administered systemically at conventional doses. therefore, in our study, to overcome these limitations, we used transferrin (trf) as a drug carrier. we compared the effect of dox and doxorubicin-transferrin conjugate (dox-trf) on human leukemic lymphoblasts (ccrf-cem). the in vitro growth-inhibition test, xtt assay, indicated that dox-trf was more cytotoxic for leukemia cells than dox alone. in our researches we also evaluated the alternations of mitochondrial transmembrane potential (dw m) , and production of reactive oxygen species (ros). we monitored the dw m using dye jc- ( , ', , '-tetrachloro- , ', , '-tetraethylbenzimidazolcarbocyanine) . the level of ros was studied using the fluorescent probe dcfh -da ( ', 'dichlorodihydrofluorescein diacetate). the results demonstrate that dox-trf induced, decrease of mitochondrial membrane potential and significantly higher production of ros compared with dox treated cells. moreover, all these results seem to be correlated with dna fragmentation analyzed by dna ladder. the tested processes were partially inhibited by the antioxidant, n -acetylocysteine (nac). the changes induced by dox-trf conjugate and free drug were suggest the different mechanism of action of dox alone and conjugated with transferrin. time-resolved detection of protein-protein interaction masahide terazima kyoto university, kyoto, - , japan revealing molecular mechanism of a protein reaction has been a central issue in biophysics. for that purpose, a variety of time-resolved spectroscopic methods have been developed. however, most of them can monitor only dynamics associated with an optical transition and it has been very difficult to trace processes without optical transition. we used the pulsed laser induced transient grating (tg) method to study spectrally silent reactions of various proteins in time-domain. here we will show studies on pixd. pixd is a kda short protein which consists of the bluf domain and additional short helices, and is involved in phototactic movement. the photochemical reaction studied by absorption spectroscopy revealed that this protein exhibits typical photochemistry of the bluf proteins. the red-shifted intermediate is generated within a ps. the spectrum does not change after this initial reaction, and returns back to the dark state with a time constant of s at room temperature. we studied the reaction of this protein by our method and found that the proteinprotein interaction is drastically changed during the reaction. the details and the biological meaning will be presented. human ileal bile acid-binding protein (i-babp) plays a key role in the enterohepatic circulation of bile salts. previously we have shown that the protein has two binding sites and triand dihydroxy bile salts bind with strong and moderate positive cooperativity, respectively. positive cooperativity is thought to be related to a slow conformational change in the protein. our current study is directed at the structural and dynamic aspects of molecular recognition in human i-babp using nmr spectroscopy and other biophysical techniques. as a first step in the investigation, n relaxation nmr experiments have been employed to characterize the backbone motion in the apo and holo protein on a wide range of timescales. our results show a moderately decreased ps-ns flexibility in the ligated protein, with most significant ordering near the portal region. in addition, the measurements indicate a slow ls-ms fluctuation at four distinct segments in the apo protein, a motion not observed in the doubly-ligated form at room temperature. our studies support the hypothesis of an allosteric mechanism of binding cooperativity in human i-babp. to shed more light on the molecular details of the binding mechanism, a site-directed mutagenesis study is in progress. cationic porphyrin-peptide conjugates were recently shown to enhance the delivery of peptide moiety to the close vicinity of nucleic acids but their interaction with dna is not yet studied. we synthesized two cationic porphyrin-peptide conjugates: tetra-peptides were linked to the tri-cationic meso-tri( -n-methylpyridyl)-mono-( -carboxyphenyl)porphyrin and bi-cationic meso- , -bis( -n-methylpyridyl)- , di-( -carboxyphenyl)porphyrin. dna binding of porphyrins, and their peptide conjugates was investigated with comprehensive spectroscopic methods. evidences provided by the decomposition of absorption spectra, fluorescence decay components, fluorescence energy transfer and cd signals reveal that peptide conjugates of di-and tricationic porphyrins bind to dna by two distinct binding modes which can be identified as intercalation and external binding. the peptide moiety does not oppose the interaction between the dna and cationic porphyrins. we compared the effect of complexation on structural stability of dna and nucleoprotein complex : hela nucleosomes and t phage. uv and cd melting studies revealed that the porphyrin binding increases the melting temperature of dna and destabilizes the dna protein interaction in the nucleosomes but not in the t phage. the wide nucleotide specificity of -phosphoglycerate kinase (pgk) allows its contribution to the effective phosphorylation (activation) of nucleotide-based pro-drugs against hiv. here the structural basis of the nucleotide-pgk interaction is characterised in comparison to other kinases, namely pyruvate kinase (pk) and creatine kinase (ck) by enzyme kinetic and structural modelling studies. the results evidenced favouring the purine vs. pyrimidine base containing nucleotides for pgk rather than for pk or ck. this is due to the exceptional ability of pgk in forming the hydrophobic contacts of the nucleotide rings that assures the appropriate positioning of the connected phosphate chain for catalysis. the unnatural l-configurations of the nucleotides (both purine and pyrimidine) are better accepted by pgk than either by pk or ck. further, for the l-forms the absence of the ribose oh-groups with pgk is better tolerated for the nucleotides with purine rather than pyrimidine base. on the other hand, positioning the phosphate chain of both purines and pyrimidines with l-configuration is even more important for pgk, as deduced from the kinetic studies with various nucleotide-site mutants. these characteristics of the kinase-nucleotide interactions can provide a guideline in drug-design. the role of the enzyme types atp-ases in the muscle contraction g. vincze-tiszay , j. vincze , e. balogh hheif, budapest, hungary, nové zá mky, slovakia the myofibrilla assuring muscle contraction gains energy to the slipping in mechanisms and the degree of efficiency of this process will decisively be determined by the velocity of recombination of the atp molecule. in this there play a particular part the na + -k + -atp-ase and mg ++ -atp-ase enzymes. chemical reactions taking place in the living organism are catalyzed by enzymes, so the recombination from adp to atp, too. this transport process can be modelled from the energetic point of view on the basis of the general transport theorem through the following formula: grad a x dx dt: from the point of view of muscle contraction it is of interest that, dependent from the type of the motions whether the length of time is very short, some seconds, or we can speak about a long lasting process. in the first case one can compare the decomposition of the atp with the avalanche effect while in the spot. its degree of efficiency is determined by the migration and linkage velocity of the ions. conclusion: the degree of efficiency of the muscle contraction is determined by the quantities of the two enzymes (na + -k + -atp-ase and mg ++ -atp-ase) as related to each other. [ ] . experiments with deletion mutants have shown that the aminoterminal domain contains a beta sheet with an ordered array of acidic residues, which mediates the attachment to basic calcium phosphates [ , ] . the inhibition is based on the formation of nanometer-sized, spherical mineral-fetuin-a colloids, denoted as calciprotein particles (cpps) [ , ] . the initially formed cpps show hydrodynamic radii in the range of nm and are only transiently stable. after a distinct lag time, they are subject to a morphological change towards larger prolate ellipsoids with hydrodynamic radii of - nm [ ] . in this context, we studied the role of fetuin-a in the formation and ripening of cpps. on the one hand, dynamic light scattering (dls) was used to study the influence of temperature, fetuin-a concentration and mineral ion supersaturation on the kinetics of cpp ripening [ ] . on the other hand, the protein fetuin-a was investigated by means of small angle x-ray scattering (saxs) and fluorescence correlation spectroscopy (fcs degradation of the mrna cap (mrna ' end) by dcps (decapping scavenger) enzyme is an important process of the gene expression regulation, but little is known about its mechanism. the biological role of dcps and its potential therapeutic applications, e.g. as a novel therapeutic target for spinal muscular atrophy, make it an interesting object for biophysical investigations. the ability of dcps to act on various short capped mrnas will be presented. we have examined the substrate specificity and binding affinity of the enzyme in a quantitative manner, employing experimental physics' resources, such as atomic force microscopy (afm) and fluorescence spectroscopy for enzyme kinetics and timesynchronized-titration method (tst ) . in this study we extended the application of mqd-ihc to investigate potential biomarkers associated with prostate cancer (pca) invasiveness and lethal progression. objectives: to establish a mqd-ihc protocol using qd light-emitting nanoparticles ) to detect the expression/activation of critical cell signaling proteins at the single cell level; ) to image the plasticity and lethal progression of human pca with specific emphasis on emt and c-met signaling; and ) to examine the utility of mqd-ihc in clinical pca specimens to determine its invasion ability and predict its metastatic capability. results: we analyzed the co-expression and activation of osteomimicry associated biomarkers: b -microlgobulin (b -m), phosphorylated cyclic amp responsive element binding protein (pcreb) and androgen receptor (ar) in , cells from localized pca tissue areas (gleason and ) of patients with known metastatic status. the overall median % triple positive for b -m + /pcreb + /ar + cells was . %. the median triple positive for the samples with metastatic potential was % compared with those without metastatic potential (median = %); p = . by a wilcoxon rank sum test. the results were confirmed in pca bone metastatic specimens. we also investigated the c-met signaling in castration-resistant human pca model or crpc xenografts and the clinical pca specimens and found that the downstream signal components including pakt and mcl- were activated. conclusion: to validate our findings, additional clinical specimens with confirmed survival data will be analyzed and the cell-signaling-network-based mqd-ihc will be automated by vectra image analysis system in a high throughput manner with the hope to predict the lethal progression of pca prior to clinical manifestation of distant metastases. protein ligand binding is an important field of biopharmaceutical research. there are many techniques for quantitative determination of the ligand binding. the combination of isothermal titration calorimetry (itc) and thermal shift assay provides a robust estimate of the binding constant. many binding reactions are coupled to the absorption or release of protons by the protein or the ligand, conformational changes of the protein and other processes. to correlate the structural features of binding with the energetics of binding one needs to carry out a detailed thermodynamic study of the binding reaction and to determine dependencies such as ph, buffer and temperature. here we present a detailed thermodynamic description of radicicol binding to human heat shock protein hsp and determined proton linkage contributions to observed binding thermodynamics. we calculated the pk a of the group responsible for proton linkage, the protonation enthalpy of this group and intrinsic thermodynamic parameters for radicicol binding. the intrinsic enthalpy of radicicol binding to hsp is one of the largest enthalpies observed for any protein -ligand binding. the structural features responsible for such large binding enthalpy and very favorable intrinsic binding gibbs free energy are discussed. neuronal systems and modelling o- optogenetic electrophysiology walther akemann, amelie perron, hiroki mutoh, and thomas knö pfel laboratory for neuronal circuit dynamics, riken brain science institute, japan the combination of optical imaging methods with targeted expression of protein-based fluorescent probes enables the functional analysis of selected cell populations within intact neuronal circuitries. we previously demonstrated optogenetic monitoring of electrical activity in isolated cells, brain slices and living animals using voltage-sensitive fluorescent proteins (vsfps) generated by fusing fluorescent proteins with a membrane-integrated voltage sensor domain. however, several properties of these voltage reporters remained suboptimal, limiting the spatiotemporal resolution of vsfpbased voltage imaging. a major limitation of vsfps had been a reduced signal-to-noise ratio arising from intracellular aggregation and poor membrane targeting upon long-term expression in vivo. to address this limitation, we generated a series of enhanced genetically-encoded sensors for membrane voltage (named vsfp-butterflies) based on a novel molecular design that combines the advantageous features of vsfp s and vsfp s with molecular trafficking strategies. the new sensors exhibit faster response kinetics at subthreshold membrane potentials and enhanced localization to neuronal plasma membranes after long-term expression in vivo, enabling the optical recording of action potentials from individual neurons in single sweeps. vsfp-butterflies provide optical readouts of population activity such as sensoryevoked responses and neocortical slow-wave oscillations with signal amplitudes exceeding % dr/r in anesthetized mice. vsfp-butterflies will empower optogenetic electrophysiology by enabling new type of experiments bridging cellular and systems neuroscience and illuminating the function of neural circuits across multiple scales. opsin molecules are a burgeoning new tool for temporally precise neuronal stimulation or inhibition. opsin properties are commonly characterized in cell culture or acute brain slice preparations using whole cell patch clamp techniques, where neuronal membrane voltage is fixed at the resting potential. however, in vivo, where neurons are firing action potentials, opsins are exposed to large fluctuations in membrane voltage and transmembrane ionic concentrations which can influence opsin function. in the case of implanted light delivery devices, stimulation light power varies as a function of brain tissue volume. we therefore investigated the stability of opsin properties across a variety of in vivo-like stimulation conditions. we find that off-kinetics of excitatory opsins vary significantly with holding membrane potential; channelrhodopsin (chr ) slowing with depolarisation and chief (chr /chr hybrid) in contrast, accelerating. new chr point mutation variants demonstrate stability across all membrane potentials. we additionally explore responses to initial and subsequent light pulses and find that chief has the unique property of accelerating kinetics after the first light stimulation. inhibitory opsins vary in sensitivity to light in a manner which correlates with their off-kinetics. slower opsins, such as mac (leptosphaeria maculans), have higher sensitivity at low light power densities, saturating early relative to fast inhibitory opsins such as arch (archaeorhodopsin) and nphr (halorhodopsin). we discuss the relative merits of stability versus versatility of opsins under variable stimulation conditions. it has been previously shown that overexpression of ndm ncrna in a sknbe -derived neuroblastoma (nb) cell line leads to cell differentiation, with a decrease of malignant potential. here we use the patch-recording technique to characterize the ionic channel apparatus of nb cells expressing ndm at its basal level (mock cells) or at . fold higher levels (s cells). the two cell lines shared very similar pools of functional k channels, but s cells displayed larger ttx-sensitive na currents and were able to generate action potentials, while mock cells were not. in addition, while mock cells barely express functional gaba receptors, in the majority of s rapid application of gaba elicited a current with a ec = . lm; this current was antagonized by bicuculline ( lm) and potentiated by zaleplon (ec = nm). in mock cells, real time pcr evidenced a high level of gaba a a subunit, while in s cells a significant expression of a and a was detected, whereas a mrna was downregulated by %, confirming the development of functional gaba a receptors. in the same cell lines, the presence of specific markers and the secretion of specific cytokines confirmed that ndm expression leads to a differentiation process toward a neuron-like, rather than glial-like, phenotype. was planned therefore to reconstitute a model of brain tumors in rats by orthotopic implantation of xenogenic transformed human cells. iron is important element used for chemical reaction catalysis and physiological cell functions. the reason of iron deposition is still unknown. under conditions prevailing in human brain it is expected the formation of an amorphous or minute crystalline phase. we used light, scanning (sem) and transmission electron microscopy (tem), energy-dispersive microanalysis, electron diffraction and electron paramagnetic resonance (epr) for investigation of iron deposits in globus pallidus of human brain. sem revealed iron rich particles with na, si, p, s, cl, ca and cu around glial cells. tem revealed bumpy, solid particles of platy and sometimes rounded shape with the size of lm to lm. these ones were identified as hematite. epr measurements showed the presence of fe(iii) and cu(ii), but little amount of fe(ii) can not be excluded. we consider low-temperature process of hematite formation in human globus pallidus in aqueous environment influenced by organic and inorganic factors. chemical processes leading to nanoparticles formation can be associated with neurodegenerations such as alzheimer or parkinson disease. over the past years our understanding of the basic biophysical mechanisms governing the spatio-temporal dynamics of neuronal membrane potentials and synaptic efficacies has significantly expanded and improved. much research has focussed on how ionic currents contribute to the generation and propagation of action potentials, how subthreshold signals propagate along dendritic trees, how the active properties of dendrites shape the integration of incoming signals in a neuron, and how pre-and postsynaptic activities â and potential heterosynaptic effects a determine the way synaptic efficacies change on the short-and long-term. yet, despite these advances, there have been no systematic efforts to relate the basic dynamical repertoire of neurons to the computational challenges neural circuits face, and in particular to explain systematically how the biophysical properties of neurons are adapted to process information efficiently under the constraints of noise and uncertainty in the nervous system. as an initial step in this direction, i will show how various biophysical properties of neurons, in particular short-term synaptic plasticity and dendritic non-linearities, can be seen as adaptations to resolve an important bottleneck in neuronal information processing: the loss of information entailed by the conversion of analogue membrane potentials to digital spike trains. the optogenetic toolbox has greatly expanded since the first demonstration of genetically-targeted optical manipulation of neural activity. in addition to the cation channel channelrhodopsin- (chr ), the panel of excitatory opsins now includes an array of chr variants with mutations in critical residues, in addition to other, related cation channels, and channel hybrids. the inhibitory opsin panel has similarly expanded beyond the first-described halorhodopsin (nphr), a chloride pump, to include trafficking-enhanced versions of nphr as well as the proton pumps mac and arch. while the expansion of available opsins offers researchers an increasingly powerful and diverse selection of tools, it has also made it increasingly difficult to select the optimal tool for a given experiment. one cannot extract a comparison of opsins from the current literature, since studies differ across multiple variables known to contribute to opsin performance (e.g. expression method, light power density, stimulation protocols, etc.). here, we provide the first empirical comparison of both excitatory and inhibitory opsins under standardized conditions. furthermore, we identify the set of parameters that describe the properties of an opsin in a way that is maximally relevant for biological application. o- subcellular compartment-specific distribution of voltage-gated ion channels zoltan nusser institute of experimental medicine, hungarian academy of sciences, budapest, hungary voltage-gated na + (nav) channels are essential for generating the output signal of nerve cells, the action potential (ap). in most nerve cells, aps are initiated in the axon initial segment (ais). in vitro electrophysiological and imaging studies have demonstrated that dendritic nav channels support active backpropagation of aps into the dendrites, but the subunit composition of these channels remained elusive. here, i will present evidence for the somato-dendritic location of nav channels in hippocampal pyramidal cells (pcs). using a highly sensitive electron microscopic immunogold localization technique, we revealed the presence of the nav . subunit in pc proximal and distal dendrites, where their density is -fold lower than that found in aiss. a gradual decrease in nav . density along the proximo-distal axis of the dendritic tree was also detected. we have also investigated the subcellular distribution of kv . voltage-gated k + channel subunit and found a somato-dendritic localization. in contrast to that of nav . channels, the density of kv . first increases then decreases as a function of distance from the somata of pcs. such subcellular compartment-specific distribution of voltage-gated ion channels increases the computational power of nerve cells. keywords: memory, extra cellular matrix, random walk we expose first a biological model of memory based on one hand of the mechanical oscillations of axons during action potential and on the other hand on the changes in the extra cellular matrix composition when a mechanical strain is applied on it. due to these changes, the stiffness of the extra cellular matrix along the most excited neurons will increase close to these neurons due to the growth of astrocytes around them and to the elastoplastic behavior of collagen. this will create preferential paths linked to a memory effect. in a second part, we expose a physical model based on random walk of the action potential on the array composed of dendrites and axons. this last model shows that repetition of the same event leads to long time memory of this event and that paradoxical sleep leads to the linking of different events put into memory. myelinated nerve fibres were studied with fluorescent microscopy and laser interference microscopy. ca + redistribution during prolonged stimulation, changes in morphology and rearrangement of cytoplasmic structures were compared in normal conditions and after membrane modification by lysolecithin and methyl-b-cyclodextrin. lysolecithin is a detergent known to provoke demyelination, and methyl-b-cyclodextrin extracts cholesterol from membranes. cholesterol extraction could lead to disruption of membrane caveolae-like microdomains or ''rafts'' and solubilisation of different proteins connected to them. our data suggest that methyl-b-cyclodextrin and lysolecithin lead to different changes in morphology and distribution of cytoplasmic structures. the effect was different for different regions of the nerve (node of ranvier, paranodal and internodal regions). the agents also altered the kinetics of ca + response to stimulation in myelinated fibres. extracellular carbonic anhydrase contributes to the regulation of ca + homeostasis and salivation in submandibular salivary gland nataliya fedirko, olga kopach, nana, voitenko lviv national university, human and animal physiology, lviv, ukraine the maintenance of ph in the oral cavity is important for the oral heath since even a minor drop in ph can result in dental caries and damage to the teeth. submandibular salivary gland (smg) is main source of fluid and electrolytes enriched saliva therefore its core for oral ph homeostasis. smg secretion is activated by acetylcholine (ach) in [ca + ] idependend manner and accompanied with oral ph acidic shifts. ph shifts could be due to changes in buffering capacity that is regulated by carbonic anhydrase (ca). despite the expression of different subtypes of ca in smg the role of ca in the regulation of smg function is unclear yet. we found that ca inhibition by benzolamide (bz) decreased of fluid secretion in vivo extracellular na + concentration in situ. the latter confirm the ability of ca to modify both primarily and final saliva secretion. we also found correlation between the secretion and ca + -homeostasis since bz-induced decrease of: in striated muscle ca + release from the sarcoplasmic reticulum (sr) occurs when ryanodine receptors (ryr-s) open either spontaneously or upon the stimulation from dihydropyridine receptors that are located in the adjacent transverse-tubular membrane and change their conformation when the cell is depolarized. recent observations demonstrated that muscles from animal models of ptdinsp phosphatase deficiency suffer from altered ca + homeostasis and excitation-contraction coupling, raising the possibility that ptdinsp-s could modulate voltage-activated sr ca + release in mammalian muscle. the openings of a single or a cluster of ryr-s can be detected as ca + release events on images recorded from fibres loaded with fluorescent ca + indicators. to elucidate the effects of ptdinsp-s on ca + release events, images were recorded from skeletal muscle fibers enzymatically isolated from the m. flexor digitorum breavis of mice utilizing a super-fast scanning technique. a wavelet-based detection method was used to automatically identify the events on the images. three different ptdinsp-s (ptdins p, ptdins p, and ptdins( , )p) were tested. all these ptdinsp compounds decreased the frequency of spontaneous ca + release events. supported by the hungarian national science fund (otka ), té t. calcium sparks elicited by mmol/l caffeine and by a depolarization to - mv were recorded at high time resolution on both x-y ( frames/s) and line-scan images ( lines/ ms) on intact skeletal muscle fibers of the frog. while a typical spark appeared in one frame only, . and . % of spark positions overlapped on consecutive frames following caffeine treatment or depolarization, respectively. while both caffeine and depolarization increased the frequency of sparks, as estimated from x-y images, the morphology of sparks was different under the two conditions. both the amplitude (in df/f ; . ± . vs. . ± . ; n = vs. ; mean ± sem, p \ . ) and the full width at half maximum (in lm; parallel with fiber axis: . ± . vs. . ± . ; perpendicular to fiber axis: . ± . vs. . ± . ) of sparks was significantly greater after caffeine treatment than on depolarized cells. these observations were confirmed on sparks identified in line-scan images. in addition, x-t images were used to analyze the time course of these events. calcium sparks had significantly slower rising phase under both conditions as compared to the control. on the other hand, while the rate of rise of signal mass was decreased after depolarization, it increased in the presence of caffeine. prolonged depolarisation of skeletal muscle cells induces entry of extracellular calcium into muscle cells, an event referred to as excitation-coupled calcium entry. skeletal muscle excitation-coupled calcium entry relies on the interaction between the , dihydropyridine receptor on the sarcolemma and the ryanodine receptor on the sarcoplasmic reticulum membrane. in this study we exploited tirf microscopy to monitor with high spatial resolution excitationcoupled calcium entry (ecce) in primary coltures of human skeletal muscle cells harbouring mutation in the ryr gene linked to malignant hyperthermia and central core disease. we found that excitation-coupled calcium entry is strongly enhanced in cells from patients with central core disease compared to individuals with malignant hyperthermia and controls. in addition, excitation-coupled calcium entry induces generation of reactive nitrogen species and causes the nuclear translocation of nfatc . the activation of nfatc dependent genes is consistent with an increase of the il secretion from primary coltures human myotubes from ccd patients and with fibre type predominance of skeletal muscle of ccd patients. membrane lipids, microdomains & signalling p- ftir and calorimetric investigation of the effects of trehalose and multivalent cations on lipid structure sawsan abu sharkh, jana oertel, and karim fahmy division of biophysics, institute of radiochemistry, helmholtz-zentrum dresden-rossendorf, germany e-mail: s.sharkh@hzdr.de the structure of membrane lipids is of fundamental importance for the integrity of cell and organelle membranes in living organisms. membrane lipids are typically hydrated and their headgroup charges counter-balanced by solvated ions. consequently, water loss can induce severe structural changes in lipid packing (lyotropic transitions) and can lead to the damage of lipid membranes even after rehydration. this can be one out of several factors that affect the viability of organisms undergoing desiccation. many organisms, however, are resistant to even extreme water loss. some of them synthesize trehalose which has been shown to be associated with survival of desiccation in phylogenetically diverse organisms (yeast, nematodes, brine shrimp, insect larvae, resurrection plants, and others). here we have studied hydration sensitive transitions in model lipids to determine the effect of trehalose and electrostatics on lipid order. hydration pulse-induced time-resolved fourier-transform infrared (ftir) difference spectroscopy was used to address hydration-dependent lipid structure as a function of trehalose. in combination with differential scanning calorimetry and studies of langmuir-blodget films we arrive at a structural and energetically consistent picture of how trehalose can affects lipidic phase behaviour and support a native lipid structure under water loss. experiments were performed on model lipids with different headgroups and native lipids from desiccation-tolerant organisms. controlled self-assembly and membrane organization of lipophilic nucleosides and nucleic acids: perspectives for applications martin loew , paula pescador , matthias schade , julian appelfeller , jü rgen liebscher , oliver seitz , daniel huster , andreas herrmann , and anna arbuzova humboldt universitä t zu berlin, institute of biology/ biophysics, berlin, germany, humboldt universitä t zu berlin, institute of chemistry, berlin, germany, universitä t leipzig, department of medical physics and biophysics, leipzig, germany lipophilic conjugates of nucleosides and nucleic acids such as dna, rna, and peptide nucleic acid (pna) -combining assembly properties of amphiphiles and specific molecular recognition properties of nucleic acids -allow numerous applications in medicine and biotechnology. we recently observed self-assembly of microtubes, stable cylindrical structures with outer diameters of nm and - lm and a length of - lm, from a cholesterol-modified nucleoside and phospholipids. morphology and properties of these microtubes and functionalization with lipophilic dna will be characterized. we also observed that lipophilic nucleic acids, pna and dna differing in their lipophilic moieties, partition into different lipid domains in model and biological membranes as visualized by hybridization with respective complementary fluorescently-labeled dna strands. upon heating, domains vanished and both lipophilic nucleic acid structures intermixed with each other. reformation of the lipid domains by cooling led again to separation of membrane-anchored nucleic acids. by linking specific functions to complementary strands, this approach offers a reversible tool for triggering interactions among the structures and for the arrangement of reactions and signaling cascades on biomimetic surfaces. conformational dependent trafficking of p-glycoprotein with rafts zsuzsanna gutayné tó th, orsolya bá rsony, katalin goda, gá bor szabó and zsolt bacsó university of debrecen, mhsc, department of biophysics and cell biology, debrecen, hungary p-glycoprotein (pgp), an abc-transporter playing a prominent role in multidrug resistance, demonstrate conformationdependent endocytosis on the surface of t -mdr cells. these cell surface transporters have a uic conformationsensitive-antibody-recognizable subpopulation, which is about one-third of the rest persisting long on the cell surface and perform fast internalization via rafts. we have identified that the rapid internalization is followed by quick exocytosis, in which the other subpopulation is not or only slightly involved. the exocytosis presents a cholesterol depletion dependent intensification, in contrast to the internalization, which is inhibited by cyclodextrin treatment. this continuous recycling examined by total internal reflection (tirf) microscopy increases the amount of the raft associated subpopulation of pgps in the plasma membrane, and it might have a role in restoring the cholesterol content of the membrane after cholesterol depletion. our presentation will summarize related endocytotic, exocytotic and recycling processes and that how does our data fit into our current notions regarding to the cholesterol and sphingomyelin trafficking. membrane nanodomains based on phase-segregating lipid mixtures have been emerged as a key organizing principle of the plasma membrane. they have been shown to play important roles in signal transduction and membrane trafficking. we have developed lipid-like probes carrying multivalent nitrilotriacetic acid (tris-nta) head groups for selective targeting of histagged proteins into liquid ordered or liquid disordered phases. in giant unilamellar vesicles strong partitioning of tris-nta lipids into different lipid phases was observed. for a saturated tris-nta lipid, at least -fold preference for the liquid ordered phase was found. in contrast, an unsaturated nta lipid shows a comparable preference for the liquid disordered phase. partitioning into submicroscopic membrane domains formed in solid supported membranes was confirmed by superresolution imaging. single molecule tracking of his-tagged proteins tethered to solid supported membranes revealed clear differences in the diffusion behavior of the different nta-lipids. by using bsa as a carrier, multivalent nta lipids were efficiently incorporated into the plasma membrane of live cells. based on this approach, we established versatile methods for probing and manipulating the spatiotemporal dynamics of membrane nano domains in live cells. il- is a multifunctional cytokine with pleiotropic effects on t cells. the il- r consists of the cytokine-specific a-subunit and the c c -chain shared with other cytokines, including il- and - , important regulators of t cells. we have previously shown the preassembly of the heterotrimeric il- and il- r, as well as their participation in common superclusters with mhc glycoproteins in lipid rafts of human t lymphoma cells. integrity of lipid rafts was shown to be important in il- signaling. we could hypothesize that other members of the c c cytokine receptor family, such as the il- r complex, may also fulfill their tasks in a similar environment, maybe in the same superclusters. co-localization of il- r with lipid rafts as well as with the il- r/mhc superclusters was determined by clsm. molecular scale interactions of il- ra with il- r and mhc molecules were determined by microscopic and flow cytometric fret experiments. the role of lipid rafts in il- r signaling was assessed by following the effect of cholesterol depletion on il- induced stat phosphorylation. our results suggest the possibility that preassembly of the receptor complexes in common membrane microdomains with mhc glycoproteins may be a general property of c c cytokines in t cells. to unravel the molecular processes leading to fas clustering in lipid rafts, a -mer peptide corresponding to the single transmembrane domain of the death receptor was reconstituted into model membranes that display liquid-disordered/ liquid-ordered phase coexistence, i.e. mimicking cells plasma membranes. using the intrinsic fluorescence of the peptide two tryptophans residues (trp and trp ), fas membrane lateral organization, conformation and dynamics was studied by steady-state and time-resolved fluorescence techniques. our results show that the fas has preferential localization to liquid disordered membrane regions, and that it undergoes a conformational change from a bilayer inserted state in liquid-disordered membranes to an interfacial state in liquidordered membranes. this is a result of the strong hydrophobic mismatch between the (hydrophobic) peptide length and the hydrophobic thickness of liquid-ordered membranes. in addition, we show that ceramide, a sphingolipid intimately involved in fas oligomerization and apoptosis triggering, does not affect fas membrane organization. overall, our results highlight ceramide role as an enhancer of fas oligomerization, and unravel the protective function of fas transmembrane domain against non-ligand induced fas apoptosis. organization and dynamics of membrane-bound bovine a-lactalbumin: a fluorescence approach arunima chaudhuri and amitabha chattopadhyay centre for cellular and molecular biology, hyderabad , india e-mail: amit@ccmb.res.in many soluble proteins are known to interact with membranes in partially disordered states, and the mechanism and relevance of such interactions in cellular processes are beginning to be understood. interestingly, apo-bovine alactalbumin (bla), a soluble protein, specifically interacts with negatively charged membranes and the membranebound protein exhibits a molten globule conformation. we have used the wavelength-selective fluorescence approach to monitor the molten globule conformation of bla upon binding to negatively charged membranes as compared to zwitterionic membranes. tryptophans in bla exhibit differential red edge excitation shift (rees) upon binding to negatively charged and zwitterionic membranes, implying differential rates of solvent relaxation around the tryptophan residues. our results utilizing fluorescence anisotropy, lifetime and depth analysis by the parallax approach of the tryptophans further support the differential organization and dynamics of the membrane-bound bla forms. in addition, dipole potential measurements and dye leakage assays are being used in our ongoing experiments to explore the mechanism of bla binding to membranes. these results assume significance in the light of antimicrobial and tumoricidal functions of a-lactalbumin. role of long-range effective protein-protein forces in the formation and stability of membrane protein nano-domains nicolas destainvill laboratoire de physique thé orique, université paul sabatier toulouse -cnrs, toulouse, france we discuss a realistic scenario, accounting for the existence of sub-micro-metric protein domains in plasma membranes. we propose that proteins embedded in bio-membranes can spontaneously self-organize into stable small clusters, due to the competition between short-range attractive and intermediate-range repulsive forces between proteins, specific to these systems. in addition, membrane domains are supposedly specialized, in order to perform a determined biological task, in the sense that they gather one or a few protein species out of the hundreds of different ones that a cell membrane may contain. by analyzing the balance between mixing entropy and protein affinities, we propose that protein sorting in distinct domains, leading to domain specialization, can be explained without appealing to preexisting lipidic micro-phase separations, as in the lipid raft scenario. we show that the proposed scenario is compatible with known physical interactions between membrane proteins, even if thousands of different species coexist. lipid rafts are cholesterol and sphingolipid-enriched functional microdomains present in biomembranes. rafts have been operationally defined as membrane fractions that are detergent insoluble at low temperature. here we have characterized drms from erythrocytes treated with the nonionic detergents brij and brij , at °c and °c, and compared them to drms obtained with triton x- (tx ). we have also investigated the effect of cholesterol depletion in drms formation. brij drms were enriched in cholesterol as well as tx drms. hptlc analysis showed a very similar distribution of phosphatidylcholine-pc, phosphatidylethanolamine-pe and sphingomyelin-sm in brij drms to that found in ghost membranes. sm-enriched drms were obtained only with tx while pe content was decreased in tx drms, in comparison to brij drms. immunoblot essays revealed that rafts markers (flotillin- and stomatin) were present in all drms. contrary to tx drms, analysis of electron paramagnetic resonance spectra (with -doxyl stearate spin label) revealed that brij drms are not in the liquid-ordered state, evincing the differential extraction of membrane lipids promoted by these detergents. supported by fapesp/cnpq (brazil). several biological membrane mimics have been built to investigate the topology of molecules in membranes. among them ''bicelles'', i.e., mixtures of long-chain and short-chain saturated phospholipids hydrated up to %, became very popular because they orient spontaneously in magnetic fields. disk-shaped systems of - nm diameter and - nm thickness have been measured by electron microscopy and solid state nmr and can be oriented by magnetic fields with the disc-plane normal perpendicular to the field. we have been developing recently lipids that contain in one of their chains a biphenyl group (tbbpc) affording an orientation parallel to the magnetic field, in the absence of lanthanides. a large number of hydrophobic molecules including membrane proteins have been successfully embedded and static nmr afforded finding the orientation of protein helices in membranes; mas nmr provided the d structure of peptides in bicelles. biphenyl bicelles keep their macroscopic orientation for days outside the field, thus leading to combined nmr and x-rays experiments. tbbpc allows also construction of lm vesicles showing a remarkable oblate deformation in magnetic fields (anisotropy of - ) and opens the way to applications for structural biology or drug delivery under mri. imaging membrane heterogeneities and domains by super-resolution sted nanoscopy christian eggeling, veronika mueller, alf honigmann, stefan w. hell department of nanobiophotonics, max planck institute for biophysical chemistry, am fassberg , gö ttingen, germany cholesterol-assisted lipid and protein interactions such as the integration into lipid nanodomains are considered to play a functional part in a whole range of membrane-associated processes, but their direct and non-invasive observation in living cells is impeded by the resolution limit of [ nm of a conventional far-field optical microscope. we report the detection of the membrane heterogeneities in nanosized areas in the plasma membrane of living cells using the superior spatial resolution of stimulated emission depletion (sted) far-field nanoscopy. by combining a (tunable) resolution of down to nm with tools such as fluorescence correlation spectroscopy (fcs), we obtain new details of molecular membrane dynamics. sphingolipids or other proteins are transiently (* ms) trapped on the nanoscale in cholesterol-mediated molecular complexes, while others diffuse freely or show a kind of hopping diffusion. the results are compared to sted experiments on model membranes, which highlight potential influences of the fluorescent tag. the novel observations shed new light on the role of lipid-protein interactions and nanodomains for membrane bioactivity. ca + controlled all-or-none like recruitment of synaptotagmin- c ab to membranes sune m. christensen, nicky ehrlich, dimitrios stamou bio-nanotechnology laboratory, department of neuroscience and pharmacology, nano-science center, lundbeck foundation center biomembranes in nanomedicine, university of copenhagen, copenhagen, denmark e-mail: ehrlich@nano.ku.dk & stamou@nano.ku.dk synaptotagmin- (syt) is the major ca + sensor that triggers the fast, synchronous fusion of synaptic vesicle with the presynaptic membrane upon ca +-mediated membrane recruitment of the cytosolic c ab domain. the ca +-dependent recruitment of syts c ab domain to membranes has so far been investigated by ensemble assays. here we revisited binding of wild type c ab and different c ab mutants of syt to lipid membranes using a recently developed single vesicle assay. the hallmark of the single vesicle approach is that it provides unique information on heterogeneous properties that would otherwise be hidden due to ensemble averaging. we found that c ab does not bind to all vesicles in a homogenous manner, but in an all-or-none like fashion to a fraction of the vesicles. the fraction of vesicles with bound c ab is regulated by the amount of negatively charged lipids in the membrane as well as by [ca +] . this ca + controlled all-ornone like recruitment of syt to membranes provides a possible explanation for the strongly heterogeneous behavior of the in vitro model system for neuronal membrane fusion. furthermore, heterogeneity in release probability among synaptic vesicles is a critical property in determining the output of a neuronal signaling event. new insights in the transport mechanism of ciprofloxacin revealed by fluorescence spectroscopy mariana ferreira, sílvia c. lopes, paula gameiro requimte, faculdade de ciê ncias da universidade do porto, porto, portugal keywords: fluoroquinolones; liposomes; proteoliposomes; ompf; ciprofloxacin; fluorescence; anisotropy. fluoroquinolones are antibiotics that have a large spectrum of action against gram negative and some gram positive bacteria. the interaction between these species and liposomes has been cited as a reference in the understanding of their diffusion through phospholipid bilayer and can be quantified by the determination of partition coefficients between a hydrophobic phase (liposomes) and an aqueous solution. it is also known that some porins of the bacterial membranes are involved in transport mechanism of many fluoroquinolones. ompf, a well characterized membrane protein characteristic of the outer membrane of gram negative bacteria assumes the conformation of homo-trimer, whose monomers have two tryptophan residues (one located at the interface of monomers and the other at the interface lipid/protein). thus, we proceeded to study the interaction of ciprofloxacin, a second generation fluoroquinolone, with unilamellar liposomal vesicles and ompf proteoliposomes of pope/popg, pope/popg/cardiolipin and e. coli total. partition coefficients (kp's) and the association with ompf proteoliposomes were determined by steady state fluorescence spectroscopy under physiological conditions (t= °c; ph . ). the membrane mimetic systems used were characterized by dls and fluorescence anisotropy. motivation is whether there exist differences in the patternforming capabilities of two adhesion molecules of different roles: cd , mediating ,,dynamic'' adhesion in cell rolling and icam- , mediating ,,static'' adhesion during the formation of immune-synapse. homo-and hetero-associations of cd , icam- and the mhci is investigated on the nm-and lmdistance levels on ls t colon carcinoma cells in two different conditions of lymphocyte homing: ( ) with ifnc and tnfa, both lymphokines up-regulating the expression level of mhci and icam- and down-regulating that of cd . ( ) crosslinking of cd and icam- representing receptor engagement. the observations are explained by assuming the existence of a kinase cascade-level crosstalk between the cd and icam- molecules which manifests in characteristic complementary changes in the properties of cell surface receptor patterns. for the characterisation of cluster morphology new colocalization approaches were developed: (i) ,,number of first neighbours'' distribution curves, (ii) ,,acceptor photobleaching fret-fluorescence intensity fluctuation product'' correlation diagrams, and (iii) ,,random gradient-kernel smoothing assisted decay'' of pearson-correlations, and (iv) k-function formalism. analyzing janus kinases in living cells with single-color fcs using confocal and tir-illumination thomas weidemann , hetvi gandhi , remigiusz worch , , robert weinmeister , christian bö kel , and petra schwille biophysics research group, technische universitä t dresden, germany, crtd, center for regenerative therapies dresden, technische universitä t dresden, germany, institute of physics, polish academy of science, warsaw, poland cytokine receptors of the hematopoietic superfamily transduce their signal through non-covalently bound janus kinases. there are only such kinases in humans (jak , , and tyk ), which associate to different cytokine receptor chains. here we study the dynamics of gfp-tagged jak and jak in epithelial cells with fluorescence correlation spectroscopy (fcs). jak and jak behave differently in various aspects: in the absence of receptors, jak still binds the membrane, whereas jak diffuses homogeneously in the cytoplasm. we used fcs under total internal reflection illumination (tir-fcs) and determined the membrane binding affinity of jak to be ± nm. the association of jak with the common gamma chain (c c ) is very tight as shown by fluorescence recovery after photobleaching (frap). molecular brightness analysis of single-point fcs shows that jak diffuses as a monomer in the rather small cytoplasmic pool, whereas jak diffuses as dimers, which undergo a defined oligomerization. the degree of oligomerization decays at higher concentrations, indicating that some unknown, saturable scaffold is involved. characterizing the binding and mobility schemes of the janus kinases may be important to further elucidate their specific and redundant effects in signal transduction. plasma membrane (pm)-enriched fraction obtained through subcellular fractioning protocols are commonly used in studies investigating the ability of a compound to bind to a receptor. however, the presence of mitochondria membranes (mi) in the pm-enriched fraction may compromise several experimental results because mi may also contain the interest binding proteins. aiming to analyze the subcellular fractioning quality of a standard sucrose density based protocol, we investigated (a) the na + k + -atpase (pm marker) and succinate dehydrogenase -sd (mi marker) activities; (b) the immunocontent of the adenine nucleotide translocator (ant -mi membrane marker) in both pm-and mi-enriched fractions. since several binding protocols may require long incubation period, we verified the quality of both fractions after hours of incubation in adequate buffer. our results show that pmand mi-enriched fractions exhibit contamination with mi or pm, respectively. we did not observe any effect of incubation on na + k + -atpase activity and ant content in both fractions. surprisingly, sd activity was preserved in the pm-but not in mi-enriched fraction after incubation. these data suggest the need of more careful use of pm-enriched fraction preparation in studies involving pm proteins characterization. human neutrophil peptide (hnp ) is a human cationic defensin that present microbial activity against various bacteria (both gram-positive and negative), fungi and viruses. hnp is stored in the cytoplasmic azurophilic granules of neutrophils and epithelial cells. in order to elucidate the mode of action of this antimicrobial peptide (amp), studies based on its lipid selectivity were carried out. large unilamellar vesicles (luv) with different lipid compositions were used as biomembranes model systems (mammal, fungal and bacterial models). changes on the intrinsic fluorescence of the tryptophan residues present in hnp upon membrane binding/insertion were followed, showing that hnp have quite distinct preferences for mammalian and fungal membrane model systems. hnp showed low interaction with glucosylceramide rich membranes, but high sterol selectivity: it has a high partition for ergosterol-containing membranes (as fungal membranes) and low interaction with cholesterolcontaining membranes (as in mammalian cells). these results reveal that lipid selectivity is the first step after interaction with the membrane. further insights on the hnp membrane interaction process were given by fluorescence quenching measurements using acrylamide, -doxylstearic acid ( ns) or ( ns). nanoparticles (np) are currently used in many industrial or research applications (paints, cosmetics, drug delivery materials…). recent papers demonstrate clearly their activity with biological membranes (nanoscale holes, membrane thinning, disruption). different studies of the np-membrane interaction suggest that parameters are particularly important, such as the np size, their surface properties or their aggregation state. composition of biological membranes being particularly complex, supported lipid bilayers (slb) composed of a restricted number of lipids are usually used as simplified membrane model. moreover, these two-dimensional systems are convenient for surface analysis techniques, such as atomic force microscopy (afm), giving information on the morphology of the slb and its mechanical properties. in this work, we study the behaviour of slbs made of lipids representative of the membrane fluid phase (popc) or of the raft phase (sphingomyelin). these slbs are deposited on planar surfaces (mica or glass) previously recovered with silica beads ( or nm in diameter) in order to mimick the np-membrane interaction. we will present in this work our first results obtained by afm and fluorescence microscopy. it is well known that the eukaryotic nuclei are the sphere of lipids active metabolism. the investigations demonstrated the existence of numerous enzymes in nuclei which modulate the changes of nuclear lipids during different cellular processes. although the nuclear membrane is accepted as the main place of the lipids localization, nearly % of nuclear lipids are discovered in chromatin fraction. the ability of chromatin phospolipids to regulate dna replication and transcription was already demonstrated. the chromatin phospolipids seems to play an important role in cell proliferation and differentiation as well as in apoptosis. it seems also possible that chromatin phospholipids may be participated in realization of cisplatin antitumor effects. the -hour in vivo effect of cisplatin on rat liver chromatin phospholipids was investigated. the phospholipids of rat liver chromatin were fractionated by microtlc technique. the quantitative estimation of fractionated phospholipids was carried out by computer program fugifilm science lab. image gauge v . . the alteration of total phospholipids content as well as the quantitative changes among the individual phospholipids fractions in rat liver chromatin after in vivo action of cisplatin was established. the total content of chromatin phospholipids was significantly decreased after the cisplatin action. four from five individual phospholipids fractions were markedly changed after the drug action. two cholin-content phospholipids, particularly phosphatidylcholine and sphingomyelin exhibit diversity in sensitivity to this drug: the increase of sphingomyelin content accompanied by quantitative decrease of phosphatidylcholine. the quantity of cardiolipin was markedly increased while the amount of phosphatidylinositol was decreased after the cisplatin treatment. the phosphatidylethanolamin content remained unchanged after the drug action. it seems that high sensitivity of chromatin phospholipids exhibited to cisplatin action may play an important role in antitumor effects of this drug. membrane lipids and drug resistance in staphylococci r. d. harvey institute of pharmaceutical science, king's college london, stanford street, london se nh, uk staphylococci express numerous resistance mechanisms against common antimicrobials, including peptide components of the innate immune system which have been trumpeted as being likely candidates to replace our increasingly ineffective antibiotics. the membrane phospholipid lysylphosphatidylglycerol (l-pg) appears to play a key role in staphylococcal drug resistance, since its absence in mutant bacteria renders them susceptible to a range of cationic antimicrobials. the current assumption about the role l-pg plays in drug resistance is that of facilitating charge neutralisation of the plasma membrane, leading to loss of affinity towards cationic moieties. we have investigated this phenomenon using a range of model membrane systems composed of both synthetic lipids and reconstituted natural lipid extracts, using such techniques as stopped-flow fluorescence, circular dichroism and neutron scattering. our conclusions indicate that the initial assumptions about the role of l-pg in drug resistance are over-simplistic and certainly do not tell the whole story of the physical and biological properties of this fascinating moderator or membrane behaviour. our findings show that l-pg does not inhibit antimicrobial drug action by charge dampening, hinting at a different protective mechanism. modulation of a-toxin binding by membrane composition m. schwiering, a. brack, c. beck, h. decker, n. hellmann institute for molecular biophysics, university of mainz, mainz, germany although the alpha-toxin from s. aureus was the first poreforming toxin identified, its mode of interaction with membranes is still not fully understood. the toxin forms heptameric pores on cellular and artificial membranes. the present hypothesis is that the initial binding to the membrane occurs with low affinity, and that an efficient oligomerisation, relying on clusters of binding sites, is the reason for the overall high affinity of the binding process. in order to separate the effects of increasing concentration of binding sites from this topological effect, we investigated the oligomer formation based on pyrene-fluorescence for a series of lipid compositions, where the fraction of toxin binding lipids (egg phospatidylcholine (epc) or egg sphingomyelin (esm)) was varied while their concentration remained constant. the results indicate that an increased local density of toxin binding sites occurring due to phase separation facilitates oligomer formation. furthermore, the change in local environment (number of neighboring cholesterol molecules) upon domain formation also enhances oligomer formation.we thank the dfg (sfb ) for financial support, s. bhakdi and a. valeva for production of the toxin and helpful discussions. we explored quercetin effects on lipid bilayers containing cholesterol using a spectrofluorimetric approach. we used the fluorescent probe laurdan which is able to detect changes in membrane phase properties. when incorporated in lipid bilayers, laurdan emits from two different excited states, a non-relaxed one when the bilayer packing is tight and a relaxed state when the bilayer packing is loose. this behavior is seen in recorded spectra as a shift of maximum emission fluorescence from nm at temperatures below lipids phase transition to nm at temperatures above lipids phase transition values. emission spectra of laurdan were analyzed as a sum of two gaussian bands, centered on the two emission wavelengths allowing a good evaluation of the relative presence of each population. our results show that both laurdan emission states are present with different shares in a wide temperature range for dmpc liposomes with cholesterol. quercetin leads to a decrease in the phase transition temperature of liposomes, acting in the same time as a quencher on laurdan fluorescence. this paper is supported by the sectorial operational programme human resources development, financed from the caveolins are essential membrane proteins found in caveolae. the caveolin scaffolding domain of caveolin- includes a short sequence containing a crac motif (v tkywfyr ) at its c-terminal end. to investigate the role of this motif in the caveolin-membrane interaction at the atomic level, we performed a detailed structural and dynamics characterization of a cav- (v -l ) nonapeptide encompassing this motif and including the first residue of cav- hydrophobic domain (l ), in dodecylphosphocholine (dpc) micelles and in dmpc/dhpc bicelles, as membrane mimics. nmr data revealed that this peptide folded as an amphipathic helix located in the polar head group region. the two tyrosine sidechains, flanked by arginine and lysine residues, are situated on one face of this helix, whereas the phenylalanine and tryptophan side-chains are located on the opposite face (le lan c. et al., , eur. biophys. j., , - ). to investigate the interactions between the crac motif and the lipids, we performed molecular dynamics simulations in two different environment: a dpc micelle and a popc bilayer. the results obtained are in good agreement with nmr data and the comparison between both systems provided insight into the orientation of the crac motif at the membrane interface and into its interactions with lipids. this work was partially supported by the strategic grant posdru/ / . our study suggests that conformation and positioning of hydroxyl groups significantly affects thermotropic properties of sphingolipids and sterol interaction. the polymorphism of a new series of bolaamphiphile molecules based on n-( -betainylamino-dodecane)-octyl b-d-glucofuranosiduronamide chloride is investigated. the length of the main bridging chain is varied in order to modify the hydrophilic/lipophilic balance. the other chemical modification was to introduce a diacetylenic unit in the middle of the bridging chain to study the influence of the pp stacking on the supramolecular organisation of these molecules. dry bolaamphiphiles self-organize in supramolecular structures such as lamellar crystalline structure, lamellar fluid structure and lamellar gel structure. the thermal dependence of these structures, as well as the phase transition is followed by smallangle and wide-angle x-ray scattering. once the thermal cycle is accomplished, the system remains in the kinetically stabilized undercooled high-temperature phase at temperature of °c. subsequently, the time dependence of the relaxation to the thermodynamically stable phase is followed and very slow relaxation on the order of hours or days is observed. the study of polymorphism and the stability of various phases of this new series of bolaamphiphiles is interesting for potential application in health, cosmetics or food industry, is undertaken in this work. alkylphospholipids have shown promising results in several clinical studies and among them perifosine (opp) is promising for breast cancer therapy. antitumor effect was much better in estrogen receptor negative (er-) than in estrogen receptor positive (er+) tumors in vivo. it is believed that apl do not target dna, but they insert in the plasma membrane and ultimately lead to cell death. liposomes made of opp and different amount of cholesterol (ch) showed diminished hemolytic activity as compared to micellar opp, but in most cases cytotoxic activity was lower. in order to find optimal liposomal composition and to understand better the difference in the response of er+ and er-cells the interaction opp liposomes with er+ and er-cells was studied. for liposomes with high amount of ch both cell types showed slow release of the liposome entrapped spin probe into the cytoplasm. liposmes with low amount of ch interact better with cells but the release is faster for er-as for er+ cells at °c. experiments with nitroxide-labeled opp (sl-opp) liposomes suggest that the exchange of sl-opp between liposomes and cellular membranes is fast. however, translocation of sl-opp across the plasma membrane is slow, but seems to be faster for opp resistent, er+ cells as for er-cells at °c. estimation of a membrane pore size based on the law of conservation of mass krystian kubica , artur wrona and olga hrydziuszko institute of biomedical engineering and instrumentation, wroclaw university of technology, wroclaw, poland, centre for systems biology, university of birmingham, birmingham, uk the size of biomembrane pores determines which solutes or active compounds may enter the cell. here, using a mathematical model of a lipid bilayer and the law of conservation of mass, we calculate the radius of a membrane pore created by rearranging the lipid molecules (the pore wall was formed out of the lipid heads taken from the membrane regions situated directly above and below the pore, prior its formation). assuming a constant number of lipid molecules per bilayer (with or without the pore) and based on the literature data ( % decrease in the area per chain for a fluid-to-gel transition and a matching change of one chain volume not exceeding %) we have shown that the pore radius can measure up to . nm (for a nm thick lipid bilayer) without the lipid molecules undergoing a phase transition. a further assumption of area per chain being modified as a consequence of the lipids conformational changes has resulted in an increase of the calculated radius up to . nm. finally, a comparison of the pore volume with the corresponding volume of the lipid bilayer has led to a conclusion that for the system under consideration the membrane pore can only be created with the lipids undergoing fluidto-gel conformational changes. the key signaling pathway involves tyrosine phosphorylation of signal transducers and activators of transcription (stat and stat ) by receptor-associated janus kinases. we aim to unveil of the very early events of signal activation including ligand-induced receptor assembly and the recruitment of the cytoplasmic effector proteins stat and stat in living cells. to this end, we have explored the spatiotemporal dynamics of stat recruitment at the membrane on a single molecule level. highly transient interaction of stats to membrane-proximal sites was detected by tirf-microscopy, allowing for localizing and tracking individual molecules beyond the diffraction limit. thus, we obtained a pattern of the spatio-temporal recruitment of stat molecules to the plasma membrane revealing distinct submicroscopic structures and hotspots of stat interaction with overlapping recruitment sites for stat and stat . strikingly, these stat binding sites were independent on receptor localization and expression level. simultaneous superresolution imaging of the cytoskeleton revealed the organization of stat recruitment sites within the cortical actin skeleton. characterization of molecular dynamics on living cell membranes at the nanoscale level is fundamental to unravel the mechanisms of membrane organization andcompartmentalization. we have recently demonstr ated the feasibility of fluorescence correlation spectroscopy (fcs) based on the nanometric illumination of near-field scanning optical microscopy (nsom) probes on intact living cells [ ] . nsom-fcs was applied to study the diffusion of fluorescent lipid analogs on living cho cells. the experiments allowed to reveal details of the diffusion hidden by larger illumination areas and associated with nanoscale membrane compartmentalization. the technique also offers the unique advantages of straightforward implementation of multiple color excitation, opening the possibility to study nanoscale molecular cross-correlation. furthermore, the nsom probe evanescent axial illumination allows to extend diffusion study to the membrane-proximal cytosolic region. as such, nsom-fcs represents a novel powerful tool to characterize the details of many biological processes in which molecular diffusion plays a relevant role. the growing interest in supported lipid bilayers (slbs) on conductive substrates, such as gold, is due to the possibility of designing lipid-based biosensor interfaces with electrochemical transduction. due to the hydrophobicity of gold it is still a challenge to deposit planar and continuous bilayers without previous surface modification. most studies on gold concern single-phase slbs without cholesterol or gangliosides, two vital components of biomembranes. in this work the experimental conditions suitable for the formation of complex slbs with phase-separation directly on gold are exploited. the mixtures dopc/dppc/cholesterol ( : : ) with or mol % of ganglioside gm , which should yield lipid raft-like domains according to reported phase diagrams, were studied. slb with lipid rafts were successfully formed onto bare au ( ), although surface modification with -mercapto-undecanoic acid sam stabilized the slbs due to its charge and hydrophilicity. the different experimental conditions tested had an impact on nano/microdomains organization observed by atomic force microscopy in buffer solution. surface characterization through the combined use of ellipsometry, cyclic voltammetry and afm allowed to optimize the conditions for the formation of more planar and compact slbs. it is widely accepted that the conversion of the soluble, nontoxic amyloid b-protein (ab) monomer to aggregated toxic ab rich in b-sheet structures is central to the development of alzheimer's disease. however, the mechanism of the abnormal aggregation of ab in vivo is not well understood. we have proposed that ganglioside clusters in lipid rafts mediate the formation of amyloid fibrils by ab, the toxicity and physicochemical properties of which are different from those of ab amyloids formed in solution [ , ] . in this presentation, we report a detailed mechanism by which ab-( - ) fibrillizes in raft-like lipid bilayers composed of gm /cholesterol/sphingomyelin. at lower concentrations, ab formed an a helix-rich structure, which was cooperatively converted to a b sheet-rich structure above a threshold concentration. the structure was further changed to a seed-prone b structure at higher concentrations. the seed recruited ab in solution to form amyloid fibrils. [ hepatitis c virus (hcv) has a great impact on public health, affecting more than million people worldwide since it is the cause of liver-related diseases such as chronic hepatitis, cirrhosis and hepatocarcinoma. hcv entry into the host cell is achieved by the fusion of viral and cellular membranes, replicates its genome in a membrane associated replication complex, and morphogenesis has been suggested to take place in the endoplasmic reticulum (er) or modified er membranes. the variability of the hcv proteins gives the virus the ability to escape the host immune surveillance system and notably hampers the development of an efficient vaccine. hcv has a single-stranded genome which encode a polyprotein, cleaved by a combination of cellular and viral proteases to produce the mature structural proteins (core, e , e , and p ) and non-structural ones (ns , ns , ns a, ns b, ns a and ns b), the latter associated with the membrane originated from the er in the emerging virus. the ns b protein, a fundamental player in the hcv replicative process and the least characterized hcv protein, is a highly hydrophobic protein associated with er membranes. it has recently been shown that the c-terminal is palmitoylated and the n-terminal region has potent polymerization activity. the expression of ns b induces the formation of the so called membranous web, which has been postulated to be the hcv rna replication complex. thus, a function of ns b might be to induce a specific membrane alteration that serves as a scaffold for the formation of the hcv replication complex and therefore has critical role in the hcv cycle. due to the high hydrophobic nature of ns b, a detailed structure determination of this protein is very difficult. the ns b protein is an integral membrane protein with four or more transmembrane domains. the c-terminal region of ns b is constituted by two a helices, h (approximately from amino acid to ) and h (approximately from amino acid to ), which have been studied as potential targets for inhibiting hcv replication. previous studies from our group, based on the study of the effect of ns b peptide libraries on model membrane integrity, have allowed us to propose the location of different segments in this protein that would be implicated in lipid-protein interaction. additionally, the h region could be an essential constituent in the interaction between protein and membrane. in this study we show that peptides derived from the c-terminal domain of ns b protein of hcv are able to interact with high affinity to biomembranes, significantly destabilizing them and affecting their biophysical properties. there were also differences in the interaction of the peptide depending on the lipid composition of the membranes studied. we have also applied fluorescence spectroscopy, infrared spectroscopy and differential scanning calorimetry which have given as a detailed biophysical study of the interaction of the peptide with model biomembranes. this work was supported by grant bfu - -bmc (ministerio de ciencia y tecnología, spain) to j.v. a semi-quantitative theory describing the adhesion kinetics between soft objects as living cells or vesicles was developed. the nucleation-like mechanism has been described in the framework of a non-equilibrium fokker-planck approach accounting for the adhesion patch growth and dissolution (a. raudino, m. pannuzzo, j. chem. phys. , ( )). a well known puzzling effect is the dramatic enhancement of the adhesion/fusion rate of lipid membranes by water-soluble polymers that do not specifically interact with the membrane surface. we extend the previous approach by molecular dynamics simulations in the framework of a coarse-grained picture of the system (lipid+polymer+ions embedded in an explicit water medium) in order to test and support our previous analytical results. simulations show that the osmotic pressure due to the polymer exclusion from the inter-membrane spacing is partially balanced by an electrostatic pressure. however, we also evidenced an interesting coupling between osmotic forces and electrostatic effects. indeed, when charged membranes are considered, polymers of low dielectric permittivity are partially excluded from the inter-membrane space because of the increased local salt concentration. the increased salt concentration means also a larger density of divalent ions which form a bridge at the contact region (stronger adhesion). the overall effect is a smaller membrane repulsion. this effect disappears when neutral membranes are considered. the model could explain the fusion kinetics between lipid vesicles, provided the short-range adhesion transition is the rate-limiting step to the whole fusion process. the role of ceramide acyl chain length and unsaturation on membrane structure sandra n. ceramide fatty acid composition selectively regulates distinct cell processes by a yet unknown mechanism. however, evidence suggests that biophysical processes are important in the activation of signalling pathways. indeed, ceramide strongly affects membrane order, induces gel/fluid phase separation and forms highly-ordered gel domains. the impact of ceramide n-acyl chain in the biophysical properties of a fluid membrane was studied in popc membranes mixed with distinct ceramides. our results show that: i) saturated ceramide has a stronger impact on the fluid membrane, increasing its order and promoting gel/fluid phase separation, while their unsaturated counterparts have a lower (c : ceramide) or no (c : ceramide) ability to form gel domains at physiological temperature, ii) differences between distinct saturated species are smaller and are related mainly to domain morphology, and iii) very long chain ceramide induces the formation of tubular structures probably associated with interdigitation. these results suggest that generation of different ceramide species in cell membranes has a distinct biophysical impact with acyl chain saturation dictating membrane lateral organization, and chain asymmetry governing interdigitation and membrane morphology. extra high content of cholesterol (chol) in fiber-cell membranes in the eye lens leads to chol saturation and formation of cholesterol bilayer domains (cbds). it is hypothesized that high enrichment in cholesterol helps to maintain lens transparency and protect against cataractogenesis. in model studies, the cbd is formed in a phospholipid bilayer when cholesterol content exceeding the cholesterol solubility threshold, thus, the cbd is surrounded by phospholipid bilayer saturated with cholesterol. in the present study, we carried out molecular dynamics (md) simulation of two bilayers: a palmitoyloleoylphosphatidylcholine (popc) bilayer (reference) and a : popc-chol bilayer, to investigate the smoothing effect of the saturating amount of cholesterol on the bilayer. to our knowledge, this effect has not been studied so far so this study is certainly providing new results. our results indicate that saturation with cholesterol significantly narrows the distribution of vertical positions of the center-of-mass of the popc molecules and the popc atoms in the bilayer and smoothes the bilayer surface. we hypothesize that this smoothing effect decreases light-scattering and helps to maintain lens transparency. the phospholipid content of staphylococcus aureus membranes displays a high degree of variability ( - ). the major phospholipids found in s. aureus are phosphatidylglycerol (pg), cardiolipin (cl) and lysylphosphatidylglycerol (lpg), ( ) the concentrations of which are environment dependent and see fluctuations on exposure to high concentrations of positively charged moieties ( ) . up regulation of lpg has a suspected role in neutralisation of the plasma membrane in response to cationic threats. studies have been conducted to probe biomimetic models of this theory however our focus is to look at atomic details of membrane extracts in the presence of magaininf w. s. aureus lipid extracts from cells grown at ph . and . , studies by neutron diffraction with and without peptide at two contrasts. d-spacings were assessed by vogt area fitting and bragg's law. bilayer separation at low ph was * - Å less than ph . . with peptide, bilayer separations of ph . and . extracts were reduced by * Å and * Å , respectively. reduced pg content of low ph extracts is suggested to reduce d-spacing, however presence of peptide further reduces this, possibly by an anion neutralisation effect. abnormal d-spacing on increased humidity may be due to the breakdown of lpg. activation of neutrophils releasing hocl and apoptosis of vein endothelial cells are the events documented to occur in the course of atherosclerosis. as lipid chlorohydrins, which are the key products of the reaction between hocl and unsaturated fatty acid residues, were found in atherosclerotic plaques, we decided to check their biological activity in the context of their ability to act as the mediators of hoclinduced oxidative stress and apoptosis in the culture of immortalized human umbilical vein endothelial cells (hu-vec-st). the concentration of reactive oxygen species was found to be elevated after h cell incubation with phospahtidylcholine chlorohydrins. this effect was at least partially caused by the leakage of superoxide anion from mitochondria and followed by depletion of gsh and total thiols. the significant decrease of antioxidant capacity of cell extracts was also observed. the intracellular red-ox imbalance was accompanied by the increase of the ratio between phosphorylated and dephosphorylated forms of p map kinase. after longer incubation a significant number of apoptotic cells appeared. summing up, phosphatidylcholine chlorohydrins may be regarded as signaling molecules, able to initiate signalling pathways by induction of oxidative stress. giant unilamellar vesicles (guvs) are a valuable tool in the study of lateral distribution of biological membrane components. guv dimensions are comparable to typical cell plasma membranes and lipid phase separation can be observed through fluorescence microscopy. guv studies frequently require immobilization of the vesicles, and several methods are available for that effect. one of the most common methodologies for vesicle immobilization is the use of avidin/streptavidin coated surfaces and biotin labeled lipids at very low concentration in the vesicles. here, we analyze the effect of using this methodology on lipid domain distribution for different lipid compositions. we show that as a result of non-homogeneous distribution of biotin labeled lipids between liquid disordered, liquid ordered and gel phases, distribution of lipid domains inside guvs can be dramatically affected. monitoring membrane permeability: development of a sicm approach christoph saßen , and claudia steinem institute for organic and biomolecular chemistry, university of gö ttingen, tammannstraße , gö ttingen, germany, ggnb doctoral program: imprs, physics of biological and complex systems scanning ion conductance microscopy (sicm) utilises a nanopipette containing an electrode as a probe for surface investigations with resolutions of / of the inner pipette diameter. experiments are conducted under physiological conditions, in situ and without mechanical contact of probe and sample. hence, sicm serves as a well-suited technique for the investigation of soft objects such as cells or artificial lipid membranes. using pore-suspending membranes (psm) as a model system, interactions of melittin as an example for cell penetrating peptides (cpps) and lipid membranes are investigated by means of sicm. formation of a range of solvent free psm from lipid vesicles has been achieved as confirmed by means of fluorescence microscopy and sicm. application of melittin results in rupturing of the lipid bilayer. putative insights gained from this assay are critical concentrations of membrane permeabilising ccps and answers to mechanistic questions, e.g. whether ccps translocate only or form pores within the lipid bilayer. positioning of the z-ring in escherichia coli prior to cell division is regulated by intracellular pole-to-pole oscillation and membrane binding of min proteins, allowing assembly of ftsz filaments only at the center plane of the cell. in order to investigate the influence of membrane geometry on the dynamic behavior of membrane binding of min proteins, we combined concepts of synthetic biology and microfabrication technology. glass slides were patterned by a gold coating with microscopic windows of different geometries, and supported lipid bilayers (slb) were formed on these microstructures. on slbs, min-proteins organize into parallel waves. confinement of the artificial membranes determined the direction of propagation.min-waves could be guided along curved membrane stripes, in circles and even along slalom-geometries. in elongated membrane structures, the protein waves always propagate along the longest axis. coupling of protein waves across spatially separated membrane patches was observed, dependent on gap size and viscosity of the aqueous media above the bilayer. this indicates the existence of an inhomogeneous and dynamic protein gradient above the membrane. minimal systems for membrane associated cellular processes petra schwille bio technology center biotec, technical university of dresden, germany the strive for identifying minimal biological systems, particularly of subcellular structures or modules, has in the past years been very successful, and crucial in vitro experiments with reduced complexity can nowadays be performed, e.g., on reconstituted cytoskeleton and membrane systems. in this overview talk, i will first discuss the virtues of minimal membrane systems, such as guvs and supported membranes, in quantitatively understand protein-lipid interactions, in particular lipid domain formation and its relevance on protein function. membrane transformations, such as vesicle fusion and fission, but also vesicle splitting, can be reconstituted in these simple subsystems, due to the inherent physical properties of selfassembled lipids, and it is compelling question how simple a protein machinery may be that is still able to regulate these transformations. as an exciting example of the power of minimal systems, i show how the interplay between a membrane and only two antagonistic proteins from the bacterial cell division machinery can result in emergence of protein self-organization and pattern formation, and discuss the possibility of reconstituting a minimal divisome. quantitative microscopic analysis reveals cell confluence regulated divergence of pdgfr-initiated signaling pathways with logically streamlined cellular outputs Á rpá d szö } or, lá szló ujalky-nagy, já nos szö ll} osi, gyö rgy vereb university of debrecen, department of biophysics and cell biology, debrecen, hungary platelet derived growth factor receptors (pdgfr) play an important role in proliferation and survival of tumor cells. pdgf-bb stimulation caused a redistribution of pdgf receptors towards gm rich domains, which was more prominent in confluent monolayers. pdgf-bb stimulation significantly increased relative receptor phosphorylation of the ras / mapk pathway specific tyr residues and the pi -kinase / akt pathway specific tyr residues in nonconfluent cultures. tyr residues that serve as adaptors for ras-gap which inactivates the mapk pathway and tyr residues feeding into the plc-gamma / camk-pkc pathway were the docking sites significantly hyperphosphorylation following ligand stimulation in confluent cells. we found that p-akt facilitated cell survival and pmapk dependent proliferation is more activated in dispersed cells, while phospholipase c-gamma mediated calcium release and pkc-dependent rhoa activation are the prominent output features pdgf stimulus achieves in confluent cultures. these observations suggest that the same stimulus is able to promote distinctly relevant signaling outputs, namely, cell division and survival in sparse cultures and inhibition of proliferation joined with promotion of migration in confluent monolayers that appear contact inhibited. a thermodynamic approach to phase coexistence in ternary cholesterol-phospholipid mixtures jean wolff, carlos m. marques and fabrice thalmann institut charles sadron, université de strasbourg, cnrs upr, , rue du loess, strasbourg cedex, f- , france e-mail: thalmann@ics-cnrs.unistra.fr we present a simple and predictive model for describing the phase stability of ternary cholesterol-phospholipid mixtures. assuming that competition between the liquid and gel organizations of the phospholipids is the main driving force behind lipid segregation, we derive a phenomenological gibbs free-energy of mixing, based on the calorimetric properties of the lipids main transition. gibbs phase diagrams are numerically obtained that reproduces the most important experimental features of dppc-dopc-chol membranes, such as regions of triple coexistence and liquid orderedliquid disordered segregation. based on this approach, we present a scenario for the evolution of the phase diagram with temperature. results for other phospholipid species, such as popc or psm will also be presented. interleukin- and - receptors play a central role in the activation, survival and death of t lymphocytes. they form supramolecular clusters with mhc i and ii glycoproteins in t cells. in damaged or inflamed tissues the extracellular k+ concentration increases, which can depolarize the membrane. the common signaling beta and gamma chains of il- / r are phosphorylated upon cytokine binding and get a permanent dipole moment, thus their conformation, interactions, mobility and activity may be sensitive to the membrane potential. we induced depolarization on ft . t lymphoma cells by increasing the ec. k+ level or by blocking kv . voltage gated k+ channels with margatoxin. fcs measuremens showed that the lateral mobility of fab-labeled il- / r and mhc i and ii decreased upon depolarization, while that of gpi-linked cd did not change. fret efficiency measured between some elements of the il-receptor/mhc cluster increased, which may reflect an increase of cluster size. il- -induced receptor activity, as monitored by measuring stat -phosphorylation, increased upon depolarization, whereas il- induced phosphorylation did not change. our results may reveal a novel regulatory mechanism of receptor function by the membrane potential. cytokines play an important role in t cell activation and immunological memory, whereas mhcs are known for the role in antigen presentation. we applied rnai to silence the expression of mhc i in order to study its possible role in receptor assembly and function. fret data indicated that the association of il- r and il- r with mhc i as well as between il- r and il- r weakened. fcs indicated an increase of receptor mobility also suggesting the partial disassembly of the clusters. mhc i gene silencing lead to a remarkable increase of il- /il- induced phosphorylation of stat transcription factors. in search for the molecular background of this inhibition of signaling by mhc i we checked il- binding and the formation of the receptor complex (il- r alpha -il- r beta association), but we did not find a difference as compared to the control. our results suggest that mhc i plays an organizing role in maintaining supramolecular receptor clusters and inhibits il- r signaling, revealing a nonclassic new function of mhc i beyond its classical role in antigen presentation. interleukin- (il- ) is an important cytokine involved in adaptive immunity. il- binds with high affinity the singlepass transmembrane receptor il- ra. the occupied complex, il- /il- ra then engages either il- rc or il- ra , to form an activated type i or ii receptor, respectively. this formation of heterodimers is believed to trigger cross-activation of intracellular janus kinases. here we follow a fluorescently labeled ligand through various stages of receptor activation in hek t: using fluorescence correlation spectroscopy (fcs), we see that the receptor chains diffuse as monomers within the plasma membrane. using dual-color fccs provides direct evidence for ligand induced co-diffusion of occupied il- ra and il- ra . in contrast, type i complexes containing il- rc could not be observed. however, ectopic expression of gfp-tagged il- rc/jak induced stable fluorescent speckles in or close to the plasma membrane. we identified these structures as early sorting endosomes by colocalization of surface markers like eea and rab gtpases. the il- ra chain is continuously trafficking into these compartments. these observations suggest that the formation of a type i il- r heterodimer may require internalization and that early endosomes serve as a platform for il- signaling. among the membrane associated proteins, the ras family, which is lipid-anchored g protein, plays a key role in a large range of physiological processes and, more importantly, is deregulated in a large variety of cancer. in this context, plasma membrane heterogeneity appears as a central concept since it ultimately tunes the specification and regulation of ras-dependent signaling processes. therefore, to investigate the dynamic and complex membrane lateral organization in living cells, we have developed an original approach based on molecule diffusion measurements performed by fluorescence correlation spectroscopy at different spatial scales (spot variable fcs, svfcs) ( ). we have shown in a variety of cell types that lipidbased nanodomains are instrumental for cell membrane compartmentalization. we have also observed that thesenanodomains are critically involved in the activation of signaling pathways and are essential for physiological responses ( - ). more recently, weextend the application of svfcs to characterizethe dynamics of k-rasproteinat the plasma membrane. as major result, we demonstrated that the rate of k-ras association/dissociation from the membrane is fast but vary as a functional of the activation state of the molecule as well as of specific intracellular protein interactions. we have so demonstrated that an helical lid sub-domain in the sbd is essential for monomeric as binding, but not for the anti-aggregation activity of the chaperone, suggesting that hsp is able to interact with pre-fibrillar oligomeric species formed during as aggregation and that, then, the mechanism of binding for these species is different from that of the monomeric protein. aggregation of the acylphosphatase from sulfolobus solfataricus (sso acp) into amyloid-like protofibrils is induced by the establishment of an intermolecular interaction between a -residue unfolded segment at the n-terminus and the globular unit of another molecule. we have used data from hydrogen/deuterium exchange experiments, intermolecular paramagnetic relaxation enhancements and isothermal titration calorimetry measurements on an aggregation-resistant sso acp variant lacking the -residue n-terminus to characterize the initial steps of the aggregation reaction. under solution conditions that favour aggregation of the wild-type protein, the truncated protein was found to interact with a peptide corresponding to the n-terminal residues of the full length protein. this interaction involves the fourth strand of the main b-sheet structure of the protein and the loop following this region and induces a slight decrease in protein flexibility. we suggest that the amyloidogenic state populated by sso acp prior to aggregation does not present local unfolding but is characterized by increased dynamics throughout the sequence that allow the protein to establish new interactions, leading to the aggregation reaction. amyloid-like aggregates alter the membrane mobility of gm gangliosides martino calamai and francesco pavone university of florence, lens -european laboratory for non-linear spectroscopy, sesto fiorentino, florence, italy neuronal dysfunction in neurodegenerative pathologies such as alzheimer's disease is currently attributed to the interaction of amyloid aggregates with the plasma membrane. amongst the variety of toxic mechanisms proposed, one involves the binding of amyloid species to gm gangliosides. gm takes part into the formation of membrane rafts, and exerts antineurotoxic, neuroprotective, and neurorestorative effects on various central neurotransmitter systems. in this study, we investigated the effects of amyloid-like aggregates formed by the highly amyloidogenic structural motif of the yeast prion sup (sup nm) on the mobility of gm on the plasmamembrane of living cells. preformed sup nm aggregates were incubated with cells and gm molecules were subsequently labeled with biotinylated ctx-b and streptavidin quantum dots (qds). single qds bound to gm were then tracked. the mobility of gm was found to decrease dramatically in the presence of sup nm aggregates, switching from brownian to mainly confined motion. the considerable interference of amyloid-like aggregates with the lateral diffusion of gm might imply a consequent loss of function of gm , thus contributing to explain the toxic mechanism ascribed to this particular interaction. insights into the early stages of fibrillogenesis of insulin using mass spectrometry harriet l. insulin is a vital hormone in metabolic processes as it regulates the glucose levels in the body. insulin is stored in the b cells of the pancreas as a hexamer, however its biologically active form is the monomer. the formation of fibrillar aggregates of insulin rarely occurs in the body; however localised amyloidosis at the site of injection for diabetes patients and aggregation of pharmaceutical insulin stocks present problems. in the current study oligomers formed early in the process of fibril assembly in vitro are observed by mass spectrometry (ms). ms is the only technique which allows early species to be characterised as it can identify different oligomeric orders by mass to charge ratio and show protein abundance and aggregation propensity. on mobility ms is used to examine rotationally averaged collision cross sections of oligomers in the aggregating solution. a wide array of oligomers is observed and the stability of specific species is remarked. the presence of multiple conformations for the highly charged oligomers is particularly noted and their assignment confirmed using fourier transform ion cyclotron resonance ms and collision induced dissociation. molecular modelling has been used to further explore the conformational space the oligomers inhabit. amyloid fibrils consisting of different proteins have been recognized as an accompanying feature of several neurodegenerative diseases. many proteins without known connection to any diseases have been found to form amyloid fibrils in vitro, leading to suggestion that the ability to form fibrils is the inherent property of polypeptide chain. the observed common character of protein amyloid formation enables to seek further clues of fibrillation mechanism by studying generalized sequenceless polypeptide models, e.g. polylysine. we have studied conformational transitions of polylysine, with different chain length at various ph, ionic strength and temperature by means of novel approachviscometric method. this polypeptide undergoes alfa-helix to beta-sheet transition and forms amyloid fibrils in special conditions. temperature induced a-helix to b-sheet transitions occurs at ph interval form to . and with increasing chain length is slightly shifted to the lower ph. we have found narrow ph interval, in which the thermal transition is fully reversible, suggesting the high sensitivity of polypeptide conformation on subtle changes in charge on its side chains. this work was supported within the projects vega , and , cex sas nanofluid and by esf project no. . understanding the mechanisms of the conversion from the native state of a protein to the amyloidal state represents a fundamental step in improving the purification, storage and delivery of protein-based drugs and it is also of great relevance for developing strategies to prevent in vivo protein aggregation. amyloid fibrils have a structural arrangement of cross b-sheet but they can also experience different packing into three dimensional superstructures, i.e. polymorphism. it is well known that, among others, both the geometric confinement of the molecules and shear forces can affect the final morphology of the aggregates. importantly, due to the complexity and crowding of the cellular region, such parameters also play a crucial role in in vivo processes. we present an experimental approach to study in vitro amyloid aggregation in a controlled and uniform shear force field and within microscale environments. in particular we focus on the effect of these two parameters on the formation of spherical aggregates, known as spherulites. using micro channels of different cross-sections from to lm x lm and flow rates in the range of hundreds of ll/min, the number and diameter of spherulites within the channels have been characterized using crossed polarizers optical microscopy. inhibition of insulin amyloid fibrillization by albumin magnetic fluid k. insulin amyloid aggregation causes serious problems for patient with insulin dependent diabetes undergoing long-term treatment by injection, in production and storage of this drug and in application of insulin pumps. recent studies indicate that protein amyloid aggregation causes the cell impairment and death; however, the prevention of amyloid aggregation is beneficial. we have investigated ability of albumin magnetic fluid (amf) to inhibit insulin amyloid aggregation by spectroscopic and microscopic techniques. albumin magnetic fluid consists of magnetic fe o nanoparticles sterically stabilized by sodium oleate and functionalized with bovine serum albumin (bsa) at various weight ratios bsa/fe o . we have found the positive correlation between inhibiting activity of afm and nanoparticle diameter and zeta potential. the ability of amf to inhibit formation of amyloid fibrils exhibits concentration dependence with ic values comparable to insulin concentration. the observed features make amf of potential interest as agents effective in the solving of problems associated with insulin amyloid aggregation. (this work was supported within the projects vega , and , cex sas nanofluid, apvv- - , sk-ro- - and esf project ). amyloid formation of peptides causes diseases like alzheimer's and parkinson's disease. however, the conditions for the onset of the neurotoxic beta-sheet formation are poorly understood. we focus on aggregation triggers and their interplay: interactions with hydrophobic-hydrophilic interfaces, orientation of peptides in d, metal ion complexation and lipid layers. the tailor-made model peptides exhibit defined secondary structure propensities and metal ion binding sites. the interactions of the peptide with the air-water interface and with metal ions are studied using surface sensitive methods connected to film balance measurements. x-ray diffraction, x-ray reflection, infrared reflection-absorption spectroscopy and total reflection x-ray fluorescence were applied to reveal the layer structure, peptide conformations and metal ion binding at the interface. we found that amyloid formation in d is dominated by the hydrophobic-hydrophilic interface and not comparable to the bulk behaviour. the interface can enhance or inhibit betasheet formation. the effect of metal ion complexation depends on the arrangement of the binding sites in the peptide and the preferred metal complexation geometry. the two triggers interface and metal ion complexation, can oppose each other. effect of apoe isoform and lipidation status on proteolytic clearance of the amyloid-beta peptide hubin, e. alzheimer's disease (ad) is the most common type of dementia in the elderly. the most important genetic risk factor identified for ad is the isoform, e , e or e , of apolipoprotein e (apoe), a lipid-carrying protein. one hallmark of ad is the accumulation of amyloid-beta peptide (ab) in the brain which is thought to result from an imbalance between the production of ab and its clearance. previous studies report an important role for apoe in ab degradation. we sought to determine the effect of apoe isoform and lipidation status on the degradation of soluble ab by proteinases such as insulin-degrading enzyme and neprilysin. in this study an in vitro ab clearance assay based on the competition between ab and a fluorogenic peptide substrate is developed to quantify ab degradation. to elucidate the proteolytic clearance mechanism, the fragments resulting from cleavage are identified by mass spectrometry and further analyzed to identify the interacting stretch of the ab sequence with the different apoe isoforms. the results suggest that apoe influences the rate of ab degradation. the aggregation of proteins into fibrillar nanostructures is a general form of behaviour encountered for a range of different polypeptide chains. the formation of these structures is associated with pathological processes in the context of alzheimer's and parkinson's diseases but is also involved in biologically beneficial roles which include functional coatings and catalytical scaffolds. this talk focuses on recent work directed at understanding the kinetics of this process through the development and application of experimental biophysical methods and their combination with kinetic theories for linear growth phenomena. lbs contains not only as, but also other proteins including - - proteins. - - proteins exist mainly as a dimer and its exact functions are remain unclear. however, recent work has shown that the association of - - (eta) with as in lbs. herein we show how - - (eta) can modulate as in vitro aggregation behavior, by rerouting it toward the formation of stable non-fibrillar aggregates. we also show that the resulting populations of fibrillar and pre-fibrillar aggregates exhibit a modified toxicity in vivo with respect to the unperturbed aggregates. interestingly, - - (eta) does not show any binding affinity for monomeric as, nor for the mature fibrillar aggregates. we provide evidence that it acts on the oligomeric species which form during the amyloidogenesis process of aggregation. since - - (eta) can influence the toxicity of amyloidogenesis without perturbing the functional as monomers, we are convinced that once fully understood, its mode of action could represent a promising model to mimic with synthetic drugs and peptides. what makes an amyloid toxic: morphology, structure or interaction with membranes? more than human diseases are related to amyloids. in order to understand why some amyloids may become toxic to their host and some others are not, we first developed a genetical approach in the yeast saccharomyces cerevisiae. we have chosen the amyloid/prion protein het-s prion forming domain ( - ) from the fungi podospora anserina, which is not toxic in yeast. some toxic amyloids mutants were generated by random mutagenesis. in vitro the most toxic mutant called ''m '' displays very peculiar nanofibers, which polymerized mainly in amyloid antiparallel b-sheets whereas the non-toxic wt exhibits a parallel polymerization. we further established the dynamic of assembling of the m toxic amyloid, in comparison to the wt non-toxic amyloid, and showed the presence of specific oligomeric intermediates also organized in antiparallel b-sheet structures. a more global structure/toxicity study on more than mutants clearly identified an antiparallel b-sheet signature for all the toxic mutants. therefore size, intermediates and antiparallel structures may account for amyloid toxicity in yeast but we still wonder what their cellular targets are. recently, we established the first evidences that toxic mutants may specifically bind in vitro to lipids, particularly negatively charged. interconnected mechanisms in abeta ( - ) we present an experimental study on the fibril formation of ab( - ) peptide at ph . . the kinetics of this process is characterized by the occurrence of multiple transient species that give rise to final aggregates whose morphology and molecular structure are strongly affected by the growth conditions. to observe in details the aggregation pathway as a function of solution conditions, we have used different experimental techniques as light scattering, thioflavin t fluorescence, circular dichroism and two-photon fluorescence microscopy. this approach gives information on the time evolution of conformational changes at molecular level, on the aggregates/fibrils growth and on their morphologies. the selected experimental conditions allowed us to highlight the existence of at least three different aggregation mechanisms acting in competition. a first assembly stage, which implies conformational conversion of native peptides, leads to the formation of small ordered oligomers representing an activated conformation to proceed towards fibril growth. this process constitutes the rate limiting step for two distinct fibril nucleation mechanisms that probably implicate spatially heterogeneous mechanisms. the formation of amyloid fibrils of amylin - was studied by means of molecular dynamics (md) and energy partition on three peptide ß-sheet stack systems with the same amino acid composition: wild type amylin - (amyl - ), reverse amylin - (rev-amyl - ) and scrambled amylin version scr-amyl - . the results show that for amylin - peptides, amino acid composition determines the propensity of a peptide to form amyloid fibrils independent of their sequence. the sequence of amino acids defines the shape and the strength of amyloid protofibril, which conforms with the atom force microscope (afm) data [ ] . md show that the x revamyl- - stack has looser selfassembly than the x amyl- - stack, which conforms with the results of fourier transform infrared spectroscopy (ftir) measurements for the peptides studied [ ] . the results of md show that x amyl- - could have a turn, which consists with ftir data [ ] . data on ab aggregation kinetics have been characterized by a large spread between experiments on identical samples that contain so many molecules that stochastic behaviour is difficult to explain unless caused by uncontrolled amounts of impurities or interfaces. we have therefore spent considerable effort to eliminate sources of inhomogeneity and reached a level of reproducibility between identical samples and between experiments on separate occasions that we can now collect data that can lead to mechanistic insights into the aggregation process per se, and into the mechanism of action of inhibitors. data on ab aggregation will be shown that give insight into the influence of physical parameters like peptide concentration, shear and ionic strength, as well as the effect of inhibitory proteins, model membranes and the effects of sequence variations. monte carlo simulations of amyloid formation from model peptides corroborate the finding from experiments and underscore that the very high level of predictability and reproducibility comes from multiple parallel processes. negatively-charged membranes were reported to catalyze ''amyloid-like'' fiber formation by non-amyloidogenic proteins [ ] . our study aims to elucidate the factors that govern the formation of these amyloid-like fibers. lysozyme was selected as a model of non-amyloidogenic protein and was fluorescently-labeled with alexa fluor (a -lz). first, a -lz partition towards phosphatidylserine-containing liposomes was characterized quantitatively using fluorescence correlation spectroscopy (fcs), in order to calculate the protein coverage of liposomes. secondly, the interaction between a -lz and negatively-charged lipid membranes was studied using both steady-state and time-resolved fluorescence techniques. this interaction was found to switch from a peripheral binding to the anionic headgroups, at high lipid/ protein molar ratio (l/p), to a partial insertion of protein into the hydrophobic core of the membrane, at low l/p. finally, the lipidprotein supramolecular complexes formed at low l/p were characterized by fluorescence lifetime imaging microscopy (flim). the mean lifetime of a -lz in these supramolecular structures is much lower compared to the values obtained for the free and bound a -lz at high l/p. the fiber characterization will be complemented by fcs studies. [ the conversion of normal prp c to its pathological isoform prp sc is a key event in prion diseases and is proposed to occur at the cell surface or more probably in acidic late endosomes. a convergence of evidence strongly suggests that the early events leading to the structural conversion of the prp seem to be in relation with more or less stable soluble oligomers, which could mediate neurotoxicity. as commonly shared by other amyloidogenic proteins, membrane-bound monomers undergo a series of lipid-dependent conformational changes, leading to the formation of oligomers of varying toxicity rich in b-sheet structures (annular pores, amyloid fibrils). here, we have used a combination of biophysical techniques (dynamic and static light scattering, fluorescence techniques, and quartz crystral microbalance) to elucidate the interaction of native monomeric prp and that of purified b-rich oligomeric prp on model lipid membranes. under well established conditions, three b-sheet-rich oligomers were generated from the partial unfolding of the monomer in solution, which were found to form in parallel. from single mutation and/or truncation of the full length prp, the polymerization pathway is strongly affected, revealing the high conformational diversity of prp. in our previous work, we identify the minimal region of the prp protein leading to the same polymerization pattern of the full length prp. soluble -subunits and -subunits oligomers were obtained depending on the single mutation or truncation and purified. we compare their structural properties (ftir, cd) when associated with anionic lipid bilayers and study their propensities to permeabilize the membrane. fluorescence kinetics suggest different mechanisms of membrane perturbation for the monomer and the prp oligomers. deciphering this complex network of lipid interactions and conformational diversity of the prp protein will help for understanding of how amyloidogenic proteins induce neurotoxicity. the traditional view of the lipid bilayer described as a ''sea'' of lipids where proteins may float freely, is going to be inadequate to describe the increasingly large number of complex phenomena which are known to take place in biological membranes. membrane-assisted protein-protein interactions, formation of lipid clusters, protein-induced variation of the membrane shape, abnormal membrane permeabilities and conformational transitions of membrane-embedded proteins are only a few examples of the variegated ensemble of events whose tightly regulated cross-talk is essential for cell structure and function. experimental work on the above mentionated problems is very difficult and some time not accessible, especially when the studied systems have a fast dynamics. due to the large size of the systems usually involved in this multifaceted framework, a detailed molecular description of these phenomena is beyond the possibilities of conventional amyloid aggregation, a generic behavior of proteins, is related to incurable human pathologies -amyloid-related diseases, associated with formation of amyloid deposits in the body. all types of amyloid aggregates possess rich bsheet structural motif. the recent data confirm the toxic effect of aggregates on the cells, however, it was found that reduction of amyloid aggregates plays important role in prevention as well as therapy of amyloidosis. we have investigated effect of phytoalexin derivatives on amyloid fibrillization of two proteins, human insulin and chicken egg lysozyme, by tht and ans fluorescence assays. we have found that amyloid aggregation of both studied proteins was significantly inhibited by phytoalexin derivates cyclobrassinin and benzocamalexin. for most effective phytoalexins the estimated ic values were at low micromolar concentration. the observed inhibiting activity was confirmed by transmission electron microscopy. our data suggest the potential therapeutic use of the most effective phytoalexins in the reduction of amyloid aggregation. (this work was supported within the projects vega , , cex sas nanofluid, apvv- - , sk-ro- - and esf project ). the amyloid pore hypothesis suggests that interactions of oligomeric alpha-synuclein (as) with membranes play an important role in parkinson's disease. oligomers are thought to permeabilize membranes and interfere with ca + pathways. permeabilization by as requires the presence of negatively charged phospholipids. whether as can bind and permeabilize membranes with physiologically relevant lipid compositions has not been extensively explored. here we report on the binding of as to giant unilamellar vesicles (guvs) with physiologically relevant lipid compositions. comparing different protocols of oligomer preparation, leakage assays on both large unilamellar vesicles (calcein release) and guvs (hpts efflux assay) show that as is not able to permeabilize these membranes. the presence of cholesterol has a stabilizing effect on these membrane systems. in agreement with these findings, we do not observe concentration dependent as toxicity using in vivo mts assays. however, in the calcein release assay, different as preparations show differences in kinetics and as concentrations that cause % leakage. these results motivate us to critically reassess the amyloid pore hypothesis, and suggest that membrane permeabilization may be attributable only to a very specific as species. alpha-synuclein oligomers impair membrane integrity-a mechanistic view martin t. stö ckl, mireille m. a. e. claessens, vinod subramaniam nanobiophysics, mesa+ institute for nanotechnology, university of twente, enschede, the netherlands one of the most prevalent neurodegenerative diseases is parkinson's disease (pd), which is accompanied with the loss of dopaminergic neurons. although the mechanisms leading to the death of these cells are still unclear, the protein alpha-synuclein (as) is one of the pivotal factors. previous studies indicate that especially oligomeric forms of as show a detrimental effect on membrane integrity. as an intact membrane is crucial to many cellular processes, the impairment of the membrane integrity is a likely pathway for neuronal death. we use different phospholipid bilayer model systems to investigate the mechanisms underlying this process. atomic force microscopy in combination with suspended asymmetric phospholipid bilayers, which closely mimic the plasma membrane, allows the identification of the binding sites, the measurement of penetration depths of the as oligomers into the phospholipid bilayer, and the detection of membrane thinning or creation of membrane defects. using an approach based on phospholipid vesicles we were able to observe for the first time that as oligomers cause an enhanced lipid-flip flop. suggesting that the loss of lipid asymmetry is a novel mechanism which may contribute to or trigger neuronal death in pd. amyloid protein-membrane interactions and structuretoxicity relationship study h.p. ta (toxic) has a much higher and more specific effect on negatively charged phospholipids (dops, dopi and dopg) than in the case of wt (non-toxic). therefore the insertion of protein into phospholipid monolayers, which occurred similarly for both wt and m , is not a key factor for these effects (h.p. ta et al. langmuir, in press). we are now using unilamellar vesicles as a membrane model to investigate the amyloid protein (toxic and nontoxic) -phospholipid interactions. results confirmed the high specific and strong effects of m on negatively-charged membrane. in this project, we study the chemical, physical and biological properties of fibrillar networks. the formation and the network mechanics are investigated by combining droplet-based microfluidics with optical microscopy and small angle x-ray scattering (saxs). the chosen system, fibrin network formation, plays an important role in blood coagulation processes. crosslinking of fibrinogen induced by an enzymatic reaction with thrombin leads to d fibrin network formation. the fibrillar networks are formed within picoliter droplets of aqueous solutions in an continuous oil phase. droplets containing fibrinogen and thrombin can be produced of different sizes and stored for fibrin network formation. the formation of the fibrillar networks is imaged by fluorescence microscopy. to analyze the elastic properties of the networks, the droplets flow through a microchannel device of alternating width in order to squeeze and stretch the networks. additionally, saxs experiments will give structural information about the molecular dimensions of the networks. the amyloid beta peptide (ab), implicated in alzheimer's disease (ad), is released from the amyloid precursor protein (app) by secretase-induced cleavage. this process results in the release of a range of ab peptides varying in length. the brains of ad patients often contain longer ab peptides while the total concentration of ab is unaffected. longer peptides are more hydrophobic having far-reaching consequences for their toxicity and aggregation. as ab is necessary for normal neuronal function, research activities into ad therapeutic development currently explore the possibilities of modulating c-secretase activity to produce short ab peptides. whether such an approach effectively ameliorates the toxic effect of ab has not been explored yet. to answer this question, we studied the impact of heterogeneity in ab pools in an in vitro biophysical and in cellulo context using microelectrode array to assay the synaptic activity of primary neurons. we show that various lengths of the ab peptide and mixtures thereof aggregate with distinct kinetics and notoriously affect synaptotoxic and cytotoxic response. we also show that small amounts of less abundant peptides ab and ab induce aggregation and toxicity of ab while the behavior of ab is unaffected. one of the most important irreversible oxidative modifications of proteins is carbonylation, a process of introducing the carbonyl group in reaction with reactive oxygen species. importantly, carbonylation increases with the age of cells and is associated with the formation of intracellular protein aggregates and the pathogenesis of age-related disorders such as neurodegenerative diseases and cancer. however, it is still largely unclear how carbonylation affects protein structure, dynamics and aggregability on the atomic level. here, we use classical molecular dynamics simulations to study structure and dynamics of the carbonylated headpiece domain of villin, a key actin-organizing protein. we perform an exhaustive set of molecular dynamics simulations of native villin headpiece together with every single combination containing carbonylated versions of its seven lysine, arginine and proline residues, the quantitatively most important carbonylable amino acids. surprisingly, our results suggest that high levels of carbonylation, far above those associated with cell death in vivo, may be required to destabilize and unfold protein structure through the disruption of specific stabilizing elements, such as salt bridges or proline kinks, or tampering with the hydrophobic effect. on the other hand, by using thermodynamic integration and molecular hydrophobicity potential approaches, we quantitatively show that carbonylation of hydrophilic lysine and arginine residues is equivalent to introducing hydrophobic, chargeneutral mutations in their place, and, by comparison with experimental results, demonstrate that this by itself significantly increases intrinsic aggregation propensity of both structured, native proteins and their unfolded states. finally, our results provide a foundation for a novel experimental strategy to study the effects of carbonylation on protein structure, dynamics and aggregability using site-directed mutagenesis. septins are an evolutionarily conserved family of gtp-binding proteins involved in important cellular processes, such as cytokinesis and exocytosis, and have been implicated in neurological diseases, such as alzheimer's and parkinson's diseases. the focus of this study was two septins of schistosoma mansoni, (the causative agent of schistosomiasis in south america) named smsept and smsept , which were produced in a recombinant system. our objective was to verify if these septins from a simpler organism display similar characteristics to human septins. analysis of protein structure by circular dichroism showed that both recombinant smseptins produced were folded. the gtpase activity assay showed that smsept was able to hydrolyze gtp, whereas smsept was not. aggregation studies for amyloid fibril detection by right angle light scattering and thioflavin t fluorescence assay were performed. both proteins showed a temperature dependent increase in light scattering and fluorescence emission of tht probe. this indicated that s. mansoni septins tend to aggregate into amyloid-like fibers in high temperatures, with thresholds of °c for smsept and °c for smsept . these results are in accordance to that previously reported for human septins. in our work we investigated the response to standard chemotherapy of blood lymphocytes of patients suffering with melanoma. dna single and double strand breaks were determined using comet assay; intracellular levels of marker proteins were detected using immunocytochemistry. ultimately this set of parameters allows to characterize two mechanisms of dna repair (base excision repair, ber and mismatch repair, mmr) which together with apoptosis proneness underlie response of tumor cells to chemotherapy. cell death caused by o mg adducts is promoted by mmr system by inducing unrepaired double strand breaks in dna. there was a linear correlation between the level of dsdna breaks in lymphocytes after -st cycle of chemotherapy and mmr efficiency in them. the level of double strand breaks in dna after -st cycle of chemotherapy is predictive of clinical outcome. otherwise damage at the level of ssdna (ap-sites and single strand breaks) and ber mechanism associated with it couldn't be a good prognostic factor of chemotherapy. high level of double strand breaks in dna in blood lymphocytes of melanoma patients hours after -st cycle of chemotherapy appears to be a marker of a good prognosis. self-assembly and stability of g-quadruplex: counterions, pressure and temperature effects e. baldassarri jr., p. mariani, f. spinozzi, m. g. ortore saifet dept. & cnism, marche polytechnic university, ancona, italy the important role of g-quadruplex in biological systems is based on two main features: composition and stability of telomeres, and activity of telomerase. the g-quadruplex structures are formed by supramolecular organization of basic units called g-quartets that are planar rings constituted by four guanosines linked by hoogsten hydrogen bonds. gquadruplex requires the presence of monovalent cations playing a key role in stabilizing these structures, since they give rise to coordination bonds needed for the stacking of more tetrads. we performed x-ray diffraction experiments at different pressures (ranging from to bar), and small angle x-ray scattering (saxs) changing the temperature (between - °c retinoic acid receptor (rar) is a member of the nuclear receptor superfamily. this ligand-inducible transcription factor binds to dna as a heterodimer with the retinoid x receptor (rxr) in the nucleus. the nucleus is a dynamic compartment and live-cell imaging techniques make it possible to investigate transcription factor action in real-time. we studied the diffusion of egfp-rar by fluorescence correlation spectroscopy (fcs) in order to uncover the molecular interactions determining receptor mobility. in the absence of ligand we identified two distinct species with different mobilities. the fast component has a diffusion coefficient of d = . - lm /s corresponding to small oligomeric forms, whereas the slow component with d = . - . lm /s corresponds to interactions of rar with the chromatin or other large structures. the rar ligand binding domain fragment also has a slow component probably as a result of indirect dna-binding via rxr, with lower affinity than the intact rar:rxr complex. importantly, rar-agonist treatment shifts the equilibrium towards the slow population of the wild type receptor, but without significantly changing the mobility of either the fast or the slow population. by using a series of mutant forms of the receptor with altered dna-or coregulator-binding capacity we found that the slow component is probably related to chromatin binding, and that coregulator exchange, specifically the binding of the coactivator complex, is the main determinant contributing to the redistribution of rar during ligand activation. formation of inactive nuclear with high level of dna compaction in sperm cells is accompanied by a substitution of linker histones h by a number of other proteins. among them sperm-specific histones (ssh), which are characterized by elongated arginine-rich polypeptide chain compared to the somatic h . the secondary and tertiary structure of the ssh and their interactions with dna were studied using spectroscopic and thermodynamic approaches. the histones were isolated from sperm of marine invertebrates and rat thymus. all studied ssh demonstrate no considerable compaction of dna in solutions of low ionic strength. however, at physiological conditions, ssh h from s.intermedius and a.japonica compact dna more intensively than other ssh. the somatic h from rat thymus revealed a minimal ability to compact the dna. we suggest that the ssh h are able to interact with dna not only in the major groove but also in the minor groove of the double helix inducing considerable structural changes in dna and facilitating the formation of the supercompact sperm chromatin. the authors are grateful for the financial support from the russian foundation for basic research (grants § - - and - - ) and from administration of saint-petersburg. ionizing radiation causes modification and destruction of nitrogenous bases in dna molecule. there are also local breakages of hydrogen bonds both in the lesion sites mentioned above and in other sites of the macromolecule. to reveal the amount of some of these damages we applied cd and uv absorption spectroscopy. radiation-induced changes in dna structure influence its uv absorption spectrum in different ways: partial denaturation causes hyperchromic effect, while destruction of the bases results in hypochromic shift. at the same time both of them result in the same changes in dna cd spectra: the decrease in intensity. we attempted to segregate the described damages in dna structure and studied the influence of dna ionic surroundings on the radiation effect. it is shown that the radiation efficiency of base destruction and partial denaturation increases with decreasing concentration of nacl in irradiated solution. udu (ugly duckling) has been first identified from a zebrafish mutant and shown to play an essential role during erythroid development; however, its roles in other cellular processes remain largely unexplored. facs analysis showed that the loss of udu function resulted in defective cell cycle progression and comet assay indicated the presence of increased dna damage in udu mutants. we further showed that the extensive p -dependent apoptosis found in udu mutants is a consequence of activation in the atm-chk pathway. udu appears not to be required for dna repair, because both wild-type and udu embryos similarly respond to and recover from uv treatment. yeast two-hybrid and coimmunoprecipitation data demonstrated that pah-l repeats and sant-l domain of udu interacts with mcm and mcm . furthermore, udu was colocalized with brdu and heterochromatin during dna replication, suggesting a role in maintaining genome integrity. recently, we started to work on the second zebrafish homolog, udu , and its mammalian counterpart, gon l. preliminary data showed that udu and gon l mrna injection can rescue zebrafish udu mutant phenotypes. furthermore, pah-l and sant-l domains of udu and gon l can bind to mcm and mcm and they are localized in the nucleus. these data suggest that udu and gon l are functionally equivalent to zebrafish udu. their molecular mechanism leading to udu phenotypes is currently under investigation. chromatin condensation: general polyelectrolyte association and histone-tail specific folding nikolay korolev, nikolay berezhnoy, abdollah allahverdi, renliang yang, chuan-fa liu, james p. tam, lars nordenskiö ld school of biological sciences, nanyang technological university, nanyang drive, , singapore the major component of chromatin, dna, is a densely charged polyanion. electrostatic interactions between dna and dnapackaging proteins contribute decisively to formation of its elementary unit, the nucleosome, and are also important in chromatin folding into higher-order structures. we investigate condensation of dna and chromatin and find that electrostatics and polyelectrolyte character of dna play dominant role in both dna and chromatin condensation. by comprehensive experimental studies and using novel oligocationic ligands, we suggest simple universal equation describing dna condensation as a function of oligocation, dna and monovalent salt concentrations and including the ligand-dna binding constant. we found that similar dependence was also observed in condensation of the nucleosome arrays. next, we studied how general electrostatic and specific structural alterations caused by lysine acetylations in the histone tails influence formation of -nm chromatin fibre and intermolecular nucleosome array association. for the first time, a structural model is suggested which explains critical dependence of chromatin fibre folding on acetylation of the single lysine at position of the histone h . exceptional importance of the h lys acetylation in general and gene specific transcriptional activation has been known for many years but no structural basis for this effect has yet been proposed. detection of specific dna sequences is central to modern molecular diagnostic. ultrasensitive raman measurements of nucleic acids are possible through exploiting the effect of surface-enhanced raman scattering (sers). in this work, the sers spectra of genomic dnas from leaves of different apple trees grown in the field, have been analyzed [ ] . a detailed comparative analysis of sers signatures of genomic dnas is given. sers wavenumbers (cm - ) are reported here for all types of vibrations of plant genomic dnas, including bands assigned to localized vibrations of the purine and pyrimidine residues, localized vibrations of the deoxyribosephosphate moiety, etc. proposed band assignments are given. a strong dependence of the sers spectra on dna concentration and on time have been observed. in biochemical fields, nucleic acids might be used to explore the interaction between dna and small molecules, which is important in connection with probing the accurate local structure of dna and with understanding the natural dnamediated biological mechanisms [ ] . the ph-dependent structure of dna studied by fourier transform infrared spectroscopy the region of the infrared spectrum studied covered the wave number range from cm - to cm - . ir spectra show that in ph . - . interval carbonyl (c=o) band at - cm (assigned to guanine) is reduced in intensity and slightly shifted to lower frequencies. at the ph . significantly decreases band intensity at cm - due to unbounded c =o of thymine and shifts to lower frequencies, indicating at the transition of this group in bounded form, supposedly by means of excess polarized hydroxyl ions. together this, in basic region a new intense absorption band has been observed in - cm - frequency interval, corresponding to o-h group in-plane bending vibration ( - cm - ). as for acidic conditions, it was observed that under the extreme ph (* . ) value carbonyl absorption region shifts to higher frequencies and absorption intensity significantly increased, indicated at releasing of c=o groups from h-bonding between base pairs. moreover, bands intensity at cm - and cm - corresponding to out-of-plan deformation of nh groups increased due to rupture of connections between the dna strands. during the last decade it was found that in many cases specific structural organization of multi-molecular protein and dna-protein complexes determines their functioning in living cells. although these functioning structures are usually unique, it is often possible to identify their common structural elements. one of the interesting examples of such universal elements are hmgb domains: structurally conservative functional domains of non-histone proteins hmgb / also identified in many nuclear proteins. using afm, thermodynamic approaches, circular dichroism and molecular absorption spectroscopy in far-uv and mid-ir regions we have studied structural organization of the complexes between dna and different proteins, including hmgb , hmgb-domain recombinant proteins and linker histone h . we have demonstrated, that interaction with dna leads to increasing both a-helicity of the proteins and thermal stability of dna. also, this interaction may result in formation of highly ordered supramolecular complexes facilitated by hmgb-domains. the c-terminal sequence of hmgb / regulates affinity of the proteins to dna and can be ''inactivated'' by interaction with histone h . based on the data obtained a model of the interaction of multy-domain hmgb-proteins with dna is suggested. darmstadt, germany, lmu biozentrum; munich, germany *these authors contributed equally to this work chromatin in living cells displays considerable mobility on a local scale. this movement is consistent with a constrained diffusion model, in that individual loci execute multiple, random jumps. to investigate the connection between local chromatin diffusion (lcd) and the changes in nuclear organization, we established a stable hela cell line expressing gfp-pcna. this protein, a core component of the replication machinery, serves as a cell-cycle marker and allows us to visualize sites of ongoing dna synthesis within the nucleus. to monitor lcd, we labeled discrete genomic loci through incorporation of cy -dutp. this experimental system, in conjunction with particle tracking analysis, has enabled us to quantitatively measure chromatin mobility throughout the cell cycle. our results demonstrate that lcd is significantly decreased in s-phase. to explore the connection between dna replication and reduced chromatin movement, we undertook a more detailed examination of lcd in s-phase nuclei, correlating chromatin mobility with sites of replication. our results demonstrate that labeled chromatin in close proximity to gfp-pcna foci exhibit significantly decreased mobility. we therefore conclude that presence of active replication forks constrains the movement of adjacent chromatin. single-molecule studies of dna replication antoine m. van oijen zernike institute for advanced materials, groningen university, nijenborgh . ag groningen, the netherlands e-mail: a.m.van.oijen@rug.nl advances in optical imaging and molecular manipulation techniques have made it possible to observe individual enzymes and record molecular movies that provide new insight into their dynamics and reaction mechanisms. in a biological context, most of these enzymes function in concert with other enzymes in multi-protein complexes, so an important future direction will be the utilization of single-molecule techniques to unravel the orchestration of large macromolecular assemblies. we are applying a single-molecule approach to study dna replication. i will present recent results of single-molecule studies of replication in bacterial and eukaryotic systems. by combining the stretching of individual dna molecules with the fluorescence observation of individual proteins, we visualize the dynamic interaction of replication factors with the fork. in the bacteriophage t replication system, we show that dna polymerases dynamically associate with and dissociate from the fork during replication. further, i will present new data from single-molecule replication studies in x. laevis oocyte extracts. we have developed a novel imaging scheme that permits single-molecule fluorescence experiments at concentrations of labeled protein that were hitherto inaccessible. using this method, we visualize, in real time, origin firing and fork movement. in force-extension diagrams of reference models of naked dna (freely jointed chain, wormlike chain) as well as extensionrotation diagrams of naked dna have been successfully recovered. of note, plectonemic structures are most efficiently simulated thanks to ode's collision detection code. new insights into nucleosome and chromatin fiber structure and dynamics will be presented. the study of the pkm. plasmid effect on the repair of dna j. vincze , i, francia , g. vincze-tiszay hheif, budapest, hungary, univ. debrecen, debrecen, hungary in our experiments was studied the effect of pkm. plasmid on repair of single strand breaks in dna induced by cogamma irradiation in e.coli k ab (wild type) and its different rec mutant cells. the pkm. resistant-factor in case of the control decreases the sensitivity of radiation, which as we suppose, is reached by the help of dna conformation change. it can be supposed from the well known effect of radiation biology that by the effect of pkm. , the ratio of dna radiation sensitive volumes by appearing its new conformation decreases. the pkm. r-factor in rec mutants in case of gamma irradiation shows effects in two ways. one is the ''chemical'' connection between the r-factor and dna, though the other relate to positive and negative ''induced'' radiation resistance from the local type effect of the connection of an r-factor and a rec mutant, and the two radiation resistant effects are added algebraically. as a result from the view of biology we have to categorize the radiation resistance and the connected repair processes as two different classes according to the change either in the chemical or in the induced radiation resistant effect. recent studies have indicated that two trimethylated peptides (k , k ), derived from the parental hybrid peptide ca( - )m( - ), strongly interact with a bacterial membrane model (mixture of zwitterionic and negatively charged lipids), but not with a membrane model of mammalian erythrocytes (zwitterionic lipids) [ ] . a reduction of the cytotoxicity effect and an improvement of the therapeutic index have also been reported for the derivatives when compared with the parental ca( - )m( - ) [ ] . in this work, with the aim of providing insight on the interaction phenomena of the indicated peptides with zwitterionic and negatively charged membrane models, a systematic molecular dynamics study was carried out. full hydrated bilayers of dmpc:dmpg ( : ) and pope:popg( : ) were studied in the presence of each peptide, and results analyzed in terms of peptide structure and membrane composition. lipid-water and lipid-lipid interactions at the membrane/water interface play important role in maintaining the bilayer structure, however, this region is not easily available for experimental studies. we performed molecular dynamics simulations of two bilayers composed of two different types of lipids: ( ) dioleoylphosphatidylcholine (dopc); ( ) galactolipid monogalactosyldiacylglycerol (mgdg). to investigate the properties of the membrane/water interface region, we performed analysis of lipid-lipid interactions: direct, via charge pairs (dopc) and hydrogen bonds (mgdg) as well as indirect, via water bridges. we also examined water-lipid interactions. existence of well-defined entities (lipids) linked by different types of interactions (hydrogen bonds, charge pairs, water bridges) makes the analysis of the membrane/ water interface region a suitable for a graph theoretical description. we applied a network analysis approach for comparative analysis of simulated systems. we note a marked difference between the organization as well as the dynamics of the interfacial region of the two bilayers. l-nucleoside analogues form an important class of antiviral and anticancer drug candidates. to be pharmacologically active, they need to be phosphorylated in multiple steps by cellular kinases. human phosphoglycerate kinase (hpgk) was shown to exhibit low specificity for nucleotide diphosphate analogues and its catalytic efficiency in phosphorylation was also affected. to elucidate the effect of ligand chirality on dynamics and catalytic efficiency, molecular dynamics simulations were performed on four different nucleotides (d-/l-adp and d-/l-cdp) in complex with hpgk and , -bisphospho-d-glycerate (bpg). the simulation results confirm high affinity for the natural substrate (d-adp), while l-adp shows only moderate affinity for hpgk. the observed short residence time of both cdp enantiomers at the active site suggests very weak binding affinity which may result in poor catalytic efficiency shown for hpgk with d-/l-cdp. analysis of the simulations unravels important dynamic conditions for efficient phosphorylation replacing the single requirement of a tight binding. using the van der waals density functional based on the semilocal exchange functional pw together with a longrange component of the correlation energy [ ] implemented in the siesta program code, we have calculated the band structure of the double stranded dna. the unit cell was built by taking together gc (or at) homogenous base pairs and we have considered the translational symmetry as the periodic boundary condition. the results obtained are compared with the oligomer calculations taking up to seven base pairs. the band structure obtained with this van der waals density functional is also compared with results obtained with other exchange-correlation functionals as well as with band structure obtained by the hartree-fock crystal-orbital method taking into account the helical symmetry of the double stranded dna. the role of different parts of dna (base pairs, sugar-phosphate backbone, na ions) is also presented. transmembrane (tm) proteins comprise some % to % of the proteome but owing to technical difficulties, relatively few of these structures have been determined experimentally. computational modeling techniques can be used to provide the essential structural data needed to shed light on structurefunction relationships in tm proteins. low-resolution electrondensity maps, obtained from cryo-electron microscopy (cryo-em) or preliminary x-ray diffraction studies, can be used to restrict the search in conformational space. at the right resolution, the locations of tm helices can be roughly determined even when the amino acids are not visible. when these data are combined with physicochemical characteristics of amino acids (such as their hydrophobicity) and with evolutionary conservation analysis of the protein family, the location of the amino acids can be modeled. the modelstructure may provide molecular interpretations of the effects of mutations. moreover, it can be used to suggest molecular mechanisms and to design new mutations to examine them. the overall approach will be demonstrated using two human proteins: copper transporter (ctr ), which is the main copper transporter in the human cell, and the kda translocator protein (tspo) of the outer mitochondria. modelstructures of these proteins and their functional implications in health and disease will be discussed. calcium channels play a crucial role in many physiological functions and their selectivity mechanism is still an unresolved question and a subject of debate. a physical model of selective ''ion binding'' in the l-type calcium channel is constructed, and consequences of the model are compared with experimental data. this reduced model treats only ions and the carboxylate oxygens of the eeee locus explicitly and restricts interactions to hard-core repulsion and ion-ion and ion-dielectric electrostatic forces. according to the charge/space competition mechanism, the charge of structural ions attracts cations into the filter, while excluded volume effects are trying to keep them out. this is a competition between energy and entropy, where the balance of these terms minimizes free energy and determines selectivity. experimental conditions involving binary mixtures of alkali and/or alkaline earth metal ions are computed. the model pore rejects alkali metal ions in the presence of biological concentrations of ca + and predicts the blockade of alkali metal ion currents by micromolar ca +. conductance patterns observed in varied mixtures containing na+ and li+, or ba + and ca +, are predicted. ca + is substantially more potent in blocking na+ current than ba +. in apparent contrast to experiments sing buffered ca + solutions, the predicted potency of ca + in blocking alkali metal ion currents depends on the species and concentration of the alkali metal ion, as is expected if these ions compete with ca + for the pore. these experiments depend on the problematic estimation of ca + activity in solutions buffered for ca + and ph in a varying background of ulk salt. equilibrium binding affinity (expressed as the occupancy of the selectivity filter by various ions) is computed by equilibrium grand canonical monte carlo (gcmc) simulations. the conductivity of the channel is estimated from the equilibrium concentration profiles using the integrated nernst-planck equation. our simulations show that the selectivity of l-type calcium channels can arise from an interplay of electrostatic and hard-core repulsion forces among ions and a few crucial channel atoms. the reduced system selects for the cation that delivers the largest charge in the smallest ion volume. we have also performed dynamic monte carlo (dmc) simulations for a model ca channel to simulate current directly and present our results for the dynamical selectivity (expressed as the flux carried by various ions). we show that the binding affinity of ca + versus na+ is always larger than the dynamical selectivity because ca + ions are tightly bound to the binding site of the selectivity filter of the channel and, at the same time, their mobility and drift velocity is smaller in this region. carotenoids are used in light-harvesting complexes with the twofold aim to extend the spectral range of the antenna and to avoid radiation damages. the effect of the polarity and conformation of the environment is supposed to be responsible for the tuning of the electronic, optical and vibrational properties of peridinin carotenoid both in solution and in protein matrix. we investigate the effect of vibrational properties of peridinin in different solvents by means of vibrational spectroscopies and qm/mm molecular dynamics simulations . the shift of vibrational fingerprints in the - cm - frequency region, due to the solvent polarity and proticity, is studied in three cases: cyclohexane (apolar/aprotic), deuterated acetonitrile (polar/aprotic) and methanol (polar/ protic). the frequencies and vibrational modes of the carbonyl, the allene, and the polyene chain were identified using effective normal mode analysis and compared with the present and previous experimental data . on the basis of our calculations and experiments in different solvents, we propose a classification of the four peridinins of the high-salt pcp form. the controlled self-assembly of functional molecular species on well defined surfaces is a promising approach toward the design of nanoscale architectures. by using this methodology, regular low-dimensional systems such as supramolecular clusters, chains, or nanoporous arrays can be fabricated. small biological molecules such as amino acids represent an important class of building blocks that are of interest for molecular architectonic on surfaces because they inherently qualify for molecular recognition and selfassembly [ ] . the interaction between amino acids and solid surfaces is decisive for the development of bioanalytical devices or biocompatible materials as well as for a fundamental understanding of protein-surface bonding. we investigate the adsorbtion mechanism of the cysteine on au( ) surfaces by means of the dft [ ] . our main concern is to describe the molecule-metal bonding mechanism. therefore we present a complex study, including the full determination of the density of states for the free and adsorbed molecule, the determination of molecule-surface bonding energy. the method of crystal orbital overlap populations is used in order to determine the contribution of specific atomic orbitals to the molecule-metal bond. it is now widely accepted that myoglobin (mb) is not simply an o storage/delivery system but, depending on oxygen concentration, it exerts other fundamental physiological roles. recent studies revealed a widespread expression and, in particular, an over-expression in response to hypoxia, in various non-muscle tissues, including tumor cells. in human five different mb isoforms are present. the two most expressed ([ %) differ only at the th position, k (mb-i) and e (mb-ii) respectively. since high-altitude natives from tibet are characterized by a higher mb concentration and locomotion efficiency, together with the observation that the mb overexpression is totally attributable to mb-ii, the idea that the latter might be one of the responses to high-altitude evolutionary adaptation, i.e. hypoxic environment, started to emerge. however, this is not yet supported by any structure/function investigation. we performed hundred nanoseconds md simulations on human mbs to investigate the structure and dynamics of both protein and surface water. important differences have been protein kinases play key roles in cell signaling and constitute crucial therapeutic targets. in normal cell, upon substrate binding, tyrosine kinase receptor kit undergoes extensive structural rearrangement leading to receptor dimerization and activation. this process is initiated by the departure of the juxta membrane region (jmr) from the active site, allowing the activation loop (aloop) deployment. the deregulation of kit activity is associated with various forms of cancer provoked by abnormalities in signal transduction pathways. mutations v g (jmr) and d h/v (a-loop) have been reported as oncogenic and/or drug-resistant. to contribute further in the understanding of kit activation/ deactivation mechanisms, we applied a multi-approach protocol combining molecular dynamics (md), normal modes analysis (nma) and pocket detection. disturbing structural effects, both local (a-loop) and long-range (jmr), were evidenced for kit d h/v in the inactive state. nma showed that jmr is able to depart its position more easily in the mutants than in the wild type. pockets analysis revealed that this detachment is sufficient to open an access to the atp binding site. our results provided a plausible conception of mutant dimerization and a way to explore putative allosteric binding sites. transmembrane association of leukocyte integrin heterodimer might be mediated by a polar interaction choon-peng chng and suet-mien tan biophysics group, a*star institute of high performance computing, and, school of biological sciences, nanyang technological university, republic of singapore the lateral association of transmembrane (tm) helices is important to the folding of membrane proteins as well as a means for signaling across the cell membrane. for integrin, a hetero-dimeric protein important for cell adhesion and migration, the association of its a-and b-subunits' tm helices plays a key role in mediating bi-directional mechanical signaling across the membrane. we found evidence from experiment and simulation for a polar interaction (hydrogenbond) across leukocyte integrin alb tm that is absent in the better-studied platelet integrin aiibb [ ] . our coarse-grained molecular dynamics simulations of tm helix-helix selfassembly showed more native-like packing achieved by alb within the simulation timescale as compared to its 'lossof-function' b t g mutant or aiibb [ ] . association free energy profiles also showed a deeper minimum at a smaller helix-helix separation for alb , suggestive of tighter packing. the likely conservation of this polar interaction across the b integrin family further reinforces its importance to the proper functioning of leukocyte integrins. active extrusion of drugs through efflux pumps constitutes one of the main mechanisms of multidrug resistance in cells. in recent years, large efforts have been devoted to the biochemical and structural characterization of rnd efflux pumps in gram-negative bacteria, in particular the acrb/a-tolc system of e.coli. specific attention has been addressed to the active part of the efflux system, constituted by the acrb unit. despite the presence of several data, crucial questions concerning its functioning are still open. the understanding of the structure-dynamics-function relationship of mexb, the analogous transporter in p. aeruginosa, encounters even more difficulties, because of the lack of structural data of the transporter in complex with substrates. to shade some light on the activity of mexb, we performed computational studies on mexb interacting with two compounds, meropenem and imipenem, the first known to be a good substrate, and the second a modest one. several techniques were used in the present work, ranging from flexible docking [ ] to standard and targeted molecular dynamics (md) simulations. starting from the published crystal structure [ ] we identified the most probable poses of the two compounds in both the original experimental and in the md-equilibrated structures. we used information from acrb binding pocket in order to find relevant binding sites of the two compounds in the analogous binding pocket of mexb. meropenem frequently lies with appropriate orientation in a pocket similar to the one identified for doxorubicin in acrb [ ] , while the occurrence of imipenem poses in the same pocket is very scarce. additionally, when present in the pocket, imipenem is located in a position that renders very unlikely its extrusion toward the oprm docking domain during the simulation of the functional peristalsis. the analysis of the trajectories has provided a complete inventory of the transporter and antibiotic hot spots, which is key information in terms of screening and design of antibiotics and inhibitors. clathrins are three-legged proteins with the intriguing ability to self-assemble into a wide variety of polyhedral cages. the nucleation and growth of a clathrin lattice against the cytosolic face of a cell membrane enables the endocytosis of membrane proteins and various external molecules, by wrapping the membrane around the cargo to produce a coated transport vesicle within the cell. clathrins can also self-assemble, in slightly acidic solutions devoid of auxiliary proteins, into empty cages. our simulations of this process, using a highly coarsegrained model, indicate that the key to self-assembly is neither calthrin's characteristic puckered triskelion shape, nor the alignment of four legs along all cage edges, but an asymmetric distribution of interaction sites around the leg's circumference. based on the critical assembly concentration, the binding strength in these cages is estimated at to k b t per clathrin. the simulations also answer the long-standing conundrum of how flat patches of purely hexagonal clathrin lattices, which in cryo electron microscopy are frequently seen to decorate cell membranes, can produce highly curved cages containing twelve pentagonal faces interdispersed between hexagonal faces. we present experimental evidence supporting this pathway. in eukaryotic cells, the exchange of macromolecules between the cytoplasm and the nucleus is mediated by specialized transport factors. by binding to these transporters, cargo molecules, which are otherwise excluded from entering the nucleus, can traverse the nuclear pore complex efficiently. most of the proteins mediating nuclear import and export exhibit a characteristic _-solenoid fold, which provides them with an unusual intrinsic flexibility. crm is an essential nuclear export receptor, which recognizes a very broad range of export cargoes. crm -dependent nuclear export is ran-gtpase-driven, and recognition of rangtp and cargo is highly cooperative. however, recent crystal structures show that the binding sites for export cargos and rangtp are located at distant parts of crm [ ] [ ] [ ] . we have used a combined approach of all-atom molecular dynamics simulations and small-angle x-ray scattering to study rangtp and cargo binding to crm . we have found that the allosteric effect in crm -dependent nuclear export arises from a combination of subtle structural rearrangements and changes in the dynamic properties of crm . light-induced phototactic responds in green algae chlamydomonas reinhardtii are mediated via microbial-type rhodopsins, termed channelrhodopsin- (chr ) and channelrhodopsin- (chr ) , which carry the chromophore retinal covalently linked to lysin via a schiff base and were shown to be directly light-gated ion-channels . the n-terminal putative seven-transmembrane region of chr was shown to be responsible for the generation of photocurrents and exhibits sequence similarity to the well understood proton pump bacteriorhodopsin (br) and the sensory rhodopsin anabaena sensory rhodopsin (asr) . as for the majority of membrane proteins, there is no d-structural data for chr available yet. here we present homology models of chr using two high-resolution x-ray template structures of br ( qhj ) respectively asr ( xio ) in order to get structural and functional insights into chr . with both homology models we performed molecular dynamics (md) simulations in a native membrane/solvent environment using gromacs . . . comparison of energetic and structural results revealed obvious advantages of the br-based homology model of chr . here we show that the br-based homology model is a reliable model of chr exhibiting structural features already found experimen-tally . our br-based homology model of chr allows predictions of putative crucial residues within chr . so we proposed several mutations within the chr sequence which are already accomplished. electrophysiologic and spectroscopic studies of these mutations are underway in order to confirm the functional relevance of these residues and to contribute to an optimized usage of chr as a powerful tool in optogenetics. ( neuroglobin is a recently discovered globin protein predominantly expressed in brain. its biological function is still elusive. despite the fact that neuroglobin shares very little sequence homology to the well-known globins as mioglobin and hemoglobin, they all have a characteristic globin fold with heme molecule bound to the distal pocket. the structural investigations and co binding kinetics revealed existence of cavities and tunnels within the protein matrix, where small ligands can be stored even for hundreds of microseconds [ ] . in human neuroglobin there is one internal disulfide bond possible which existence is proved to have significant effect on ligand affinity [ ] . in this study effects of temperature, ph, distal histidine mutation and presence of disulfide bond on co rebinding to neuroglobin are investigated by flash photolysis experiments. in parallel, the molecular dynamics simulations are performed in corresponding conditions in order to investigate structural change of neuroglobin and especially changes in distribution of internal tunnels and cavities able to bind diatomic ligands in response to different physical conditions listed above. the thrombospondin family, being extracelluar proteins, is known to be implicated in various physiological processes such as wound healing, inflammation, angiogenesis and neoplasia. the signature domain of thrombospondins shows high sequence identities and thus allows us to transfer results obtained, studying this complex calcium reach part of the proteins, from one member of the family to the other. the domain is known to play a key role in hereditary diseases such as psach or med. in this part of thrombospondins lies a binding site to integrins, important for cell attachment. it is further known that the lectin like globe binds to cd- , a feature known to be important in cancer research. as the theoretical unit we are trying to resolve these problems by numerical means and are constantly challenged by the size, where thrombospondin can be a huge trimeric protein as one strand can measure kda, and the large variety of subdomains found in this proteins. we are thus facing a multiscale problem which can range from solving, by means of quantum mechanics a specific ion binding site, to large scale abstraction by continuum mechanics. in our talk we will show you our newest results that we obtained by simulating calcium rich c-terminal domain which is known to be conserved across the entire family, and give you an outlook into the future of our research. the process of swift heavy ions energy deposition while penetrating a solid or scattering on its surface can result in a strong and nonequilibrium excitation of matter. an extremely localized character of this excitation, meanwhile, can make possible both selective changes in chemistry of matter and its surface nanomodifications . since possible applications have been found in bio-and it-technologies (cancer curing and nanostructuring respectively) in the last decade, the heavy ion bombardment technique has attracted a lot of scientific interest , . the processes of fast energy deposition into the solid and its further dissipation, however, are essentially perturbed with highly excited and nonequilibrium state of both lattice and electron systems. at such conditions therefore, the precision in treatment of processes of electron thermalization, fast electron heat conduction, and phase transformation of the overheated solid becomes crucial. having several physical models to handle the mentioned processes, it is nevertheless difficult to describe all of them within a scale of a single computational approach. our work is aimed on elaboration of the atomistic-continuum model of heavy ion bombardment of solids. in particular, the model will be applied to study the formation of nanohillocks in the experiments on swift heavy ion xe + scattering on srtio surface . [ ] g. aquaporins are protein channels located across the cell membrane with the role of conducting water or other small sugar alcohol molecules (aquaglyceroporins). the presence of the human aquaporin (hsaqp ) in cells proximal to airinteracting surfaces (eyes, lacrimal glands, salivary glands, lungs, stomach etc.) suggest its potentially important role in ''wetting'' these surfaces. the high-resolution x-ray structure of the hsaqp tetramer (pdb code d s) exhibits two important features: (i) lack of the four fold symmetry, common in most of the aquaporins, and (ii) occlusion of the central pore by a phosphatidylserine lipid tail. in this study we investigate the importance of these two features on the transport properties of the human aqp by means of molecular dynamics simulations. we found that the asymmetry in the tetramer leads to a distribution of monomeric channel structures characterized by different free energy landscapes felt by the water molecules passing through the channel. furthermore, the structures' distribution is influenced both by the presence/absence of the lipid tail in the central pore, and by the lipid composition of the bilayer that solvates the hsaqp tetramer. elucidating the modular structure of the protein g c fragment and human igg fc domain binding site using computer simulations hiqmet kamberaj faculty of technical sciences, international balkan university, skopje, r. of macedonia protein-protein recognition plays an important role in most biological processes. although the structures of many protein-protein complexes have been solved in molecular detail, general rules describing affinity and selectivity of proteinprotein interactions break down when applied to a larger set of protein-protein complexes with extremely diverse nature of the interfaces. in this work, we will analyze the non-linear clustering of the residues at the interface between proteins. the boundaries between clusters are defined by clustering the mutual information of the protein-protein interface. we will show that the mutations in one module do not affect residues located in a neighboring module by studying the structural and energetic consequences of the mutation. to the contrary, within their module, we will show that the mutations cause complex energetic and structural consequences. in this study, this is shown on the interaction between protein g c fragment and human igg fc domain by combining molecular dynamics simulations and mutual information theory, and computational alanine scanning technique. the modular architecture of binding sites, which resembles human engineering design, greatly simplifies the design of new protein interactions and provides a feasible view of how these interactions evolved. the results test our understanding of the dominant contributions to the free energy of protein-protein interactions, can guide experiments aimed at the design of protein interaction inhibitors, and provide a stepping-stone to important applications such as interface redesign. membrane proteins can form large multimeric assemblies in native membranes that are implicated in a wide range of biological processes, from signal transduction to organelle structure. hydrophobic mismatch and membrane curvature are involved in long range forces largely contributing to such segregation. however, the existing assembly specificity is thought to be coded in the atomic details of protein surface and topology. these are best described in high resolution structures and atomistic molecular dynamics simulations. in order to explore more systematically such forces and energetics arising at intermediate time scales and resolution, we use coarse grained molecular dynamics simulations applied to membrane systems spanning over to us. as a first glimpse we study how proteins induce lipid perturbations using a previously developed conformational entropy estimator. we show that in the model membrane where hydrophobic mismatch is present, lipid perturbations extend up to * a from the protein surface. however, significant variations in perturbation profiles are seen. parameters such as protein shape, surface topology, and amino acid physicochemical properties are studied to discover the parameters governing such perturbations. crossing energy barriers with self-guided langevin dynamics gerhard kö nig, xiongwu wu, bernard brooks national institutes of health, national heart, lung and blood institute, laboratory of computational biology, rockville, md, usa even with modern computer power, the applicability of molecular dynamics simulations is restricted by their ability to sample the conformational space of biomolecules. often high energy barriers cause normal molecular dynamics simulations to stay trapped in local energy minima, leading to biased results. to address this problem, self-guided langevin dynamics (sgld) were developed. it enhances conformational transitions by accelerating the slow systematic motions in the system. this is achieved by calculating the the local average of velocities and adding a guiding force along the direction of systematic motions. thus, the efficiency of sgld is governed by three factors: a.) the friction constant involved in the langevin dynamics b.) the local averaging time and c.) the guiding factor that determines the guiding force. however, the guiding force also causes deviations from the original ensemble that have to be corrected by reweighting the data, thus decreasing the efficiency. here, we explore the three-dimensional parameter space of sgld for several benchmark systems with particularly rough energy surfaces. based on our data, we supply guidelines for the optimal selection of sgld parameters, to allow the extension of our method to other biological problems of interest. propagation of d v/h mutation effects across kit receptor e. laine, i. c. de beauchê ne, c. auclair and l. tchertanov lbpa, cnrs -ens de cachan, france receptor tyrosine kinases (rtks) regulate critical biological processes. constitutive activation of rtks provokes cancers, inflammatory diseases and neuronal disorders. biological data evidenced that oncogenic mutations of the rtk kit, located either in the juxtamembrane region (jmr) or in the activation loop (a-loop) -as is the case of d v/h, displace the equilibrium of conformational states toward the active form. we present a multi-approach study that combines molecular dynamics (md), normal modes (nm) and pocket detection to characterize and compare the impact of d v/h on the structure, dynamics and thermodynamics of kit. we have evidenced a local structural destabilization of a-loop induced by the mutation and a long-range effect on the structure and position of jmr. we have further correlated these observations with experimental data and decipher some details about the activation mechanisms of the mutants, involving leverage of the jmr negative regulation and release of an access to the catalytic site. through the identification of ''local dynamic domains'' and the recording of interactions within the protein, we propose a model of the mutational effects propagation, which highlights the importance of both structural distortion and local conformational fluctuations. investigation of biological active azolidinones and related heterocycles refer to one of the most successful scientific projects of dh lnmu. it is based on three strategic vectors: organic synthesis, pharmacological research, rational design of ''drug-like'' molecules (including in silico approaches). while applying the research strategy we succeeded in gaining a number of interesting results that make possible to extend the field, especially in the scope of ''drug-like'' molecules design, notably it has focused on the search of new anticancer agents. anticancer activity screening was carried out for more than compounds (us nci protocol (dtp) based on obtained directed library over new compounds, among them compounds showed high activity level. for the purpose of optimization and rational design directions of highly active molecules with optimal ''drug-like'' characteristics and discovering of possible mechanism of action sar, compare analysis, molecular docking and qsa(p)r were carried out. obtained results allowed to form main directions of possible anticancer activity mechanisms, which probable are apoptosis-related. nowadays the investigation of cellular and molecular aspects of anticancer effects is in progress. regulation of (bio)chemical reactions by mechanical force has been proposed to be fundamental to cellular functions [ , , ] . atomic force microscopy experiments have identified the effect of mechanical force on the reactivity of thiol/disulfide exchange, a biologically important reaction [ ] . in order to understand the influence of the force at an atomistic level, we have performed hybrid quantum mechanicsmolecular mechanics (qm/mm) transition path sampling simulation of the reaction under external forces. the results of the simulations supported the experimental findings and demonstrated that the location of the transition state on the free energy surface was shifted due to force [ ] . in contrast to our findings, however, a recent experimental study suggests only a weak coupling between the mechanical force and the reaction rate [ ] . in this study, the reactants were covalently linked to a stilbene molecule. in this system a force can be applied by photo-isomerization from the relaxed trans to the strained cis configuration. a drawback of this system is that one cannot easily determine the forces that acting on the reaction coordinate. therefore, we have developed a force distribution analysis method for quantum mechanical molecular dynamics simulations. the results of the analysis show how isomerization of stilbene alters the forces acting on the reacting atoms. the force distribution is essential for understanding how chemistry is controlled by external forces. [ conformational space modelling (csm) is a promising method for membrane protein structure determination. it is based on the concept of the side-chain conformational space (sccs), which is formed by the allowed side-chain conformations of a given residue. each sccs can be calculated from a d structure or measured via epr-sdsl experiments. for structure determination a set of singly spinlabelled mutants is needed. the final structure is obtained by altering an initial (possibly random) d structure until the best fit between the calculated and measured sccs for the whole set is found. such optimization is computationally intensive; therefore csm includes several empirical approximations. one of them describes the effect of the lipid tails on the sccs. the implementation is not trivial as lipids diffuse in the plane of the membrane and the lipid tails behave differently at different membrane depths. to unravel this relationship adaptive biasing force md simulations were used. an alanine peptide helix was made in silico, spin-labelled at the middle and inserted perpendicularly into a dmpc membrane. the free energy of the spin-label orientation at various membrane depths was calculated. a d free energy surface describing the membrane ''depth'' effect was obtained. it is known that b-cyclodextrins (bcds) are able to modify the cholesterol content of cell or model membranes. however the molecular mechanism of this process is still not resolved. using molecular dynamics simulations, we have been able to study the bcd-mediated cholesterol extraction from cholesterol monolayers and lipid-cholesterol monolayer mixtures. we have investigated many conditions that would affect this process (e.g. lipid-cholesterol ratio, lipid chain unsaturation level) our results can be summarized as follow: i) dimerization of bcds, ii) binding of the dimers to the membrane surface assuming either a tilted (parallel to the membrane normal) or untilted ( °respect to the normal of the membrane) configuration, iii) the latter configuration is suitable to extract cholesterol at a reasonable computational time scale ( - ns), however, this process may be affected by unfavorable energy barriers (from to kj mol - ), iv) desorption of the complex brings cholesterol in solution, v) the bcd-cholesterol complex is able to transfer cholesterol to other membranes. with a clearer understanding of the basic molecular mechanism of the bcd mediated process of cholesterol extraction, we can begin to rationalize the design of more efficient bcds in numerous applications. the mechanism of the complex formation of biopolymers with ligands including the solvent molecules is an actual problem of modern biophysical and biological science. polypeptides form a secondary structure and mimic the motifs of the protein architecture. the study of complexation between polypeptides and solvent molecules leads to deeper understanding of the basic interaction of proteins with environment at atomic level. besides polypeptides are promising for the development of applications which encompass some of the following desirable features: anti-fouling, biocompatibility, biodegradability, specific biomolecular sensitivity. on this account polypeptides have a great significance for a variety modern applications ranging from the nanoscale medicine devises up to food technology and others. we compare the results of calculations of complexes between helical polypeptides (polyglutamic acid in neutral form and poly-c-benzyl-l-glutamate) and water molecules at dft pbe level and the results of ftir-spectroscopic study of the film of wetted polypeptides. vibrational spectroscopy is one of the most useful experimental tools to study non-covalent bonded complexes, and calculated spectra in comparison with experimental data are reliable test for reality of simulated complexes. platelet aggregation at the site of vascular injury is vital to prevent bleeding. excessive platelet function, however, may lead to thrombus formation after surgery. therefore, an accurate measure and control of platelet aggregation is important. in vitro platelet aggregometry monitors aggregate formation in platelet rich plasma triggered by agonists such as adp, epinephrine or collagen. the fraction of aggregated platelets is plotted versus time, and platelet function is assessed by analyzing the plot's morphology. we propose new measures of platelet function based on a compartmental kinetic model of platelet aggregation induced by adp. our model includes three compartments: single, aggregated and deaggregated platelets. it is simpler than earlier models and agrees with experimental data. the model parameters were determined by non-linear least squares fitting of published data. we associated the kinetic parameters with the activity of the adp receptors p y and p y . to this end, we studied published data obtained in the presence and in the absence of specific antagonists of these receptors. comparison of kinetic parameters of healthy subjects with those of patients with myeloproliferative disorder (mpd) shows that the function of p y is significantly reduced in mpd. coarse-grained modeling of drug-membrane interactions manuel n. melo, durba sengupta, siewert-jan marrink groningen biomolecular sciences and biotechnology institute, university of groningen, groningen, the netherlands the martini coarse-grained (cg) forcefield was used to simulate the actions of the antimicrobial peptide alamethicin and of the anti-tumoral drug doxorubicin. both drugs were shown to interact strongly with a fluid phospholipid bilayer, and aggregate there, in agreement with experimental results. because doxorubicin may establish intermolecular h-bonding, and thus lower its dipole moment, the cg representation of a doxorubicin dimer was adjusted. this less polar dimer was then tested for translocation and/or pore formation. contrary to results of atomistic simulations, alamethicin aggregates did not spontaneously open pores. they did so, however, when the size of the water beads was decreased. several small independent pores could then be observed. the magnitude of the permeability of these pores is analyzed and compared to experimental values. the occurrence of multiple small pores could indicate that the different conductance levels experimentally observed for alamethicin might simply result from the association of different numbers of these small pores. polarization refers to the asymmetric changes in cellular organization in response to external or internal signals. neuronal polarization begins with the growth of a single neurite shortly after cell division, followed by the growth of a second neurite at the opposite pole. this early bipolar shape is critical for brain function, as it defines axis of migration and consequently proper three dimensional organization and nerve circuitry. however, it is not known if a direct relationship exists between the formation of the second, opposite, neurite and the mechanisms involved in the formation of the first. we tackled this issue through mathematical modeling, based on membrane traffic (exocytosis-endocytosis), and lateral diffusion. with this approach, we demonstrated that a single pole of molecular asymmetry is sufficient to induce a second one at the opposite side, upon induction of growth from the first pole. our work gives mathematical proof that the occurrence of a single asymmetry in a round cell is sufficient to warrant morphological bipolarism. trypsin is one of the best characterized serine proteases. enzyme acylation process is required for substrate degradation. there is a lot of information about how this process undergoes. however, in order to obtain a more detailed description of the catalytic triad mechanism, a qm/mm approach was used. we used the hybrid qm/mm potential implemented in amber package. in the qm calculations a dft hamiltonian was used. we develop an approach based on the adaptively biased md in order to obtain the free energy surface of the conformational space defined by the reaction coordinates. this approach presents some characteristics of steered md and umbrella sampling procedures. our results offer information about the lowest energy trajectory, the barrier profile of the reaction, and the geometry of the transition state. this method also provides a further insight into the role of specific residues in the reaction. substituting asp , member of the catalytic triad, for ala we were able to detect an increase of the barrier profile. this was due to the loss of the interaction of carbonyl group of asp with nd of his , which make ne of this residue a worst proton acceptor. this results show our approach as a valuable method in the study of enzymatic mechanisms. the intracellular media comprise a great variety of macromolecular species that are not individually concentrated, but being preset in the same compartment they exclude each other's volume and produce crowding. crowding has a profound impact on protein structure and determines conformational transitions and macromolecular association. we investigated macromolecular association on a % w/w bovine serum albumin (bsa) solution by time-domain terahertz (thz) spectroscopy and molecular modeling. molecular crowding was simulated by including two bsa molecules in the same water box. we generated * such dimeric models, computed their thz spectra by normal modes analysis and compared them with the experimental data. the best bsa dimer model was selected based on the agreement with the experiment in the lowest frequencies domain of up to thz. symmetry constraints improve accuracy of ion channels models. application to two-pore-domain channels adina l. ion channels are important drug targets. structural information required for structure-based drug design is often filled by homology models (hm). making accurate hm is challenging because few templates are available and these often have substantial structural differences. besides, in molecular dynamics (md) simulations channels deviate from ideal symmetry and accumulate thermal defects, which complicate hm refinement using md. we evaluate the ability of symmetry-constrained md simulations to improve hm accuracy, using an approach conceptually similar to casp competition: build hm of channels with known structure and evaluate the efficiency of various symmetry-constrained md methods in improving hms accuracy (measured as deviation from x-ray structure). results indicate that unrestrained md does not improve accuracy, instantaneous symmetrization improves accuracy but not stability during subsequent md, while gradually imposing symmetry constraints improves both accuracy (by - %) and stability. moreover, accuracy and stability are strongly correlated, making stability a reliable criterion in predicting hm accuracy. we further used this method to refine hm of trek channels. we also propose a gating mechanism for mechanosensitive channels that was further experimentally confirmed. nucleotide modifications and trna anticodon-mrna codon interactions on the ribosome olof allné r and lennart nilsson karolinska institutet, stockholm, sweden molecular dynamics simulations of the trna anticodon and mrna codon have been used to study the effect of the common trna modifications cmo u and m a . in trna val these modifications allow all four nucleotides to be successfully read at the wobble position in a codon. previous data suggest entropic effects are mainly responsible for the extended reading capabilities but detailed mechanisms have remained unknown. the aim of this paper is to elucidate the details of these mechanisms on an atomic level and quantify their effects. we have applied: extensive free energy perturbation coupled with umbrella sampling, entropic calculations of trna (free and bound to the ribosome) and thorough structural analysis of the ribosomal decoding center. human neuroserpin (hns) is a serine protease inhibitor (serpin) of a tissue-type plasminogen activator (tpa). the conformational flexibility and the metastable state of this proteins underlies to misfolding and to dysfunctional mutations causing a class of rare genetic diseases which share the same molecular basis. the conformational transition of the native form, triggered upon the cleavage at reactive center loop (rlc), releases a complex of the cleaved form bound to the inactivated target protease. without rlc cleavage a stable inactive latent form can be obtained by intra/inter molecular loop insertion leading to polymerization. this work concerns the study of the three above mentioned forms of hns by md simulations to investigate the relation between their conformational stability and. the starting native and cleaved configurations are based on the x-ray structure, while the latent form is here modelled. the results of the simulation reveal a striking conformational stability along with the intrinsic flexibility of selected regions of the fold.the analysis of the essential collective modes of the native hns shows that the initial opening of the b-sheet a coincides with several changes in the local pattern of salt bridges and of hydrogen bonds. regulation of ubiquitin-conjugating enzymes: a common mechanism based on a pattern of hydrophobic and acidic residues enzyme temperature adaptation generally involves a modulation of intramolecular interactions, affecting proteins dynamics, stability and activity [ ] [ ] . in this contribution, we discuss studies of different classes of extremophilic enzymes, focusing on cold-adapted variants, as well as their mesophilic-like mutants, performed by all-atom molecular dynamics simulations with particular attention to structural communication among residues within the threedimensional architecture [ ] [ ] . common adaptation strategies turned out to be based on improved local flexibility in the proximity of the functional sites, decrease in interconnected electrostatic interactions, and modulation of correlated motions and networks of communicating residues. specific differences related to the diverse protein folds can also be detected. bneurexins and neuroligins are cell adhesion molecules and play important role in synapse junction formation, maturation and signal transduction between neurons. mutations in genes coding these proteins occurs in patients with cognitive diseases like autism disorders, asperger syndrome and mental retardation [ ] . it has been found that the complex bneurexin-neuroligin has also an important role in angiogenesis [ ] . herein we will present molecular foundations of bneurexin-neuroligin interactions obtained from all-atom molecular dynamics simulations of bneurexin, neuroligin and their complex ( b q) [ ] . ns md trajectories (charmm force field) were analyzed and roles of ca + and n-actetyl-d-glucosamine posttranslational modifications in intermolecular interactions were scrutinized. advances in hardware and software have enabled increasingly long atomistic molecular dynamics simulations of biomolecules, allowing the exploration of processes occurring on timescales of hundreds of microseconds to a few milliseconds. increasing the length of simulations beyond the microsecond time scale has exposed a number of limitations in the accuracy of commonly employed force fields. such limitations become more severe as the size of the systems investigated and the length of the simulations increase. here i will describe the force field problems that we have encountered in our studies, how we identified and addressed them, and what we have learned in the process about the biophysics of the systems we are investigating. while the quest for a ''perfect'' force field is not over (and may never be), our work has greatly improved the accuracy and range of applicability of simple physics-based force fields, to the point that reliable predictions can now be obtained from millisecond-timescale simulations of biomolecules. local anesthetics (la) are pain-relief drugs, widely used in medicine and dentistry. the relatively short duration of analgesia still restricts their clinical use for the treatment of chronic pain. nowadays, intensive research is focused on anesthetics entrapped into liposomes to enhance their activity and pharmacokinetic properties [ ] . in this work, we investigated the encapsulation of prilocaine (plc), an aminoamide local anesthetic, into a small unilamellar liposome. on the line of our previous work [ ] , we have carried out molecular dynamics (md) simulations using a coarse grain model up the microsecond time scale. in this way, we compare the effects of the concentration of la at fisiological ph. we were able to capture important features of the plc-vesicle interactions. the behavior of plcs at fisiological ph is essentially a combination of high and low ph: we found that all neutral plc molecules rapidly diffuse into the hydrophobic region of the vesicle adopting an asymmetric bimodal density distribution. protonated plc molecules (pplc) initially placed in water were instead only found on the external monolayer, with a high rate of exchange with the water phase and no access to the inner part of the liposome in a concentration dependent way. we focus on applications of molecular and mesoscale simulation methodologies to the cellular transport process of endocytosis, i.e., active transport mechanisms characterized by vesicle nucleation and budding of the cell membrane orchestrated by protein-interaction networks, and functionalized carrier adhesion to cell surfaces. we discuss theoretical and computational methodologies for quantitatively describing how cell-membrane topologies are actively mediated and manipulated by intracellular protein assemblies. we also discuss methods for computing absolute binding free energies for carrier adhesion. we present rigorous validation of our models by comparing to diverse range of experiments. the importance of delta-opioid receptors as target of a large number of drugs is well recognized, but the molecular details of interaction and action of the compounds are largely unknown. in an effort to shade some light on this important issue we performed an extensive computational study on the interaction of two compounds, clozapine and desmetilclozapine, with a delta-opioid receptor. according to experiments, the lacking of a single methyl group in desmetilclozapine with respect of clozapine makes the former more active than the latter, providing a system well suited for a comparative study. we investigated stable configurations of the two drugs inside the receptor by simulating their escape routes by metadynamics, an algorithm that allows the simulation of events that are otherwise out of range for standard molecular dynamics simulations. our results point out that the action of the compound is related to the spatial distribution of the affinity sites it visits during its permanency. desmetilclozapine has a larger distribution of residues, which is interacting with, than clozapine. however, large conformational changes of the receptor were not observed in the presence of both compounds. thus, a more dynamical picture of ligand-receptor affinity is proposed on the basis of the results obtained, involving the competition among different stable states as well as the interaction with the solvents. such information might be useful to provide hints and insights that can be exploited in more structure-and-dynamics-oriented drug design. the coupling between the mechanical properties of enzymes and their biological activity is a well-established feature that has been the object of numerous experimental and theoretical works. in particular, recent experiments show that enzymatic function can be modulated anisotropically by mechanical stress. we study such phenomena using a method or investigating local flexibility on the residue scale, which combines a reduced protein representation with brownian dynamics simulations. we performed calculations on the enzyme guanylate kinase in order to study its mechanical response when submitted to anisotropic deformations. the resulting modifications of the protein's rigidity profile can be related to the changes in substrate binding affinities that were observed experimentally. further analysis of the principal components of motion of the trajectories shows how the application of a mechanical constraint on the protein can disrupt its dynamics, thus leading to a decrease of the enzyme's catalytic rate. eventually, a systematic probe of the protein surface led to the prediction of potential hotspots where the application of an external constraint would produce a large functional response both from the mechan- hiv- protease autocatalyses its own release from gag and gagpol precursor polyproteins into mature functional proteins. as it is functional in the dimeric form, whilst initially, only a single monomer is embedded within each gagpol chain, the question arises as to what cut's the cutter. two individual monomers in different gagpol chains are known to come together to form an embedded-dimer precursor protease. mature-like protease activity is concomitant with n-terminal intramolecular cleavage of this transient embedded-dimer precursor, but how this crucial maturation-initiating step is physically achieved, has remained unknown. here, we show via all-atom explicit solvent molecular dynamics simulation runs of ns each that the n-terminal of an immature-like protease, with the n-terminal initially unbound as in the gagpol polyprotein, can self-associate to the active site and therefore be cleaved under conditions of thermodynamic equilibrium, identifying possible binding pathways at atomic resolution, in agreement with previous indirect experimental evidence [ ] . the binding pathway predominantly makes use of the open conformation of the beta-hairpin flaps characterised by us previously [ ] , and the n-terminal binds across the entire active site in good agreement with crystal structures of a cleavage-site peptidebound protease. the n-terminus serves two roles, firstly in the maturation of the protease itself by self-associating to the protein and then as a stabilizing component of the dimer interface in a mature protease. targeting the prior mechanism could be the focus of a novel therapeutic strategy involving immature protease inhibition. [ knotted proteins are the object of an increasing number of experimental and theoretical studies, because of their ability to fold reversibly in the same topologically entangled conformation. the topological constraint significantly affects their folding landscape, thus providing new insight and challenges into the funnel folding theory [ ] . recently developed experimental methods to trap and detect knots have suggested that denaturated ensembles of the knotted proteins may be knotted [ ] . we present numerical simulations of the early stage of folding of the knotted proteins belonging to protein families mtase (methyltransferase) and sotcase (succinyl-ornithine transcarbamylase), and of their unknotted homologues [ ] . our results show that native interactions are not sufficient to generate the knot in the denaturated configurations. however, when non-native interactions are included we observe formation of knots only in the chains whose native state is knotted. in addition, we find that the knots are formed through a universal mechanism. such a knot formation mechanism correctly predicts the fraction of the knotted proteins found in nature and can be used to make qualitative prediction on their folding times. shape and motility and also for numerous signaling processes. adhesion is based on non-covalent interactions between transmembrane proteins and the extracellular matrix. cells are able to create two-dimensional assemblies of integrins, so called focal adhesions, which they use to stick to the substrate and collect information about the environmental properties. the goal of this work is a deeper understanding of the formation and the stability of these adhesion clusters. bond cluster formation and disintegration is dynamically modeled with the aid of monte carlo simulations. in the model, a membrane is attached to a flat surface via a variable number of adhesion bonds. the spacial configuration of these adhesion points subjected to an inhomogeneous stress field maps into a distribution of local membrane/ surface distances. we introduce a model which explicitly accounts for the membrane elasticity and demonstrate that such models are able to explain the spontaneous formation of adhesion bond clusters. structure based models are successful at conjugating the essence of the energy landscape theory of protein folding [ ] with an easy and efficient implementation. recently their realm expanded beyond single protein structure, been used profitably to widely study large conformational transitions [ ] [ ] . still, when dealing with conformational transition between two well-defined structures an unbiased and realistic description of the local backbone and sidechain interactions is necessary. the proposed model merges a precise description of these interactions with a structure-based long range potential that takes into account different conformers. we present the results of the activation of the catalytic domain of human csrc tyrosine kinase for which we reconstructed the transition free energy and the description of the activation loop flexibility. the excellent model performances in terms of speed and the satisfactory accuracy of the description of the system and its flexibility are promising for a more systematic study of the tyrosine kinase family activation mechanisms. [ we introduce a previously undescribed technique for modelling the kinetics of stochastic chemical systems. we apply richardson extrapolation, a sequence acceleration method for ordinary differential equations, to a fixed-step tau-leaping algorithm, to produce an extrapolated tau-leaping method which has weak order of accuracy two. we prove this mathematically for the case of linear propensity functions. we use four numerical examples, two linear and two nonlinear, to show the higher accuracy of our technique in practice. we illustrate this by using plots of absolute error for a fixed-step tau-leap and the extrapolated tau-leap. in all cases, the errors for our method are lower than for a fixedstep tau-leap; in most cases they are second order of accuracy. the major tripartite efflux pump acrab-tolc is responsible for the intrinsic and acquired multidrug resistance in escherichia coli. at heart of the extrusion machinery there is the homotrimeric transporter acrb, which is in charge of the selective binding of structurally and chemically different substrates and energy transduction. the effects of conformational changes, which have been proposed as the key features of the extrusion of drugs, are investigated at molecular level using different computational methods like targeted molecular dynamics. simulations, including almost half a million atoms, have been used to assess several hypotheses concerning the structure-dynamics-function relationship of the acrb protein. the results indicate that, upon induction of conformational changes, the substrate detaches from the binding pocket and approaches the gate to the central funnel. in addition, we provide evidence for the proposed peristaltic transport involving a zipper-like closure of the binding pocket, responsible for the displacement of the drug. using these atomistic simulations the role of specific amino acids during the transitions can be identified, providing an interpretation of sitedirected mutagenesis experiments. additionally, we discuss a possible role of water molecules in the extrusion process. virus inhibitory peptide (virip), a amino acid peptide, binds to the fusion peptide (fp) of human immunodeficiency virus type (hiv- ) gp and blocks viral entry. molecular dynamics (md) and molecular mechanics/poisson-boltzmann surface area (mm/pbsa) free energy calculations were executed to explore the binding interaction between several virip derivatives and gp fp. a promising correlation between antiviral activity and simulated binding free energy was established thanks to restriction of the flexibility of the peptides, inclusion of configurational entropy calculations and the use of multiple internal dielectric constants for the mm/pbsa calculations depending on the amino acids sequence. bases on these results, a virtual screening experiment was carried out to design enhanced virip analogues. a selection of peptides was tested for inhibitory activity and several improved virip derivatives were identified. these results demonstrate that computational modelling strategies using an improved mm/pbsa methodology can be used for the simulation of peptide complexes. as such, we were able to obtain enhanced hiv- entry inhibitor peptides via an mm/pbsa based virtual screening approach. an essential step during the hiv life cycle is the integration of the viral cdna into the human genome. hiv- integrase mediates integration in a tight complex with the cellular cofactor: ledgf/p [ ] . disruption of the interaction interferes with hiv replication and therefore provides an interesting new drug target for antiretroviral therapy [ , ] . here we present the structure based discovery and optimization of a series of small molecule inhibitors that bind to hiv- integrase and block the interaction with ledgf/p . the work flow was set up according to a funnel principle in which a series of virtual screening tools were applied in such way to discard at each step molecules unlikely to be active against the desired target (including d filtering, pharmacophore modelling and molecular docking) the activity and selectivity of the selected molecules were confirmed in an alpha screen based assay, that measure protein-protein interaction in vitro, and furthermore by in vivo experiments. active compounds proceeded towards crystallographic soaking into the receptor protein crystals. these crystal structures not only validated the binding mode and activity of the hit compounds, but furthermore were used as a platform for structure based drug design which resulted in the rational discovery of new hit compounds and optimized lead compounds. in vitro and in vivo experiments validated the mechanism of action of these compounds and show that they are a novel class of antiretroviral compounds with in vivo inhibitory activity by targeting the interaction between ledgf/p and hiv- integrase. cross resistance profiling indicate that these compounds are active against current resistant viral strains. [ ] currently the most potent inhibitors show an in vivo ic of nm. these compounds are promising for future pharmaceutical optimizations to be used in the clinic as new antiretroviral agents. crystallography was used to validate the binding mode of the discovered inhibitors. insights in the interaction of the ligand-protein complex allowed for rational design of optimized inhibitors. ligand-induced structural changes are small, thermal fluctuations can play a dominant role in determining allosteric signalling. in thermodynamic terms, the entropy change for subsequent binding is influenced by global vibrational modes being either damped or activated by an initial binding event. one advantage of such a mechanism is the possibility for long range allosteric signalling. here, changes to slow internal motion can be harnessed to provide signalling across long distances. this paper considers homotropic allostery in homodimeric proteins, and presents results from a theoretical approach designed to understand the mechanisms responsible for both cooperativity and anticooperativity. theoretical results are presented for the binding of camp to the catabolite activator protein (cap) [ ] , where it is shown that coupling strength within a dimer is of key importance in determining the nature of the allosteric response. results from theory are presented along side both atomistic simulations and simple coarse-grained models, designed to show how fluctuations can play a key role in allosteric signalling in homodimeric proteins. [ reversibly switchable fluorescent proteins (rsfps) can be switched between a fluorescent (on) and a nonfluorescent (off) state which is accompanied by a cis-trans isomerization of the chromophore on the molecular level , . this unique property has already provided new aspects to various microscopy techniques, such as high resolution microscopy, fcs or monochromatic multicolor microscopy - . despite of their established potential, rsfps still have a major drawback: the wavelength for fluorescence excitation is always one of the two switching wavelengths. the imaging process thus inevitably results in the switching of a small fraction of the rsfps, which might hinder or complicate some experiments. we developed a new reversibly switchable fluorescent protein which eliminates the problem of the coupling between switching and fluorescence excitation. this fluorescent protein follows an unusual and currently unknown mechanism of switching between a fluorescent and a nonfluorescent state. it is brightly fluorescent and exhibits an excellent signal to noise ratio. in parallel studies [ ] , qd-based ligands (egf, mabs) were targeted to egfr in gliomas. cell-cultures, animal models and ex vivo biopsies of human high-grade as well as low-grade gliomas showed high probe specificity.. the aim is to define more precisely the tumor boundaries at the time of resection. we used the programmable array microscope designed for sensitive, high-speed optical sectioning, particularly of living cells. the pam is based on structured illumination and conjugate detection using a digital micromirror device (dmd) [ ] located in a primary image plane. the unique feature is the rapid, (re)programmable adjustment of local excitation intensity, dynamic, on-the-fly optimization is thus achieved, e.g. multipoint frap [ ] , light exposure minimization and object tracking [ ] , or super-resolution strategies. the features and operation of the rd generation pam will be presented. contraction of muscle cells, motility of microorganisms, neuronal activity, and other fast cellular processes require microscopic imaging of a three-dimensional ( d) volume with a video-rate scanning. we present d video-rate investigations of structural dynamics in biological samples with the multicontrast third-and second-harmonic generation as well as fluorescence microscope. the multidepth scanning is achieved by two combined laser beams with staggered femtosecond pulses. each of the beams is equipped with a pair of deformable mirrors for dynamic wavefront manipulation enabling multidepth refocusing with simultaneous corrections for optical aberrations. combined, more than frames per second lateral scanning with fast refocusing enables the d video-rate imaging of dynamically moving structures. in addition, combination of two laser beams is accomplished at two perpendicular polarizations enabling live imaging of sample anisotropy, which is important for structural studies particularly with the second harmonic generation microscopy. investigations of beating chick embryo hearts with the d video-rate scanning microscope revealed multidirectional cardiomyocyte contraction dynamics in myocardial tissue. intricate synchronization of contractions between different layers of myocytes in the tissue will be presented. the video-rate d microscopy opens new possibilities of imaging fast biological processes in living organisms. confocal fluorescence microscopy is an invaluable tool to study biological systems at cellular level also thanks to the synthesis of always new specific fluorescent probes, e.g. multiprobe labelling enables complex system characterization. however, only the recent employment of narrowband tunable filters overcomes the problems due to the use of the broadband ones. the possibility of acquiring the emission spectra in a spatially resolved way extends simple image intensity studies into characterization of complex probeenvironment relationship through the sensitivity of fluorescence spectra to the local molecular environment differences. consequently, fluorescence microspectroscopy (fms) is able to provide the spectral information in a well defined spatial region allowing the researcher to simultaneously obtain spatial and spectroscopic information. our instrument has been specially built to study live cells and their interaction with nanomaterials, drug carriers and modified cell environment. other main characteristics are reducing the bleaching effect and employing a white light source that does not limit the use to specific probes. graphical tools, such as colour coded images, have also been introduced to provide explicit and straightforward visual information. high speed fpga based multi-tau correlation for singlephoton avalanche diode arrays jan buchholz , , jan krieger we demonstrate the use of fret-imaging (forster resonance energy transfer) as an assay to directly monitor the dynamics of cross-bridge conformational changes in single fibres of skeletal muscle. we measured nm-distances of several fret pairs located at strategic positions to sense myosin head conformational changes: we focused our attention on the essential light chain, elc (specifically labelling a modified elc and exchanging it with the natural elc of the fibre) and we investigated its interaction with the sh helix, with the nucleotide binding pocket and with actin. we characterized fret in single rigor muscle fibres, determining distances in agreement with those from the crystallographic data. the results demonstrate the viability of the approach in sensing different fret efficiencies over a few nanometres, an essential requirement to follow the expected small fret variations in contracting muscle fibres. we are now performing dynamic experiments on rigor and active fibres by applying small stretch/release cycles to alter the interaction distances (estimated time resolution of nearly ms/frame). in this configuration, it will be possible to measure functional changes, shedding light on the myosin head dynamics during contraction. focal stimulation of cultured neurons is crucial since it mimics physiological molecules release. indeed, the nervous system finely tunes the activity of each synapse by regulating the secretion of molecules spatially and temporally. currently used techniques have some drawbacks such as a poor spatial resolution or a low flexibility. we propose a novel approach based on optical tweezers (ot) [ ] to overcome these limitations. ot allow an ease manipulation with sub-micrometric precision of silica beads, which can be functionalized with any protein. for a proof-of-principle study we coated , lm large beads with brain-derived neurotrophic factor (bdnf) or bovine serum album (bsa) as control. we showed that a single bead was able to activate the bdnf receptor trkb, inducing its phosphorylation. moreover bdnf beads but not control beads were able to induce c-fos translocation into the nucleus [ ] , indicating that the whole pathway was activated. finally, we positioned vectors in proximity to the growth cones of cultured hippocampal neurons [ ] . control beads didn't affect the normal development of these structures while bdnf beads significantly did. these findings support the use of the ot technology for long-term, localized stimulation of specific subcellular neuronal compartments. a key role of its photoactivity, due to the singlet oxygen production, which has a very short lifetime (ns-ls, depending of hyp environment). hyp sub-cellular localization depends on its concentration in the medium, incubation time and used delivery system. variations in activity of protein kinase c, (anti-apoptotic pkca and pro-apoptotic pkcd) in correlation with the activity of bcl- protein, cytochrome c release from mitochondria and decreasing of mitochondrial membrane potential after photodynamic action were monitored. the study was performed for two different delivery modes of hyp to u- mg glioma cells-hyp alone (membrane diffusion) vs. hyp loaded in low density lipoprotein (ldl) (endocytosis). confocal fluorescence microscopy, flow-cytometry and specific fluorescence labeling were used as main experimental techniques. our results show that hyp photoaction strongly affects apoptotic response of the cells and that the dynamics of this action significantly depends on used delivery system. correlation analysis between the monitored parameters (see above) determined for both delivery system is presented and critically discussed. surface contamination by bacteria is a natural and spontaneous process occurring in any natural environment on biotic (mucosa, tissues…) and abiotic surfaces (medical equipments, food surfaces…). whatever the bacterial nature (gram-positive or -negative), the environmental fluid (air, water, blood…) and the receptor surface (implants, medical equipments, food surfaces…), the surface contamination initiated by the first adherent bacteria can evolve to a three dimensional structure named biofilm (cohesive bacteria assembly ensured by an autoproduced extracellular organic matrix). the mechanisms by which these biofilms offer protective environment to viral particles or hypertolerance to antimicrobial action are not yet elucidated. to reach a better understanding of biofilm reactivity, we reported for the first time successful applications of correlative time-resolved optical microscopy approach by time-lapse (tl), frap, fcs, flim, for real-time analysis of molecular mobility and reaction inside biofilms. by means of non-biological or biological (virus, biocides and antibiotics) reactive compounds, significant advances to understand the roles of the extracellular matrix and bacteria physiological properties were obtained, an important step to improve pathogenic biofilm inactivation. here we present a feasibility study to develop two-photon microscopy ( pm) into a standard diagnostic tool for noninvasive, skin cancer diagnosis. the goal is defining experimental parameters to maximize image quality of optical biopsies in human skin, while avoiding tissue damage. possible diagnostic indicators will be compared for healthy tissue, benign, and malignant melanocytic lesions. we report on preliminary results of a study on pm imaging of ''ex-vivo'' biopsy samples, were autofluorescence intensity and contrast between lesion and surrounding tissues were optimised varying excitation wavelength, detection band, and dispersion pre-compensation. moreover, we determined modulation functions for laser power and detector gain to compensate losses in deep tissue imaging. as the main process of photo-damage, thermo-mechanical modifications were quantified and damage threshold powers were determined. in order to image structural changes in ordered tissue like collagen fibres, second-harmonic generation signals were recorded and optimised. in-vivo two-photon imaging of the honeybee antennal we adapted a two-photon microscope for in-vivo imaging of the honeybee olfactory system, focusing on its primary centres, the antennal lobes. the setup allowed obtaining both d-tomographic measurements of the antennal lobe morphology and time-resolved in-vivo calcium imaging of its neuronal activity. the morphological data were used to precisely measure the glomerular volume in both sides of the brain, investigating the question of volumetric lateralization. functional calcium imaging allowed to record the characteristic glomerular response maps to external odour stimuli applied to the bees' antennae. compared to previous neural imaging experiments in the honeybee, this work enhanced spatial and temporal resolution, penetration depth, and it minimized photo-damage. final goal of this study is the extension of the existing functional atlases of the antennal lobe to d and into the temporal dimension by investigating the time-resolved activity pattern. the use of voltage sensitive fluorescent dyes (vsd) for noninvasive measurement of the action potential (ap) in blood perfused heart have been hindered by low interrogation depth, high absorption and auto-fluorescence of cardiac tissue. these limitations are diminished using new near infrared (nir) vsd (di- -anbdqbs). here we validated toxicity and photo-toxicity of these dyes in guinea pig and human cardiac muscle slabs. application of nir vsd showed no effect on cardiac muscle contraction force or relaxation. optical action potentials closely tracked kinetics of microelectrode recorded aps in both field and electrode stimulated preparations. for phototoxicity assessment dye ( lm) preloaded cardiac slabs were exposed to prolonged laser radiation of various power. microelectrode ap recordings show that exposure to prolonged laser radiation ( min.; mw/mm ) of dye loaded tissue had no statistically significant effect on apd or conduction velocity, thus indicating no or weak photo-toxicity on the nir vsd. in contrast, exposure to min. laser radiation of phototoxic dyes (mitotracker deep-red) preloaded tissue caused significant reduction in apd (by %) and conduction velocity ( %). thus, due to the low photo-toxicity, nir vsd are well suited for in vivo cardiac imaging. streptomycesetes are filamentous gram-positive soil bacteria well known for their complex morphological development and secondary metabolite production. during their life cycle spores germinate to form a network of hyphae, which later develops into aerial mycelium when cross-walls are generated and spores are formed. we have examined and compared the last stage of the differentiation process in a wild-type s. coelicolor (m ) and its dcabb mutant lacking a calmodulin-like calcium binding protein. the strains were grown on four kinds of media: smms, smms with % saccharose, r and r with reduced calcium in order to study the effect of environment and osmotic stress on the sporulation of the two strains to assess the function of cabb protein. from the cultures pictures were taken at hours and after days using phase contrast, atomic force and confocal laser scanning microscope and the sizes of spores were measured. our results showed that the dcabb mutant made smaller spores, its differentiation and stress response were slower. we could conclude from it that the aberrant protein slows the metabolism, the signal transduction and has effects on sporulation, septation and air-mycelium formation. based on it we can tell that the cabb has a significant role in normal development. the mobility and reaction parameters of molecules inside living cells can be conveniently measured using fluorescent probes. typically fluorescence correlation spectroscopy (fcs) based on confocal microscopy is used for such measurements. this implies high time-resolution but only for a single spot at a time. in order to achieve a high time-resolution at multiple spots, we built a single plane illumination microscope (spim) equipped with high-speed image acquisition devices and a high-na detection optics. this allows us to do parallel fcs measurements in a thin plane (width * - lm) of the sample. our setup is equipped with a fast emccd camera (full frame time resolution ls) and a pixel array of spads. the spad array has a full frame time resolution of - ls, which is even fast enough to resolve the typical motion time-scale of small molecules (like egfp) inside living cells. the performance of the system is characterized by diffusion measurements of water-soluble fluorescent beads, as well as fcs measurements in living cells. our data acquisition system uses programmable hardware for some tasks and is fast enough to allow real-time correlation of pixels, as well as saving the complete dataset for later evaluation. electron cryo-microscopy (cryo-em) covers a larger size range than any other technique in structural biology, from atomic resolution structures of membrane proteins, to large noncrystalline single molecules, entire organelles or even cells. electron crystallography of two-dimensional ( d) crystals makes it possible to examine membrane proteins in the quasi-native environment of a lipid bilayer at high to moderately high resolution ( . - Å ). recently, we have used electron crystallography to investigate functionally important conformational changes in membrane transport proteins such as the sodium/proton antiporters nhaa and nhap, or the structure of channelrhodopsin. ''single particle'' cryo-em is well suited to study the structure of large macromolecular assemblies in the . to Å resolution range. a recent example is our Å map of a mitochondrial respiratory chain supercomplex consisting of one copy of complex , two copies of complex iii, and one of complex iv. the fit of the x-ray structures to our map indicates short pathways for efficient electron shuttling between complex i and iii by ubiquinol, and between complex iii and iv by cytochrome c. electron cryo-tomography can visualize large protein complexes in their cellular context at - Å resolution, and thus bridges the gap between protein crystallography and light microscopy. cryo-et is particularly suitable for studying biological membranes and large membrane protein complexes in situ. we found that long rows of atp synthase dimers along ridges of inner membrane cristae are an ubiquitous feature of mitochondria from all species we investigated ( mammals, fungi, plant). the proton pumps of the respiratory chain appear to be confined to the flat membrane regions on either side of the ridges. this highly conserved pattern suggests a fundamental role of the mitochondrial cristae as proton traps for efficient atp synthesis. single-particle analysis: advanced fluorescence imaging, including subdiffraction microscopy, relies on fluorophores with controllable emission properties. chief among these fluorophores are the on/off photoswitchable and green-to-red photoconvertible fluorescent proteins. irisfp was recently reported as the first fluorescent protein to combine irreversible photoconversion from a green-emitting to a red-emitting state with reversible on/off photoswitching in both the green and red states. the introduction of this protein resulted in new applications such as super-resolution pulse-chase imaging, but the properties of irisfp are far from being optimal from a spectroscopic point of view and its tetrameric organization complicates its use as a fusion tag. we have demonstrated how four-state optical highlighting can be rationally introduced into photoconvertible fluorescent proteins by developing and characterizing a new set of such enhanced optical highlighters derived from meo-sfp and dendra . one of these, which we called nijifp, was identified as a promising new multi-photoactivatable fluorescent protein with optical properties that make it ideal for advanced fluorescence-based imaging applications. introducing to medicine and biology concept of optical markers in tremendous way has changed the recent status of these two important disciplines. this was mainly due to strong development in imaging techniques which recently allow us to investigate both static as well dynamic properties of living cells, their components and their interactions with external factors. recently used molecular markers including organic dyes, fluorescent proteins or chelates containing lanthanide ions have several significant limitations. one of the alternatives for molecular markers are inorganic quantum dots (ie. cdse, cds) which are recently commonly used in many academic works. however, even if they are much better from physicochemical point of view, from the application point of view at this moment they are rather useless mainly because of their high risk of toxicity. one of the solution combining advantages of both concepts is to make nontoxic inorganic nanocrystals doped by lanthanide ions. in this work, we will present optical results obtained for nayf nanocrystals doped by different lanthanide ions. the aim of this work was to design and to synthesize these markers and to understand physical processes responsible for their emission and to optimize these processes to the physical limits. intravital microscopy has fostered a full blown of publication regarding the behavior of cells in different tissues and physiological conditions. however, a few papers describe how the motility parameters can be used to understand whether an interaction is occurring and, on balance, the distinction between interacting and non interacting cells is performed visually on the image time stack. here we describe a multi-parameter approach that allows to discern among different cell behaviors on an objective ground and we demonstrate its effectiveness valuating the mutual fate of natural killer (nk) and dendritic (dc) cells at the draining lymph-nodes in inflammatory and stationary conditions. the method is time saving and allows a wide scale characterization of the lymphocyte tracks and to build up statistics of the cell-cell interaction duration. this has allowed the development of a numerical model of the nk-dc interaction, based on a molecular-stochastic dynamic approach, whose output can be directly compared to the data. hemozoin is formed during malaria infection of red blood cells, the malaria parasite cleaves hemoglobin, leaving free heme which is then toxic to the parasite. the free heme is then bio-crystalized to form hemozoin which allows the parasite to remain viable. the hemozoin is released during the breakdown of the red blood cells, is small and can be difficult to resolve spatially. since it contains an abundance of heme protein, which has a strong absorbance at nm, it can be readily detected and tracked by using resonant raman scattering spectroscopy. here we use slit-scanning confocal raman imaging to detect the hemozoin and resolve it against the background molecules. inside a red blood cell, hemoglobin is the strongest background signal since it also contains large amounts of heme. nevertheless, the discrimination is possible, and the time-resolved observation of hemozoin is an important tool to understand effects of malaria since the hemozoin can trigger the immune response and cause inflammation in tissue. muscle performance at the molecular level is determined by the elementary displacement (working stroke) produced by the motor protein myosin ii, and its dependence on load. we developed a laser trap assay (the optical leash) capable of applying controlled loads to a single myosin head before the working stroke is initiated and probing actin-myosin interaction on the microsecond time scale. we found that the working stroke size depends both on load and on the detachment pathway followed by myosin. in a first pathway, myosin detaches very rapidly from actin (\ ms) without producing any movement. in a second pathway, myosin steps and remains bound to actin for a time inversely proportional to atp concentration; the working stroke remains constant (* nm) as the load is increased, until it suddenly vanishes as the isometric force is reached ( . ± . pn). a third dissociation pathway becomes more populated as the force is increased, due to premature unbinding of myosin from actin, resulting in a working stroke that decreases with load. taken together, these results give a new insight into the molecular mechanism of load dependence of the myosin working stroke, which is a primary determinant of skeletal muscle performance and efficiency. previously we have deleted either or both of these terminal helices genetically. surprisingly, all mutants rotated in the correct direction, showing that the shaft portion is dispensable. here we inquire if the rest of the c rotor, the globular protrusion that accounts for * % of the c residues, is also dispensable. keeping the n-and c-terminal helices that constitute the shaft, we have replaced the middle * residues with a short helix-turn-helix motif borrowed from a different protein. the protrusion-less mutant thus made retained a high atpase activity and rotated fast in the correct direction. this may not be unexpected because, in crystal structures, most of the removed residues do not contact with the a b ring. combined with the previous results, however, the present results indicate that none of the c residues are needed for rotation. the rotary mechanism of a molecular engine, the vacuolar proton-atpase, working in a biomembrane csilla ferencz , pá l petrovszki , zoltá n kó ta the rotary mechanism of vacuolar proton-atpase (v-atpase) couples atp hydrolysis and trans-membrane proton translocation. we tested the effect of oscillating electric (ac) field on v-atpase activity in yeast vacuoles. the ac technique has several advantages over direct observations: it can be applied on native membranes, there are no labels and attachments involved, and the target protein is in its natural environment. this was/is the first of its kind of experiment on v-atpase, and we got strikingly different results from previous studies on other proteins: both low and high frequency ac field reduces atpase activity in a wide frequency range. a sharp resonance is seen at . hz, where the atpase activity reaches or exceeds the control (no ac) level. we think that the resonance happens at that of the degrees rotor steps, meaning that the rotation rate of the rotor is around hz, under the given conditions. synchronisation of individual atpases by slow or matching, but not fast ac is likely via a hold-and-release mechanism. we can explain the above observations by assuming that the ac field interacts with the proton movements, and if we consider the estimated geometry of the hydrophilic proton channels and the proton binding cites on the rotor. [ ] . the ttss constitutes a continuous protein transport channel of constant length through the bacterial envelope [ ] . the needle of the type three secretion system is made of a single small protein (protomer). we analyzed the assembly and the structure of the ttss needle using different biophysical methods including fourier transform-infrared spectroscopy, nmr spectroscopy and x-ray crystallography. we show that the ttss needle protomer refolds spontaneously to extend the needle from the distal end. the protomer refolds from a-helix into b-strand conformation to form the ttss needle [ ] . regulated secretion of virulence factors requires the presence of additional protein at the ttss needle tip. x-ray crystal structure analysis of the tip complex revealed major conformational changes in both the needle and the tip proteins during assembly of the s. typhimurium ttss. our structural analysis provides the first detailed insight into both the open state of the ttss needle tip and the conformational changes occurring at the pathogen-host interface [ ] . [ the membrane-bound component f o of the atp synthase works as a rotary motor and plays the central role of driving the f component to transform chemi-osmotic energy into atp synthesis. we have conducted molecular dynamics simulations of the membrane-bound f o sector with explicit lipid bilayer, in which the particular interest was to observe the onset of helix motion in the c ring upon the change of the protonation state of asp of the c subunit, which is the essential element of the boyer's binding-change mechanism. to investigate the influence of transmembrane potential and ph gradient, i.e., the proton motive force, on the structure and dynamics of the a-c complex, different electric fields have been applied along the membrane normal. correlation map analysis indicated that the correlated motions of residues in the interface of the a-c complex were significantly reduced by external electric fields. the deuterium order parameter (s cd ) profile calculated by averaging all the lipids in the f o -bound bilayer was not very different from that of the pure bilayer system, which agrees with recent h solid-state nmr experiments. however, by delineating the lipid properties according to their vicinity to f o , we found that the s cd profiles of different lipid shells are prominently different. lipids close to f o formed a more ordered structure. similarly, the lateral diffusion of lipids on the membrane surface also followed a shell-dependent behavior. the lipids in the proximity of f o exhibited very significantly reduced diffusional motion. the numerical value of s cd was anti-correlated with that of the diffusion coefficient, i.e., the more ordered lipid structures led to slower lipid diffusion. our findings will not only help for elucidating the dynamics of f o depending on the protonation states and electric fields, but may also shed some light to the interactions between the motor f o and its surrounding lipids under physiological condition, which could help to rationalize its extraordinary energy conversion efficiency. this work has been published in march , and it was selected as one of the two featured articles of that issue. the research project is directed toward the construction of a synthetic bio-inorganic machine that consists in a single actin filament that interacts with a linear array of myosin ii motors regularly disposed on a nano-structured device. the motor array is intended to simulate the unique properties of the ensemble of motor proteins in the half-sarcomere of the muscle by providing the condition for developing steady force and shortening by cyclic interactions with the actin filament. the mechanical outputs in the range . - pn force and - , nm shortening will be measured and controlled the bacterial flagellar motor is a membrane-embedded molecular machine that rotates filaments, providing a propulsive force for bacteria to swim. the molecular mechanism of torque (turning force) generation is being investigated through the study of the properties and three-dimensional structure of the motor's stator unit. we are taking both topdown and bottom-up approaches, combining data from the molecular genetics studies, cross-linking, x-ray protein crystallography and molecular dynamics simulations. we have recently determined the first crystal structure of the protein domain that anchors the proton-motive-force-generating mechanism of the flagellar motor to the cell wall, and formulated a model of how the stator attaches to peptidoglycan. the work presented at the meeting will inform the audience on our latest work that establishes the relationship between the structure, dynamics and function of a key component of the bacterial flagellar motor, the motility protein b (motb). this work will be put in the perspective of the mechanism of rotation, stator assembly, anchoring to peptidoglycan and interaction with the rotor, and discussed in the light of the elementary events composing the cycle of electrochemical-to-mechanical energy conversion that drives flagellar rotation. members of the conserved kinesin- family fulfill essential roles in mitotic spindle morphogenesis and dynamics and were thought to be slow, processive microtubule (mt)-plusend directed motors. the mechanisms that regulate kinesin- function are still not well understood. we have examined in vitro and in vivo functions of the saccharomyces cerevisiae kinesin- cin using single-molecule motility assays and single-molecule fluorescence microscopy and found that cin motility is exceptional in the kinesin- family. in vitro, individual cin motors could be switched by ionic conditions from rapid (up to lm/min) and processive minus-end, to bidirectional, to slow plus-end motion. deletion of the uniquely large insert of amino acids in loop of cin induced bias towards minus-end motility and strongly affected the directional switching of cin both in vivo and in vitro. we further found that deletion of the functionally overlapping kinesin- kip and of the spindle-organizing protein ase affected cin velocity and processivity, but directionality was not affected. the entirely unexpected finding of switching of cin directionality in vivo and in vitro demonstrates that the ''gear box'' of kinesins is much more complex and versatile than thought. many biological motor molecules move within cells using stepsizes predictable from their structures. myosin-vi, however, has much larger and more broadly-distributed stepsizes than those predicted from its short lever arms. we explain the discrepancy by monitoring qdots and gold nano-particles attached to the myosin vi motor domains using high-sensitivity nano-imaging. the large stepsizes were attributed to an extended and relatively rigid lever arm; their variability to two stepsizes, one large ( nm) and one small ( nm). these results suggest there exist two tilt-angles during myosin-vi stepping, which correspond to the pre-and post-powerstrokes states and regulate the leading head. the large steps are consistent with the previously reported hand-over-hand mechanism, while the small steps follow an inchworm-like mechanism and increase in frequency with adp. switching between these two mechanisms in a strain sensitive, adpdependent manner allows myosin-vi to fulfill its multiple cellular tasks including vesicle transport and membrane anchoring. http://www.fbs.osaka-u.ac.jp/labs/yanagida/, http:// www.qbic.riken.jp/. ferritin deposits iron in oxyhydroxide iron core surrounded by protein shell. the iron core structure may vary in different ferritins in both normal and pathological cases. to study iron core variations the mö ssbauer spectroscopy with a high velocity resolution was applied for comparative analysis of normal and leukemia chicken liver and spleen tissues, human liver ferritin and commercial pharmaceutical products imferon, maltoferÒ and ferrum lek as ferritin models. mö ssbauer spectra of these samples measured with a high velocity resolution at room temperature were fitted using two models: homogenous iron core (one quadrupole doublet) and heterogeneous iron core (several quadrupole doublets). the results of both fits demonstrated small variations of mö ssbauer hyperfine parameters related to structural variations of the iron cores. these small structural variations may be a result of different degree of crystallinity, the way of iron package, nonequivalent iron position, etc. obtained small differences for normal and leukemia tissues may be useful for distinguishing ferritins from normal and pathological cases. this work was supported in part by the russian foundation for basic research (grant # - - -a). invasion of epithelial cells by salmonella enterica is mediated by bacterial ''effector'' proteins that are delivered into the host cell by a type iii secretion system (ttss). the collaborative action of these translocated effectors modulates a variety of cellular processes leading to bacterial uptake into mammalian cells. type iii effectors require the presence in the bacterial cytosol of specific tts chaperones. effectors are known to interact with their chaperone via a chaperone binding domain (cbd) situated at their n-terminus. this work focus on sopb, an effector with phosphoinositide phosphatase activity and particularly its interaction with the specific chaperone sige by biochemical, biophysical and structural approaches. we have co-expressed sopb with its specific chaperone sige and purified the complex, determined the limits of the cbd and purified the sopb cbd /sige complex. the structure of sige has been solved previously but no crystals could be obtained for structure determination of both complexes. we used saxs experiment combined with biophysical approach to analyse the interaction between sopb and its chaperone as well as the quaternary structure on the complex that will be described in this presentation. guanylate monophosphate kinase (gmpk) is a cytosolic enzyme involved in nucleotide metabolic pathways. one of the physiological roles of gmpks is the reversible phosphoryl group transfer from atp to gmp (its specific ligand), yielding adp and gdp. the gmpk from haemophilus influenzae is a small protein, with a number of amino acids in the primary structure. in order to determine the secondary structure changes of this enzyme, as well as some physical characteristics of its complexes with gmp and atp ligands, circular dichroism (cd) and atr -ftir studies were performed. the enzyme and its ligands were dissolved in tris -hcl buffer, at ph . and °c. from the cd spectra the content of the secondary structure elements of gmpk and gmpk/gmp, gmpk/atp (with and without mg + ) were determined. the major secondary structure elements of gmpk from haemophilus influenzae were a-helix (* %) and b-sheet (* %). atr -ftir experiments show that the amide i and amide ii bands of the gmpk are typical for a protein with great a-helix content. from the second derivative spectra, the content of the secondary structure elements were estimated. these data were in agreement with those obtained by cd. assembly of the mature human immunodeficiency virus type capsid involves the oligomerization of the capsid protein, ca. the c-terminal domain of ca, ctd, participates both in the formation of ca hexamers, and in the joining of hexamers through homodimerization. intact ca and the isolated ctd are able to homodimerize in solution with similar affinity (dissociation constant in the order of lm); ctd homodimerization involves mainly an a-helical region. in this work, we show that peptides derived from the dimerization helix (which keep residues energetically important for dimerization and with higher helical propensities than the wild-type sequence) are able to self-associate with affinities similar to that of the whole ctd. moreover, the peptides have a higher helicity than that of the wild-type sequence, although is not as high as the theoretically predicted. interesting enough, the peptides bind to ctd, but binding in all peptides, but one, does not occur at the dimerization interface of ctd (helix ). rather, binding occurs at the last helical region of ctd, even for the wild-type peptide, as shown by hsqc-nmr. as a consequence, all peptides, but one, are unable to inhibit capsid assembly of the whole ca in vitro. the peptide whose binding occurs at the ctd dimerization helix has an val?arg mutation in position , which has been involved in dimer-dimer contacts. these findings suggest that event keeping the energetically important residues to attain ctd dimerization within a more largely populated helical structure is not enough to hamper dimerization of ctd. putp is an integral membrane protein located in the cytoplasmic membrane of escherichia coli, being responsible for the coupled transport of na + and proline. it belongs to the family of sodium solute symporters (sss). structural data for putp is not available, but secondary structure predictions together with biochemical and biophysical analyses suggest a transmembrane motif. from a recent homology model based on the x-ray structure of the related na + /galactose symporter vsglt, previously published electron paramagnetic resonance (epr) studies, and recent crystallographic and epr studies on the cognate bacterial homolog of a neurotransmitter:na + symporter, leut, it has been proposed that helices viii and ix as well as the interconnecting ''loop '' region determine the accessibility of the periplasmic cavities which bind sodium and proline. we performed site-directed spin labeling of ''loop '' in combination with epr spectroscopy to investigate the structural features of this region and possible conformational changes induced by sodium and proline. analyses of spin label mobility and polarity as well as accessibility to paramagnetic quenchers allow us to refine this region in the present homology model. furthermore, our data suggest conformational changes in this region upon substrate binding including an overall motion of a helical segment. fatty acid-binding proteins (fabp) are a family of low molecular weight proteins that share structural homology and the ability to bind fatty acids. the common structural feature is a b-barrel of antiparallel b-strands forming a large inner cavity that accommodates nonpolar ligands, capped by a portal region, comprising two short a-helices. b-fabp exhibits high affinity for the docosahexaenoic (dha) and oleic acid (oa). it is also postulated that b-fabp may interact with nuclear receptors from ppar family. in the present work, we used molecular biology and spectroscopic techniques to correlate structure, dynamics and function. site-directed mutagenesis was used to produce mutants of b-fabp with a nitroxide spin probe (mtsl) selectively attached to residues located at the portal region. esr spectra of the labeled b-fabp mutants were sensitive to the location of the mutation and were able to monitor interactions in three cases: ( ) shsp are ubiquitous proteins involved in cellular resistance to various stress (oxidative, heat, osmotic…). they are able to prevent aggregation of non-native proteins through the ability of forming large soluble complexes and preventing their nonspecific and insoluble aggregation. in consequence to this molecular chaperone function, they can regulate many processes (resistance to chemotherapy, modulation of the cellular adhesion and invasion, inflammatory response in skin), and the modulation of their expression has been found to be a molecular marker in cancers, spermatogenesis, or cartilage degeneration. furthermore, they are involved in several pathologies: myopathies, neuropathies, cancers, cataracts. among the human members (hspb - ), this study focused on hspb (hsp involved in some cancers), hspb (lens specific), hspb (lens, muscle, heart, lung) and hspb -r g (responsible for a desmin-related myopathy and a cataract). as shsp form large, soluble (but polydisperse in mammals) hetero-oligomers, molecular biology, biochemistry, biophysics and bioinformatics were successfully combined to compare the functional/dysfunctional assemblies in order to understand the critical parameters between shsp members depending upon their tissue and cellular localization. ionizing radiation is a type of radiation that contains enough energy to displace electrons and break chemical bonds. this can promote the removal of at least one electron from an atom or molecule, creating radical species, namely, reactive oxygen species (ros) [ , ] . these are often associated with damages at cellular level, such as, dna mutations, cell cycle modifications and, in animal cells, cancer. to overcome this problem, organisms developed different protection/repair mechanisms that enable them to survive to these threats. dna glycosylases are enzymes that are part of base excision repair (ber) system, mainly responsible for dna repair. they can recognize a dna lesion and, in some cases, are able to remove the mutated base. here we propose to study one of those enzymes, endonuclease iii, which contains a [ fe- s] cluster [ , ] . samples were exposed to different doses of uv-c radiation and the effects were studied by electrophoretic and spectroscopic methods. [ na,k-atpase is an integral protein present in the plasma membrane of animal cells, and consists of two main subunits: the a and b. cholesterol is an essential constituent of the animal membrane cells. in order to study the interaction between na,k-atpase and cholesterol, we have used the dsc technique, and a proteoliposome system composed by the enzyme and dppc:dppe, with different percentage in mol of cholesterol. the heat capacity of purified na,k-atpase profile exhibits three transitions with , and kcal/mol at , and °c. multiple components in the unfolding transition could be attributed either to different steps in the pathway or to independent unfolding of different domains. this denaturation of na,k-atpase is an irreversible process. for the proteoliposome, we also observed three peaks, with , and kcal/mol and , and °c. this increase in dh indicates that the lipids stabilize the protein. when cholesterol was used (from to mol %), the first transition was shifted to a lower temperature value around °c. these results confirm that cholesterol has an influence on the packing and fluidity of lipid bilayer and changes in lipid microenvironment alter the thermostability as well as the activity of na,k-atpase. financial support: fapesp. we have undertaken to study the structure and function of peroxisomal multifunctional enzyme type (mfe- ) from different organisms. mfe- is a key enzyme in long-and branched-chain fatty acids breakdown in peroxisomes. it contains two enzymes in the same polypeptide and also consists of differing amount of domains depending on the species. crystal structure and enzyme kinetics of drosophila melanogaster mfe- has revealed the domain assembly and raised a question about existence of a substrate channeling mechanism. small-angle x-ray scattering studies have further resolved the assembly of domains in the human mfe- . mutations in the mfe- -coding gene in humans may cause d-bifunctional protein deficiency -a metabolic disease characterized by accumulation of fatty acyl-coa intermediates due to inactive or residually active mfe- protein. we have also studied the structure, stability and dynamics of such mutant proteins both experimentally and in silico. the latest results on all these studies will be presented. ftsz is a protein that plays a key role in bacterial division, forming a protein ring directly related to the constriction of the membrane. this process has been observed to occur without the help of molecular motors. nonetheless, the details of the self-assembly and subsequent force generation of the septal ring are still obscure. afm observations allows the study of the behaviour of ftsz solutions on a substrate with unprecedented resolution, permitting the identification of individual protein filaments. the different resulting structures can be compared to monte carlo models for a d lattice accounting for the essential interactions between monomers. these include a strong longitudinal bond that allows a limited flexibility (i.e., curvature of the filaments) and a weaker lateral interaction. the work we present follows this approach, focussing on the latest experiments with ftsz mutants. by using these mutants, it is possible to choose the specific region of the monomer that will anchor to the substrate, thus generating new structures that provide an insight into monomer-monomer interactions. in this way, we explore the anisotropy of the lateral bond in ftsz, a factor that has not been taken into account before but may prove to be of importance in fstz behaviour in vivo. modeling protein structures and their complexes with limited experimental data dominik gront university of warsaw, pasteura , - warsaw, poland conventional methods for protein structure determination require collecting huge amounts of high-quality experimental data. in many cases the data (possibly fragmentary and/or ambiguous) on itself cannot discriminate between alternative conformations and a unique structure cannot be determined. small angle xray scattering is an example of such a ''weak'' experiment. the spectrum encodes only several independent degrees of freedom that provide a global description of a molecular geometry in a very synthetic way. in this contribution we utilized both local information obtained from nmr measurements and global description of a macromolecule as given by saxs profile combined with a knowledge-based bimolecular force field to determine tertiary and quaternary structure of model protein systems. saxs curve as well as various kinds of local nmr data such as isotropic chemical shifts and their tensors, j-couplings, rdc, backbone noe and redor from nmr in solid phase are parsed with the ''experimental'' module of bioshell toolkit and utilized by rosetta modeling suite to generate plausible conformations. obtained results show the new protocol is capable to deliver very accurate models. noenet-use of noe networks for nmr resonance assignment of proteins with known d structure dirk stratmann, carine van heijenoort and eric guittet structural genomics programs today yield an increasing number of protein structures, obtained by x-ray diffraction, whose functions remain to be elucidated. nmr plays here a crucial role through its ability to readily identify binding sites in their complexes or to map dynamic features on the structure. an important nmr limiting step is the often fastidious assignment of the nmr spectra. for proteins whose d structures are already known, the matching of experimental and back-calculated data allows a straightforward assignment of the nmr spectra. we developed noenet, a structure-based assignment approach. it is based on a complete search algorithm, robust against assignment errors, even for sparse input data. it allows functional studies, like modeling of protein-complexes or protein dynamics studies for proteins as large as kd. almost any type of additional restraints can be used as filters to speed up the procedure or restrict the assignment ensemble. noenet being mainly based on nmr data (noes) orthogonal to those used in triple resonance experiments (jcouplings), its combination even with a low number of ambiguous j-coupling based sequential connectivities yields a high precision assignment ensemble. we observed, that t. thermophilus isopropylmalate dehydrogenase (ipmdh) has higher rigidity and lower enzyme activity at room temperature than its mesophilic counterpart (e. coli), while the enzymes have nearly identical flexibilities under their respective physiological working conditions, suggesting that evolutionary adaptation tends to maintain optimum activity by adjusting a ''corresponding state'' regarding conformational flexibility. in order to reveal the nature of the conformational flexibility change related to enzymatic activity, we designed a series of mutations involving non conserved prolines specific to thermophilic ipmdh. proline to glycine mutations substantially increased conformational flexibility and decreased conformational stability. the mutant enzyme variants did not show enhanced catalytic activity, but the non arrhenius temperature dependence of enzyme activity of the wild type was abolished. this phenomenon underlines the fact that the delicate balance between flexibility, stability and activity which is required for the environmental adaptation of enzymes can be easily disrupted with mutations even distant from the active site, providing further evidence that optimization of proper functional motions are also a selective force in the evolution of enzymes. the kinetoplastids trypanosoma brucei, t. cruzi and leishmania major are responsible for causing great morbidity and mortality in developing countries. the all a-helical dimeric dutpases from these organisms represent promising drug targets due to their essential nature and markedly different structural and biochemical properties compared to the trimeric human enzyme. to aid in the development of dutpase inhibitors we have been structurally characterizing the enzymes from these species. here we present the structure of the t. brucei enzyme in open and closed conformations, completing the view of the enzymes from the kinetoplastids. furthermore, we sought to probe the reaction mechanism for this family of enzymes as a mechanism has been proposed based on previous structural work but has not received any further verification. the proposed scheme is similar to that of the trimeric enzyme but differs in detail. using tryptophan fluorescence quenching in the presence of the transition state mimic alf we have been able to identify which species is the likely transition state in the reaction. the crystal structure of t. brucei in complex with this transition state analogue confirms the nature of the nucleophilic attack clearly showing how it differs from trimeric enzymes. the structure of factor h-c d complex explains regulation of immune complement alternative pathway circular dichroism (cd) spectroscopy is a widely used technique for studying the secondary structure (ss) of proteins. numerous algorithms have been developed for the estimation of the ss composition from the cd spectra. although, these methods give more or less accurate estimation for proteins rich in a-helical structure, they often fail to provide acceptable results on mixed or b-rich proteins. the problem arises from the diversity of b-structures, which is thought to be an intrinsic limitation of the technique. the worst predictions are provided for proteins of unusual b-structures and for amyloid fibrils. our aim was to develop a new algorithm for the more accurate estimation of ss contents for a broader range of protein folds with special interest to amyloid fibrils. using synchrotron radiation cd (srcd), we were able to collect high quality spectra of amyloid fibrils with good s/n ratios down to nm. the novel reference dataset with spectra that significantly differ from present reference sets, extends the information content for ss determination. our algorithm takes into account the diverse twist of the b-sheets that has a great influence on the spectral features. for the first time, we can reliably distinguish parallel and antiparallel b-structure using cd spectroscopy. monitoring the assembly of the membrane protein insertase alexej kedrov , marko sustarsic , arnold j.m. driessen groningen biomolecular sciences and bioengineering institute, university of groningen, the netherlands, university of oxford, uk molecular forces that govern membrane protein integration and folding remain a major question in current molecular biology and biophysics. each nascent polypeptide chain should acquire its unique three-dimensional folded state within a complex environment formed by the anisotropic lipid membrane and the membrane-water interface. secyeg translocase and members of a recently described yidc/oxa /alb chaperone family are recognized as primary players in the membrane protein genesis. these proteins, so called insertases, serve as membraneembedded molecular pores where the newly synthesized protein is loaded prior its release into the bilayer. here we apply fluorescence correlation spectroscopy to monitor the assembly of the insertase:ribosome:nascent polypeptide chain complexes in solution and reconstituted into nanodiscs and model membranes. results provide insights on molecular mechanisms and dynamics of the insertase functioning. conformational changes during gtpase activity induced self-assembly of human guanylate binding protein revealed by epr spectroscopy correct assembly and regulation of multi-component molecular machines is essential for many biological activities. the type iii secretion system (t ss) is a complex molecular machine that is a key virulence determinant for important gram-negative pathogens including shigella, yersinia and salmonella species [ ] [ ] . the t ss consists of multiple copies of * different proteins (totalling * mda), spans both bacterial membranes and drives insertion of a contiguous pore into the host-cell membrane. virulence factors are secreted via this apparatus directly into the host cell. in all t ss various levels of regulation occur with switching between secretion off and on states overlaid on control of which substrates are secreted. genes involved in a variety of these switches have been identified but the molecular mechanisms underlying these functions is poorly understood. we are studying the t ss of shigella flexneri, the causative agent of dysentery and will present the structure of the so-called ''gatekeeper protein'' mxia. the diacylglycerolacyltransferase (dgat ) is an integral protein from the reticulum endoplasmic membrane that plays an essential role in triacylglyceride synthesis. in cattle, this enzyme is associated to the fat content regulation on milk and meat. in this study, synthetic peptides corresponding to both dgat binding sites (sit and sit ) were designed, purified and employed to investigate the enzyme interaction with substrates and membrane models. different binding specificities in the interaction with phospholipid vesicles and micelles were noted. sit showed to bind more strongly in nonpolar membrane models, while sit was electrostatically attracted to negative phospholipid surfaces. the binding of both peptides was followed by significant conformational changes (like unordered to helix transition) in circular dichroism spectra and a nm blue shift in fluorescence emission. the binding of sit and sit peptides to negative liposome gave dissociation constants (k d ) of and . lm, respectively, and a leakage action -fold higher to sit . the difference in specificity is related to the features of the putative substrates (acyl-coas and diacylglycerol) and can be attributed to the distinct role of each dgat binding site during lipid synthesis. supported by fapesp. ftsz, the bacterial homologue of tubulin, assembles into polymers in the bacterial division ring. the interfaces between monomers include a gtp molecule, although the relationship between polymerization and gtpase activity is still controversial. a set of short ftsz polymers were modelled and the formation of active gtpase structures was monitored using molecular dynamics. only the interfaces nearest the polymer ends exhibited geometry adequate for gtp hydrolysis. conversion of a mid-polymer interface to a close-to-end interface resulted in its spontaneous rearrangement from an inactive to an active conformation. fluorescent proteins (fps) have become extremely valuable tools in the life sciences. due to the latest advances in the light microscopy, there is a steady need for fps with improved spectral properties. mirisfp is a monomeric fp that can be switched reversibly between a bright green fluorescent and a dark state by illumination with light of specific wavelengths [ ] . structurally, this photo-switching is based on a cis-trans isomerization of the chromophore. upon illumination with violet light, mirisfp can be irreversibly photoconverted from the green-emitting to a red-emitting form. the red form can again be switched reversibly between a fluorescent and a dark state. to elucidate the mechanistic details of the photoinduced reactions, we have generated mirisgfp . this variant can still undergo reversible photoswitching, but lacks the ability to photoconvert to the red state so that the photoinduced transitions of the green form can be studied without 'artifacts' due to green-to-red photoconversion. using uv/visible spectroscopy, we have characterized the on-and off-switching processes in great detail. several light-activated reaction pathways have been identified. they are highly intertwined so that the net effect achieved with light of a particular wavelength depends on the relative probabilities to photoinduce the various processes. phosducin (pd) is a g t bc-binding protein that is highly expressed in photoreceptors. pd is phosphorylated in dark-adapted retina and is dephosphorylated in response to light. dephosphorylated pd binds g t protein bc-heterodimer with high affinity and inhibits its interaction with g t a or other effectors, whereas phosphorylated pd does not. therefore pd down-regulates the light response in photoreceptors. phosphorylation of pd at s and s leads to the binding of the - - protein. the - - proteins function as scaffolds modulating the activity of their binding partners and their role in pd regulation is still unclear. the - - protein binding may serve to sequester phosphorylated pd from g t bc or to decrease the rate of pd dephosphorylation and degradation. we performed several biophysical studies of the - - :pd complex. analytical ultracentrifugation was used to determine the complex stoichiometry and dissociation constant. conformational changes of pd induced both by the phosphorylation itself and by - - binding were studied using the time-resolved fluorescence spectroscopy techniques. mö ssbauer spectroscopy with a high velocity resolution was used for comparative study of various oxyhemoglobins for analysis of the heme iron electronic structure and protein structure-function relationship. samples of pig, rabbit and normal human oxyhemoglobins and oxyhemoglobins from patients with chronic myeloleukemia and multiple myeloma were measured using mö ssbauer spectrometric system with a high velocity resolution at k. mö ssbauer spectra were fitted using two models: one quadrupole doublet (model of equivalent iron electronic structure in a-and b-subunits of hemoglobins) and superposition of two quadrupole doublets (model of non-equivalent iron electronic structure in a-and b-subunits of hemoglobins). in both models small variations of mö ssbauer hyperfine parameters (quadrupole splitting and isomer shift) were observed for normal human, rabbit and pig oxyhemoglobins and related to different heme iron stereochemistry and oxygen affinity. small variations of mö ssbauer hyperfine parameters for oxyhemoglobins from patients were related to possible variations in the heme iron stereochemistry and function. the different types of silk produced by orb-weaving spiders display various mechanical properties to fulfill diverse functions. for example, the dragline silk produced by the major ampulate glands exhibits high toughness that comes from a good trade-off between stiffness and extensibility. on the other hand, flagelliform silk of the capture spirals of the web is highly elastic due to the presence of proline and glycine residues. these properties are completely dictated by the structural organization of the fiber (crystallinity, degree of molecular orientation, secondary structure, microstructure), which in turn results from the protein primary structure and the mechanism of spinning. although the spinning process of dragline silk begins to be understood, the molecular events occurring in the secretory glands and leading to the formation of other silk fibers are unknown, mainly due to a lack of information regarding their initial and final structures. taking advantage of the efficiency of raman spectromicroscopy to investigate micrometer-sized biological samples, we have determined the conformation of proteins in the complete set of glands of the orb-weaving spider nephila clavipes as well as in the fibers that are spun from these glands. domain of rgs at . Å resolution was solved. the stoichiometry of - - f /rgs protein complex was elucidated using the analytical ultracentrifugation. to map the interaction between - - f and rgs protein we performed a wide range of biophysical measurements: h/d exchange and cross link experiments coupled to mass spectrometry, fret (fö rster resonance energy transfer) time-resolved fluorescence experiments, time-resolved tryptophan fluorescence spectroscopy and saxs (small angle x-ray scattering) measurements. based on all these results we build d model of - - f /rgs protein complex. our model revealed new details on architecture of complex formed by - - proteins. to date all known structure of - - proteins complexes suggests that the ligand is docked in the central channel of - - protein. our results indicate that the rgs domain of rgs protein is located outside the central channel of - - f protein interacting with less-conserved residues of - - f. the receptor for advanced glycation end-products (rage) is a multiligand cell surface receptor involved in various human diseases. the major alternative splice product of rage comprises its extracellular region that occurs as a soluble protein (srage). although the structures of srage domains were available, their assembly into the functional full length protein remained unknown. here we employed synchrotron small-angle x-ray scattering to characterize the solution structure of human srage. the protein revealed concentration-dependent oligomerization behaviour, which was also mediated by the presence of ca + ions. rigid body models of monomeric and dimeric srage were obtained from the scattering data recorded at different solvent conditions. the monomer displays a j-like shape while the dimer is formed through the association of the two n-terminal domains and has an elongated structure. the results provided insight into the assembly of i) the heterodimer srage:rage, which is responsible for blockage of the receptor signalling, and ii) rage homodimer, which is necessary for signal transduction, paving the way for the design of therapeutical strategies for a large number of different pathologies. clpb is a hexameric aaa? atpase that extracts unfolded polypeptides from aggregates by threading them through its central pore. the contribution of coiled-coil m domains is fundamental for the functional mechanism of this chaperone, and its location within the protein structure in previous structural models is contradictory. we present cryo-electron microscopy structural analysis of clpb from e. coli in several nucleotide states. the study reveals a novel architecture for clpb and shows that m domains form an internal scaffold located in the central chamber of clpb hexamers. this inner structure transmits local signals due to atp binding and hydrolysis by aaa? domains. surprisingly, coiled-coil m domains are seen to bend significantly around a hinge region that separates two structural motifs. our results present a new framework to understand clpb-mediated protein disaggregation. streptomyces clavuligerus isoenzymes involved in clavulanic acid biosynthesis: a structural approach clavulanic acid (ca) is a potent b-lactamase inhibitor produced by streptomyces clavuligerus. n -( -carboxyethyl) arginine synthase (ceas) and proclavaminate amidino hydrolase (pah) catalyze the initial steps in the biosynthesis of ca. recently ceas and pah genes (paralogous of ceas and pah) were related to the ca biosynthesis but their products have not been studied yet. here we present the initial structural analysis of ceas and pah using biophysical techniques. pah and ceas genes were isolated from the genomic dna of s. clavuligerus and overexpressed in e. coli. the recombinant proteins were purified by affinity chromatography and analyzed by size exclusion chromatography, non-denaturing page, dynamic light scattering, far-uv circular dichroism (cd) and fluorescence spectroscopy. our results showed that pah and ceas were obtained as hexamer and dimer respectively. both proteins showed an a/b folding, being stable up to °c. above this temperature protein unfolding was observed but the complete unfolding was not observed, even at °c. moreover ceas and pah showed to be stable over a wide ph range (ph . - . ). we are currently working on improving ceas crystals which are a promising step towards the elucidation of the ceas structure. supported by fapesp. • synchrotron radiation circular dichroism • mass spectrometry following vuv photoionisation • fluorescence imaging with lifetime and spectral measurements here we will present the srcd experiment. high photon flux of photons / sec, improved detector performances as well as user-orientated software developments have proven to be the garants for successful data collections,which considerably increased the information content obtained. the exploration into the charge transfer region of the peptide bonds is adding specifically new insights. low sample volumes of as little as ll per spectra as well as convenient sample chamber handling allow for economic and efficient data collections. typical spectra acquisition from to nm, last for min for three scans with a nm step size. prior to high resolution based techniques, srcd spectrawill answer questions about folding states of macromolecules including dna, rna and sugar macromolecules as well as their complexes with proteins and specially membrane proteins. sporulation in bacillus subtilis begins with an asymmetric cell division producing a smaller cell called the forespore, which initially lies side-by-side with the larger mother cell. in a phagocytosis-like event, the mother cell engulfs the forespore so that the latter is internalised as a cell-within-a-cell. engulfment involves the migration of the mother cell membrane around the forespore until the leading edges of this engulfing membrane meet and fuse. this releases the forespore, now surrounded by a double membrane, into the mother cell cytoplasm. membrane migration during engulfment is facilitated by the interacting proteins spoiiq and spoiiiah that are membrane-associated and expressed in the forespore and the mother cell respectively . they interact in the intercellular space and function initially as a molecular zipper and later they participate in a more elaborate complex in which spoiiq and spoiiiah are integral components of an intercellular channel. this channel is a topic of much current interest, having initially been proposed as a conduit for the passage from the mother cell to the forespore of a specific, but putative, regulator of the rna polymerase sigma factor, r g and later as a gap junction-like feeding tube through which the mother cell supplies molecules for the biosynthetic needs of the forespore. here we present data on the structure and interactions of spoiiq and spoiiiah gleaned from biophysical methods and protein crystallography. these data lead to a plausible model for the intercellular channel. the glycine receptor (glyr) is a chloride permeable ligand gated ion channel and that can mediate synaptic inhibition. due to a possible involvement in the pathophysiology of temporal lobe epilepsy, the different properties of glyrs containing alpha l and k subunit isoforms are currently investigated. previous characterizations of homomeric receptors consisting of these isoforms have shown a difference in their electrophysiological properties and their membrane distribution as observed by diffraction-limited fluorescence microscopy. we studied these isoforms, when separately expressed in transfected hek t cells, by using single molecule tracking (smt) in living cells and direct stochastic optical reconstruction microscopy (dstorm) on fixated cells. for both techniques the glyrs are stained using a primary antibody directly labeled with alexa fluor dyes. the dstorm experiments support the observation that alpha l glyr are clustered, while the alpha k glyrs are more uniformly spread. the analysis of the short range diffusion coefficients obtained by smt reveals the presence of heterogeneous motion for both isoforms. the k-isoform has a higher fraction of fast diffusion. in contrast, the l-isoform is more associated with slow diffusion and appears to undergo hindered diffusion. since nanoparticles are suitable for tumor therapy due to their passive targeting to cancer cells by enhanced permeability and retention effect [ ] , it is important to understand mechanisms of their delivery into the living cancer cells. in this respect we have developed a modular spectral imaging system based on a white light spinning disk confocal fluorescence microscope and a narrow tunable emission filter. firstly, interaction of polymer nanoparticles and cells labeled with spectrally overlapping probes was examined. the use of fluorescence microspectroscopy (fms) allowed co-localization, which showed that the size of polymer nanoparticles strongly influences their transfer across the cell plasma membrane. next, the delivery of liposomes (composed of cancerostatic alkylphospholipid (opp) and cholesterol) labeled with environment-sensitive fluorescent probe was monitored. we were able to detect a very small shift in emission spectra of cholesterol-poor opp liposomes inside and outside the cells, which would not be possible without the use of fms. this shift implies that the delivery of these liposomes into cancer cells is based on fusion with the cell membrane [ ] . [ high-resolution optical imaging techniques make now accessible the detection of nanofeatures in bio-and soft-matter by non-ionizing visible radiation. however, high-resolution imaging is critically dependent by the fluorescent probes used for reporting on the nano-environment. on account of our long-standing interest in the development of fluorescent probes, we set out to design and engineer new fluorescent systems for nanoscale imaging and sensing of biological specimens and soft-matter. these fluorophores report on fast subtle changes of their nanoscale environment at excited state and are meant to fulfill these requirements: a) optical responses (intensity, wavelength-shift, lifetime, anisotropy) predictably related to the environmental polarity, viscosity, macromolecular structure, b) high brightness allowing for single-molecule detection, c) easily conjugable to biomolecules or macromolecules of interest. notably, we aim at conjugating these properties with the capability of nanoscopy imaging based on stimulated emission depletion or stochastic reconstruction optical microscopy. in this lecture the main features and applications of the engineered probes will be reviewed and future developments in this exciting field will be discussed. foamy virus (fv) is an atypical retrovirus which shares similarities with hiv and hepatitis b viruses. despite numerous biochemical studies, its entry pathway remains unclear, namely membrane fusion or endocytosis. to tackle this issue, dual color fluorescent viruses were engineered with a gfp labeled capsid and a mcherry labeled envelope. using high resolution d imaging and d single virus tracing, we followed the entry of the fluorescent viruses in living cells with a precision of nm in the plane and nm along the optical axis. to distinguish between the two possible pathways, we developed a novel colocalization analysis method for determining the moment along every single trace where the colors separate, i.e. the fusion event. the combination of this dynamical colocalization information with the instantaneous velocity of the particle and its position within the reconstructed d cell shape allows us to determine whether the separation of capsid and envelope happens at the cell membrane or from endosomes. we then compared two types of fv and demonstrated, consistently with previous ph-dependency studies, that the prototype fv can enter the cell by endocytosis and membrane fusion, whereas the simian fv was only observed to fuse after endocytosis. phosphatidylinositol , -bisphosphate (pi( , )p ) is a minor component of the plasma membrane known to a critical agent in the regulation of synaptic transmission. clustering of pi( , )p in synaptic active zones is important for synaptic transmission. however, pi( , )p does not spontaneously segregate in fluid lipid membranes and another mechanism must be responsible for the lateral segregation of this lipid in active zones. clustering of pi( , )p is expected to be associated with lipid-protein interactions and possibly partition towards lipid rafts in the plasma membrane. here we analyze the influence of protein palmitoylation on the formation of pi( , )p clusters and on synaptic protein-pi( , )p interaction by means of fö rster resonance energy transfer measurements by fluorescence lifetime imaging (fret-flim) and fret confocal microscopy. . during sporulation an entire chromosome is transferred into the forespore. this process starts by the formation of an asymmetrically located division septum that leads to the formation of two unequally sized compartments: a large mothercell and a smaller forespore. the septum traps about % of the chromosome to be transferred into the forespore. the remaining (* mbp) are then translocated from the mother cell into the forespore by an active mechanism involving the spo-iiie dna translocase. the mechanisms of translocation, particularly the control of the directionality, still remains unknown and various models have been proposed so far. since each model predicts very different distribution of spoiiie proteins at the sporulation septum, we used palm microscopy (photoactivated localization microscopy) to investigate proteins localization in live-sporulating bacteria. using this technique, we showed that spoiiie proteins are forming a single tight focus at the septum with a characteristic size of around nm. more surprising, the focus is usually localized in the mother cell compartment and the mean distance between the spoiiie focus and the septum is nm. our data suggest that during the translocation process, spoiiie proteins are only forming stable complexes on the mother cell side, allowing then for a control of the chromosome translocation from the mother cell to the forespore. morphogenetic gradients determine cell identity in a concentration-dependent manner and do so in a way that is both incredibly precise and remarkably robust. in order to understand how they achieve this feat, one needs to establish the sequence of molecular mechanisms starting with morphogen gradient formation and leading to the expression of downstream target genes. in fruit flies, the transcription factor bicoid (bcd) is a crucial morphogen that forms an exponential concentration gradient along the embryo ap axis and turns on cascades of target genes in distinct anterior domains. we measured bcd-egfp mobility in live d. melanogaster embryos using fluorescence correlation spectroscopy and fluorescence recovery after photobleaching. we found that bcd-egfp molecules had a diffusion coefficient on the order of * lm /s during nuclear cycles - , both in the cytoplasm and in nuclei. this value is large enough to explain the stable establishment of the bcd gradient simply by diffusion. on the other hand, in the context of the extremely precise orchestration of the transcription of the hunchback bcd target gene, it is too slow to explain how a precise reading of bicoid concentration could be achieved at each interphase without the existence of a memorization process. single molecule studies of key processes during the initiation of innate and adaptive immune response the two pillars of the vertebrate immune system are the innate and adaptive immune response, which confer resistance to pathogens and play a role in numerous diseases. here we exploit single molecule fluorescence imaging on live cells to study the key molecular processes that underpin these responses. the first project looks at the changes in the organisation of toll-like receptor (tlr ) on the cell surface of macrophages upon activation via lipopolysaccharide (lps), as it is currently not known whether a higher level of tlr organisation is required for the signalling process. macrophages natively express tlr at a low level which allows for oligomerisation to be analysed in live cells by dynamic single molecule colocalisation (dysco) using data obtained by totalinternal reflection fluorescence (tirf) microscopy. the experiments of the second project aim at determining the critical initial events in t-cell triggering by labelling key proteins like the tcr receptor and cd on the surface of live t-cells and following how their spatial distribution changes following the binding of the t-cell to a surface. this enables us to distinguish between the different models of t-cell triggering which are based on aggregation, segregation or a conformational change of the tcr. the study of cells using scanning force microscopy the motility of unicellular parasites in mammalians seems very interesting, yet very complex. in a world, were inertia cannot be used for propulsion, in a world at low reynolds numbers, most of our everyday strategies of self-propulsion do not work. one class of parasites that know their way around, the flagellate trypansome, manage not only to survive in the blood stream, which is a lot faster than its own propulsion velocity and where the trypanosome is constantly attacked by its host's immune response, but also to penetrate the bloodbrain-barrier, which actually should be to tight to enter. even though trypanosomes are known for more than years, their motility behaviour is not completely elucidated yet. now, using high-speed darkfield-microscopy in combination with optical tweezers in microfluidic devices and analyzing the recorded data, new light has been shed on the motility of these parasites. astonishing results show that trypanosomes are very well adapted to their hosts environment, they even can abuse red blood cells for their self-propulsion and use the bloodstream itself to drag antibodies bound to their surface to their cell mouth, where the antibodies are endocytosed and digested. the first part of the presentation will discuss nanoparticle (quantum dot, qd) biosensors and nanoactuators that exploit novel and unusual fret phenomena in the induction/detection of protein aggregation [ ] , reversible on-off qd photoswitching [ ] , and ph sensing [ ] . the second part of the presentation will feature the application of an integrated chemical biological fret approach for the in situ (in/on living cells) detection of conformational changes in the ectodomain of a receptor tyrosine kinase (the receptor for the growth factor egf) induced by ligand binding [ ] . the measurements were conducted with a two-photon scanning microscope equipped with tcspc detection; novel methods for lifetime analysis ad interpretation were employed to confirm the concerted domain rearrangements predicted from x-ray crystallography. the study of protein-protein interactions in vivo is often hindered by the limited acquisition speed of typical instrumentation used, for instance, for lifetime imaging microscopy. anisotropy polarization is altered by the occurrence of foerster resonance energy transfer (fret) and anisotropy imaging was shown to be comparatively fast and simple to implement. here, we present the adaptation of a spinning disc confocal microscope for fluorescence anisotropy imaging that allowed to achieve in vivo imaging at high spatial and temporal resolution. we demonstrate the capabilities of this system and in-house developed analysis software by imaging living caenorhabditis elegans expressing constitutive dimeric and monomeric proteins that are tagged with gfp. measuring intracellular viscosity: from molecular rotors to photodynamic therapy of cancer marina k. kuimova department of chemistry, imperial college london, exhibition road, sw az, uk viscosity is one of the main factors which influence diffusion in condensed media and is crucial for cell function. however, mapping viscosity on a single-cell scale is a challenge. we have imaged viscosity inside live cells using fluorescent probes, called molecular rotors, in which the speed of rotation about a sterically hindered bond is viscosity-dependent [ ] [ ] [ ] . this approach enabled us to demonstrate that viscosity distribution in a cell is highly heterogeneous and that the local microviscosity in hydrophobic cell domains can be up to higher than that of water. we demonstrated that the intracellular viscosity increases dramatically during light activated cancer treatment, called photodynamic therapy (pdt) [ ] . we also demonstrated that the ability of a fluorophore to induce apoptosis in cells during pdt [ ] , or to act as a benign molecular rotor, measuring viscosity, can be controlled by carefully selecting the excitation wavelength in viscous medium [ ] . in the field of biophysics and nanomedicine, the cellular reaction and the kinetics of gene expression after transfection of live cells with plasmid dna or gene-silencing sirna is of great interest. in a previous study on the transfection kinetics of non-viral gene-transfer [ ] we realised that the development of single-cell arrays would be a great step towards easy-to-analyse, high-throughput transfection studies. the regular arrangement of single cells would overcome the limitations in image-analysis that arise from whole populations of cells. in addition to that, the analysis of expression kinetics at the single-cell can help to identify the cell-to-cell variability within a cell population. in order to develop suitable single-cell arrays, we are currently adjusting the different parameters of such a microenvironment (e.g. size, shape, surface-functionalisation) in order to end up with a defined surrounding for single-cell transfection studies. in addition to that, we try to find the optimal uptake pathway for each of the different applications. the neurodegenerative disorder alzheimer's disease (ad) causes cognitive impairment such as loss of episodic memory with ultimately fatal consequences. accumulation and aggregation of two proteins in the brain -amyloid beta and tau -is a characteristic feature. these soluble proteins aggregate during the course of the disease and assemble into amyloid-like filaments. recently it was found that the toxicity of soluble amyloid beta oligomers must also be taken into account for the pathogenesis of cognitive failure in ad. if oligomers are the predominant toxic species it would be pertinent to determine how they disrupt and impair neuronal function. the prion protein (prp) receptor has been proposed to mediate amyloid beta binding to neuronal cells. we have characterised the interaction of the amyloid beta and the prp receptor expressed on hippocampal and neuroblastoma cells at the single-molecule level. we do not detect any colocalisation of either the or amino acids variants with the prp receptor. bacterial biofilms are of the utmost importance in the study of environmental bioremediation and the design of materials for medical applications. the understanding of the mechanisms that govern cell adhesion must be analysed from the physics point of view in order to obtain quantitative descriptors. the genus rhodococcus is widely spread in natural environments. the species are metabolically diverse and thus they can degrade a wide range of pollutants. due to their high hydrophobicity, these cells are very resistant to harsh conditions, are able to degrade hydrophobic substances (e.g. oil) and attach to high-contact angle surfaces. the hydrophobicity of several strains of rhodococcus is measured and mapped using chemical force microscopy (cfm) in the present study. cfm relies on the functionalisation of scanning force microscopy (sfm) tips using hydrophobic or hydrophilic groups. in cfm, the microscope is operated in the forcevolume mode, which combines adhesion data with topographic images. the careful control of the tip chemistry permits the study of interactions between the functional groups on the tip and the bacterial surface, thus allowing the assessment of hydrophobicity. in order to perform a cfm study, the cells need to be firmly anchored to a substrate under physiological conditions (i.e. under a nutrient media or a saline buffer). to this end, several adhesive surfaces have been tested in order to find the one that gives the best results. optical microscopy is arguably the most important technique for the study of living systems because it allows d imaging of cells and tissues under physiological conditions under minimally invasive conditions. conventional far-field microscopy is diffraction-limited; only structures larger than * nm can be resolved, which is insufficient for many applications. recently, techniques featuring image resolutions down to * nm have been introduced such as localization microscopy (palm, storm) and reversible saturable optical fluorescent transition microscopy (resolft, sted). these methods are well suited for live-cell imaging and narrow the resolution gap between light and electron microscopy significantly. we have used palm imaging to study the formation and disassembly of focal adhesions of live hela cells in a high resolution pulse-chase experiment using monomeric irisfp [ ] . mirisfp is a photoactivatable fluorescent protein that combines irreversible photoconversion from a green-to a red-emitting form with reversible photoswitching between a fluorescent and a nonfluorescent state in both forms. in our experiments a subpopulation of mirisfp molecules is photoconverted to the red form by irradiating a specified region of the cell with a pulse of violet light. migration of tagged proteins out of the conversion region can be studied by subsequently localizing the proteins in other regions of the cell by palm imaging, now using the photoswitching capability of the red species. real-time image reconstruction developed in our lab [ ] allowed instant control imaging parameters. live cell imaging of cancer cells is often used for in-vitro studies in connection with photodynamic diagnostic and therapy (pdd and pdt). especially in presence of a photosensitizer, this live cell imaging can only be performed over relatively short duration (at most hour). this restriction comes from the light-induced cell damages (photodamages) that result from rapid fluorescence photobleaching of photosensitizer. while these studies reveal exciting results, it takes several hours to discover the detailed effects of the photosensitizer on cell damage. up to our knowledge, however, there is no general guideline for modification of excitation light dose to achieve that. in this paper, the relation between excitation light doses, photobleaching of photosensitizer (pvp-hyperycin) and cell vitality are investigated using human lung epithelial carcinoma cells (a ). the strategy of this paper is to reduce the excitation light dose by using a low-power pulsed blue led such that the structures are visible in time-lapse images. fluorescence signals and image quality are improved by labelling the cells with an additional non-toxic marker called carboxyfluorescein-diacetate-succinimidyl-ester (cfse). in total we collected time-lapse images (time intervals min) of dual-marked a cells under three different light intensities ( . , . and . mw/cm ) and a variety of pulse lengths ( . , . , , . and ms) over five hours. we have found that there is a nonlinear relationship between the amount of excitation light dose and cell vitality. cells are healthy, i.e. they commence and complete mitosis, when exposed to low light intensities and brief pulses of light. light intensities higher than . mw/cm together with pulse durations longer than ms often cause cell vesiculation, blebbing and apoptosis. in all other cases, however, we found no cell death. in the future, this striking nonlinearity will be studied in more detail. progressive advances in scanning ion conductance microscopy (sicm) [ ] enabled us to convert ordinary scanning probe microscope (spm) in to versatile multifunctional technique. as an imaging tool, ion conductance microscopy is capable to deliver highest possible topographical resolution on living cell membranes among any other microscopy techniques [ ] . also, it can visualize surfaces complexity of those makes them impossible to image by other spms [ ] . ion conductance microscopy combined with a battery of powerful methods such as fluorescence resonance energy transfer (fret) [ ] , patch-clamp, force mapping, localized drug delivery, nano-deposition and nano-sensing is unique among current imaging techniques. the rich combination of ion conductance imaging with other imaging techniques such as laser confocal and electrochemical [ ] will facilitate the study of living cells and tissues at nanoscale. ) and coleoptiles of wheat (triticum aestivum l.) seedlings, which were growing in light and dark conditions, were used to determine fluorescence of whole cells. fluorescence emission spectrum was monitored by fluorescent microscopy using the spectrometer usb . fluorescence intensity f , f , f and f was determined and data was statistically analyzed in annova. we observed that bgf, rf and frf intensity increased in the first leaves with the age of the seedlings. in the coleoptiles was observed great bgf intensity increase with the age of the seedlings. in the coleoptiles decreased rf intensity of the and hours old seedlings, and bgf intensity decreased of hours old seedlings. it was found that emission spectrum and fluorescence intensity changes are induced by the lack of light and salt (nacl) stress. analysis of fluorescence spectrum can quickly and accurately indicate the outset of light and salt stress in plants. there are analogical changes in fluorescence emission spectrum of plant cells in senescence and stress conditions. it was assumed that environmental stress and senescence have common mechanisms in plants. this changes can be monitored by fluorescent microscopy. triple-colour super-resolution imaging in living cells markus staufenbiel, stephan wilmes, domenik lisse, friedrich roder, oliver beutel, christian richter and jacob piehler universitä t osnabrü ck, fachbereich biologie, barbarastraße , , germany, markus.staufenbiel@biologie.uniosnabrü ck.de super-resolution fluorescence imaging techniques based on single molecule localisation has opened tremendous insight into the sub-micrometre organisation of the cell. live cell imaging techniques such as fluorescence photoactivation localization microscopy (fpalm) are currently limited to dual-colour detection due to the restricted availability of red-fluorescent photoswitchable proteins. we employed photoswitching of the oxazine dye atto under reducing conditions for super-resolution imaging in the cytoplasm of living cells. for efficient and specific covalent labelling of target proteins, we have made use of the halotag system. atto was coupled to the halotag ligand (htl) and fast reaction of htl-atto with the halotag enyzme was confirmed in vitro by solid phase binding assays. efficient labelling of the membrane cytoskeleton using lifeact fused to the halotag was observed and super-resolution imaging was readily achieved. based on this approach, we managed to follow the nanoscale dynamics of the actin cytoskeleton as well as clathrincoated pits using clathrin light chain fused to the halotag. we combined this technique with fpalm for triplecolour super-resolution imaging of the spatial distribution of membrane receptors in context of the membrane skeleton. the erbb family of receptor tyrosine kinases consists of four transmembrane proteins that transduce signals across the membrane to control cell fate. growth factor binding results in homo-and hetero-interactions between these receptors at the membrane. erbb receptors are implicated in many cancers, making them a target for therapeutic drugs. to date, studies of erbb interactions have been limited to individual family members or specific pairs, giving an incomplete picture of the highly complex behaviour controlling positive and negative feedback loops and signalling outcomes. to investigate erbb receptor interactions, we have developed tirf-based single molecule fluorescence microscopes capable of simultaneously imaging three, and soon five, fluorescence probes in live cells. we have also developed a catalogue of extrinsic fluorescent probes for : labelling of both endogenous and transfected erbb family members in mammalian cells, plus a bayesian approach to the analysis of single molecule data. this allows us to track active and inactive erbb family members at the basal surface of a model breast cancer cell line that expresses physiological levels of all four receptors. we present here initial characterisation of the entire erbb family together in the cell membrane. the human genome contains more than g proteincoupled receptors (gpcrs); overall, - % of the mammalian genome encodes these molecules. processes controlled by gpcrs include neurotransmission, cellular metabolism, secretion, and immune responses. however it is the stoichiometry of these receptors that is the most controversial. the starting point for understanding gpcr function was the idea that these receptors are monomeric. on the other hand a lot of recent studies favour the concept that gpcr form dimers and are not capable of signalling as independent monomers. recent single molecule studies try to solve this dilemma by suggesting that gpcrs form transient dimers with a lifetime of * ms. however questions remain about the physiological relevance of the preparations necessary for these studies, since they have not been performed on endogenous receptors. here, we directly image individual endogenous receptors using an equimolar mixture of two colour fluorescent fab fragments. we can then determine the receptors stoichiometry by quantifying its dynamic single molecule colocalisation (dy-sco) recorded by total-internal reflection fluorescence (tirf) microscopy. we have recently investigated the domain dynamics of pgk ( ) . structural analysis by small angle neutron scattering revealed that the structure of the holoprotein in solution is more compact as compared to the crystal structure, but would not allow the functionally important phosphoryl transfer between the substrates, if the protein would be static. brownian large scale domain fluctuations on a timescale of ns was revealed by neutron spin echo spectroscopy. the observed dynamics shows that the protein has the flexibility to allow fluctuations and displacements that seem to enable function. [ many physiological and pathological processes involve insertion and translocation of soluble proteins into and across biological membranes. however, the molecular mechanisms of protein membrane insertion and translocation remain poorly understood. here, we describe the ph-dependent membrane insertion of the diphtheria toxin t domain in lipid bilayers by specular neutron reflectometry and solid-state nmr spectroscopy. we gained unprecedented structural resolution using contrast-variation techniques that allow us to propose a sequential model of the membrane-insertion process at angstrom resolution along the perpendicular axis of the membrane. at ph , the native tertiary structure of the t domain unfolds, allowing its binding to the membrane. the membrane-bound state is characterized by a localization of the c-terminal hydrophobic helices within the outer third of the cis fatty acyl-chain region, and these helices are oriented predominantly parallel to the plane of the membrane. in contrast, the amphiphilic n-terminal helices remain in the buffer, above the polar headgroups due to repulsive electrostatic interactions. at ph , repulsive interactions vanish; the n-terminal helices penetrate the headgroup region and are oriented parallel to the plane of the membrane. the c-terminal helices penetrate deeper into the bilayer and occupy about two thirds of the acyl-chain region. these helices do not adopt a transmembrane orientation. interestingly, the t domain induces disorder in the surrounding phospholipids and creates a continuum of water molecules spanning the membrane. we propose that this local destabilization permeabilizes the lipid bilayer and facilitates the translocation of the catalytic domain across the membrane. the limited stability in vitro of mps motivates the search of new surfactants ( ) ( ) ( ) ( ) . fss with a polymeric hydrophilic head proved to be mild towards mps ( ) .new fss were designed with chemically defined polar heads for structural applications. lac-derivative was efficient in keeping several mps water soluble and active but formed elongated rods ( ) . the glu-family was synthesized, characterized in by sans and auc and for its biochemical interest. the formation of rods is related to the low volumetric ratio between the polar head and hydrophobic tail. the surfactant bearing two glucose moieties is the most promising one, leading to both homogeneous and stable complexes for both br and the b f. it was also shown be of particular interest for the structural investigation of membrane proteins using sans ( ). by combining elastic and quasi-elastic neutron scattering data, and by applying theory originally developed to investigate dynamics in glassy polymers, we have shown that in lyophilised apoferritin above t * k the dynamic response observed in the pico-to nano-second time regime is driven by ch dynamics alone, where the methyl species exhibit a distribution of activation energies. our results suggest that over the temporal and spatial range studied the main apoferritin peptide chain remains rigid. interestingly, similar results are reported for other smaller, more flexible lyophilised bio-materials. we believe this work elucidates fundamental aspects of the dynamic landscape in apoferritin which will aid development of complex molecular dynamic model simulations of super-molecules. a detailed appreciation of the relationships between dynamics and biological function will require analysis based on such models that realize the full complexity of macromolecular material. biological systems must often be stored for extended periods of time. this is done by lyophilisation in the presence of lyoprotectants, such as sugars, which results in stable products at ambient conditions. [ ] in an effort to understand the mechanism of preservation and stabilization, the interactions between sugars and liposome vesicles, which serve as a simple membrane model, have been studied extensively. amongst the common sugars, trehalose has superior preservative effects [ ] and accumulates to high concentrations in many anhydrobiotic organisms. despite many experimental and numerical studies three mechanisms are proposed: vitrification [ ] , preferential exclusion [ ] and water replacement [ ] . to gain more insight into the stabilization mechanism we have recently investigated the effect of trehalose on the bending elasticity of fully hydrated unilamellar vesicles of , -dipalmitoyl-phosphatidylcholine (dppc) in d o at temperatures below and above the lipid melting transition (tm) using neutron spin-echo. the data was analyzed using the zilman-granek theory. at all temperatures measured, trehalose stiffens the bilayer suggesting strong interactions between trehalose and the lipid. trehalose appears to broaden the melting transition but does not change the tm. this agrees with observations using differential scanning calorimetry. influence of macromolecular crowding on protein stability sté phane longeville, clé mence le coeur laboratoire lé on brillouin, gif-sur-yvette, france cell interior is a complex environment filled with a variety of different objects with respect to shape and size. macromolecules are present at a total concentration up to several hundred grams per litre and the overall occupied volume fraction can reach ö & . - . . under crowding environment protein-protein interaction play a fundamental role. the crowding environment can affect some physical, chemical, and biological properties of biological macromolecules [ , ] . traditionally, protein folding is studied in vitro at very low concentration of proteins. under such conditions, small globular single chain proteins can unfold and refold quite rapidly depending mainly to the nature of the solvent. such processes have been very intensively studied, since folding of proteins into their native structure is the mechanism, which transforms polypeptide into its biologically active structure. protein misfolding is involved in a very high number of diseases [ ] (e.g. alzheimer, parkinson, and kreuzfeld-jacob diseases, type ii diabetes, …). theoretically, the problem was studied by the introduction of the concept of excluded volume [ ] . in recent papers [ , ] , minton uses statistical thermodynamic models to address the question. he predicted that inert cosolutes stabilize the native state of proteins against unfolding state mainly by destabilizing the unfolded state and that the dimension of the unfolded state decreases with increasing the concentration of cosolute in a measurable way. small angle neutron scattering (sans) is a technique of choice for such study because, by using appropriate mixtures of light and heavy water, it is possible to match the scattering length density of the solvent to the one of the cosolute and thus to measure the conformation of a molecule at low concentration in a presence of a high concentration of another one. we will present a complete experimental study of the mechanism that leads to protein stabilization by macromolecular crowding [ , ] . coupled dynamics of protein and hydration water studied by inelastic neutron scattering and molecular dynamics simulation hiroshi nakagawa , , mikio kataoka , japan atomic energy agency, tokai, japan, juelich centre for neutron science, forschungszentrum juelich gmbh, nara institute of science and technology, ikoma, japan proteins work in an aqueous environment at ambient temperature. it is widely accepted that the proteins are flexible and mobile. the flexibility and mobility, that is, protein dynamics are essential for protein functions. neutron incoherent scattering is one of the most powerful techniques to observe protein dynamics quantitatively. here i will talk about dynamics of protein and its hydration water. the structure of a soluble protein thermally fluctuates in the solvated environment of a living cell. understanding the effects of hydration water on protein dynamics is essential to determine the molecular basis of life. however, the precise relationship between hydration water and protein dynamics is still unknown because hydration water is ubiquitously configured on the protein surface. we found that hydration level dependence of the onset of the protein dynamical transition is correlated with the hydration water network. hydration water dynamics change above the threshold hydration level, and water dynamics control protein dynamics. these findings lead to the conclusion that the hydration water network formation is an essential property that activates the anharmonic motions of a protein, which are responsible for protein function. thermal motions and stability of hemoglobin of three endotherms (platypus -ornithorhynchus anatinus, domestic chicken -gallus gallus domesticus and human -homo sapiens) and an ectotherm (salt water crocodile -crocodylus porosus) were investigated using neutron scattering and circular dichroism. the results revealed a direct correlation between the dynamic parameters, melting -, and body temperatures. on one hand, a certain flexibility of the protein is mandatory for biological function and activity. on the other hand, intramolecular forces must be strong enough to stabilize the structure of the protein and to prevent unfolding. our study presents experimental evidence which support the hypothesis that the specific amino acid composition of hb has a significant influence on thermal fluctuations of the protein. the amino acid sequence of hb seems to have evolved to permit an optimal flexibility of the protein at body temperature. macromolecular resilience was found to increase with body and melting temperatures, thus regulating hb dynamics. where k is the trap spring constant, a is the subdiffusion exponent and e a is the mittag-leffler function. the parameters obtained by fitting this equation to the experimental msds are summarized in table . at short lag times we have not found any difference between the two cell types, contrarily to the previous results obtained by afm . for both cell lines the subdiffusion exponent, a was found close to , the value predicted by the theory of semiflexible polymers. but the crossover frequency x , was found smaller for the cancerous cells for all datasets. it corresponds to passage to the confined regime at longer times. we attribute it to the bigger impact of molecular motors. we study the spatiotemporal evolution of fibrous protein networks present in the intracellular and extracellular matrices. here, we focus on the in vitro actin network dynamics and evolution. in order to study the hierarchical self-assembly of the network formation in a confined environment and provide external stimuli affecting the system minimally, we introduce a new microfluidic design. the microfluidic setup consists of a controlling channel to which microchambers of different shapes and sizes are connected through narrow channels. this design results in having mainly convective transport in the controlling channel and diffusive transport into the microchamber. rhodamine labeled actin monomers diffuse into the chamber. after polymerization is induced, they form a confined entangled network. cross-linking proteins can then be added to increase the network complexity. moreover, we can generate gradients of reactants across the microchambers and vasodilator-stimulated phosphoprotein (vasp) is a crucial regulator of actin dynamics. it is important in cellular processes such as axon guidance and migration, promoting assembly of filopodia and lamellipodia. vasp's multiple domain structure increases the range of interactions it has with actin monomers, filaments, and other proteins and it displays multiple binding modes both in vitro and in vivo, including barbed end elongation and filament bundling. however, it is not fully understood how vasp affects the structural and mechanical properties of actin networks. we characterize vasp-mediated bundling of actin networks in a simplified in vitro system using confocal microscopy and quantify mechanical properties with rheology measurements. we show that the network properties differ from other actin bundling proteins and reflect vasp's multiple domain structure, displaying a complex bundling phase space that depends upon solution conditions. we observe the formation of large bundle aggregates accompanied by a reduction in network elasticity at high protein ratios. in addition, we change vasp's actin binding mode and eliminate bundling by introducing free actin monomers. finally, we show preliminary results from a biomimetic system that extends the range of actin-vasp interaction. cell migration or proliferation? the go or grow hypothesis in cancer cell cultures tamá s garay, É va juhá sz, jó zsef tímá r, balá zs heged} us nd department of pathology, semmelweis university, budapest, hungary background: cancer related death is constantly growing in the past decades. the mortality of solid tumors is mostly due to the metastatic potential of tumor cells which requires a fine adjustment between cell migration and cell proliferation. as the metabolic processes in the cell provide a limited amount of available energy (i.e. atp) the various biological processes like cell motility or dna synthesis compete for the atp available. the go or grow hypothesis postulates that tumor cells show either high migration or proliferation potential. in our study we investigate on a large series of tumor cell lines whether this assumption stands for malignant cells. materials and methods: twenty tumor cell lines derived from malignant mesothelioma (mesodermal origin) and malignant melanoma (neuroectodermal origin) were subjected to three-days-long time-lapse videomicroscopic recordings. cell motility and proliferation were characterized by the probability of cell division within hours and the -hour migration distance of the cells. results: we found a wide range in both the cell migratory activity and the proliferation capacity in our series. the -hour migration distance ranged from to micron and from to micron in mesothelioma and melanoma cells, respectively. the lowest -hour cell division probability was found to be . in both the melanoma and mesothelioma series while the highest proliferation activity reached . and . in melanoma and mesothelioma, respectively. interestingly, in the melanoma cell lines we found a significant positive correlation (r= , ; p= , ) between cell proliferation and cell migration. in contrast our mesothelioma cell lines displayed no correlation between these two cellular processes. conclusions: in summary our findings demonstrate that the investigated tumor cells do not defer cell proliferation for cell migration. important to note the tumor cells derived from various organ systems may differ in terms of regulation of cell migration and cell proliferation. furthermore our observation is in line with the general observation of pathologists that the highly proliferative tumors often display significant invasion of the surrounding normal tissue. many cell types are sensitive to mechanical signals. one striking example is the modulation of cell proliferation, morphology, motility, and protein expression in response to substrate stiffness. changing the elastic moduli of substrates alters the formation of focal adhesions, the formation of actin filament bundles, and the stability of intermediate filaments. the range of stiffness over which different primary cell types respond can vary over a wide range and generally reflects the elastic modulus of the tissue from which these cells were isolated. mechanosensing also depends on the type of adhesion receptor by which the cell binds, and therefore on the molecular composition of the specific extracellular matrix. the viscoelastic properties of different extracellular matrices and cytoskeletal elements also influence the response of cells to mechanical signals, and the unusual non-linear elasticity of many biopolymer gels, characterized by strain-stiffening leads to novel mechanisms by which cells alter their stiffness by engagement of molecular motors that produce internal stresses. the molecular mechanisms by which cells detect substrate stiffness are largely uncharacterized, but simultaneous control of substrate stiffness and adhesive patterns suggests that stiffness sensing occurs on a length scale much larger than single molecular linkages and that the time needed for mechanosensing is on the order of a few seconds. to explore potential role of cytoskeletal component in cardiomyocyte for adaptation to extreme conditions was carried out the comparative study of expression of cytoskeletal sarcomeric protein titin in myocardium of ground squirrels during hibernation and gerbils after spaceflight. we have revealed a two-fold increase in content of long n ba titin isoform as compared to short n b titin isoform in different heart chambers of hibernating ground squirrels. the prevalence of the long titin isoform is known to determine the larger extensibility of heart muscle that promotes, according to frank-starling law, the increase in force of heart contractility for pumping higher viscous blood during torpor and adapting the myocardium to greater mechanical loads during awakening. moreover, titin mrna level showed seasonal downregulation in which all hibernating stages differed significantly from summer active level. it is possible that the decline of mrna and protein synthesis during hibernation may be regarded as the accommodation for minimization of energetic expenditures. we have not revealed differences in titin mrna levels between control gerbils and gerbils after spaceflight. but we have also observed the two-fold growth in the amount of n ba titin isoform in left ventricle of gerbils after spaceflight that is likely to be directed to the restoration of the reduced heart contractility at zero-gravity. these results suggest that the increase of the content of the long n ba titin isoform may serve as universal adaptive mechanism for regulating of heart function in response to the extreme conditions. nuclear migration is a general term for a non-random movement of the nucleus toward specific sites in the cell. this phenomenon has been described throughout the eukaryotes from yeast to mammals. the process is however still poorly understood in mammalian cells. by using microcontact printing we are able to regulate the geometry and spreading of cultured cells. adhesive micropatterns of fibronectin provide an attachment surface for the cells whereas the passivation of the surface by pll-peg prevents protein, thus cell adhesion. live cell imaging by time-lapse microscopy has shown that under these conditions cells gain a bipolar shape, and more interestingly, the nuclei of the cells showed auto-reversed motion. our research tries to understand the molecular cues and mechanisms behind the observed cellular and nuclear movement. we have already shown that the cytoskeleton plays an important role in this phenomena but the exact players and the detailed mechanism remain to be clarified. in order to identify the most important components and their relationship have drug treatments and sirna experiments have been applied. although our research focuses mainly on the motility of the nucleus, it may also help to get a better understanding of the general theme of cell migration. cell motility involves a number of strategies that cells use to move in their environments in order to seek nutrients, escape danger and fulfil morphogenetic roles. when these processes become uncontrolled, pathological behaviours, like cancer or metastasis of cancerous cells, can occur. here we present a new method for the contextual quantification of cellular motility, membrane fluidity and intracellular redox state, by using the ratiometric, redox-sensitive protein rxyfp and the ratiometric fluidity-sensitive probe laurdan. we provide evidence that dynamic redox and fluidity changes are correlated with signaling processes involved in cellular motility. these findings may pave the way to novel approaches for the pharmacological control of cell invasiveness and metastasis. manipulation of cellular mechanics anna pietuch, andreas janshoff georg-august university, tammanstraße , gö ttingen, germany, e-mail: anna.pietuch@chemie.unigoettingen.de rheological properties of cells determined by the underlying cytoskeleton (cortex) are key features in cellular processes like cell migration, cell division, and cell morphology. today it is possible to investigate local cellular elastic properties under almost physiological conditions using the afm. by performing force indentation curves on local areas on a cell surface the use of contact mechanic models provides the young's modulus, comprising information about the elastic properties of cells. the administration of cytoskeleton modifying substances into the cell is achieved by microinjection. we are also investigating morphological changes and rearrangements of the cytoskeleton in time resolved impedance measurements. electric cell-substrate impedance sensing is a label-free and minimal invasive technique which allows monitoring morphological changes of cells in real time. readout of the impedance is sensitive to changes in cell-substrate contacts as well as density of cell-cell contacts yielding important information about the integrity of the cell layer and changes in the properties of the cell membrane. we are studying the cellular response to modification of the cytoskeleton e.g. by introducing proteins which affect directly the organization of the actin structure like ezrin. mechanical characterization of actin gels by a magnetic colloids technique thomas pujol, olivia du roure, julien heuvingh pmmh, espci-cnrs-umpc-p . paris, france the actin polymer is central in cell biology: it is a major component of cytoskeleton and it plays a fundamental role in motility, division, mechanotransduction…. its polymerization just beneath the cell membrane generates forces responsible for cell movement. actin filaments form a network whose architecture is defined by the nature of the binding proteins and depends on the location in the cell. for example, in the structure which leads cell migration, the lamellipodium, the gel is branched due to its interaction with arp / protein complex. determining the mechanical properties of such actin network is a crucial interest to understand how forces are generated and transmitted in living cells. we grow a branched actin network from the surface of colloids using the arp / machinery. the particles are super paramagnetic and they attract each other via dipole-dipole interaction to form chain. by increasing the magnetic field we apply an increasing force to the gel and we optically measure the resulting deformation. from those measurements we deduce a young modulus for a large amount of data. we are characterizing different networks by varying the concentration of the capping and branching proteins and we show how mechanics can be regulated by the different proteins. microtubules (mts) are central to the organization of the eukaryotic intracellular space and are involved in the control of cell morphology. in fission yeasts cells mts transport polarity factors to poles where growth is located, thus ensuring the establishment and maintenance of the characteristic spherocylindrical shape. for this purpose, mt polymerization dynamics is tightly regulated. using automated image analysis software, we investigated the spatial dependence of mt dynamics in interphase fission yeast cells. we evidenced that compressive forces generated by mts growing against the cell pole locally reduce mt growth velocities and enhance catastrophe frequencies. in addition, our systematic and quantitative analysis (in combination with genetic modifications) provides a tool to study the role of ?tips (plus-end tracking proteins) such as mal and tip in the spatial regulation of mt dynamics. we further use this system to decipher how the linear transport by mt interferes with the feedback circuitry that assures the correct spatial distribution of tea , the main polarity factor in fission yeast cells. the dynamics of the cytoskeleton are largely driven by cytoskeletal motor proteins. complex cellular functions, such as mitotsis, need a high degree of control of these motors. the versatility and sophistication of biological nanomachines still challenges our understanding. kinesin- motors fulfill essential roles in mitotic spindle morphogenesis and dynamics and were thought to be slow, processive microtubule (mt)-plus-end directed motors. here we have examined in vitro and in vivo functions of the saccharomyces cerevisiae kinesin- cin using single-molecule motility assays and single-molecule fluorescence microscopy. in vivo, the majority of cin motors moved slowly towards mt plus-ends, but we also observed occasional minus-end directed motility episodes. in vitro, individual cin motors could be switched by ionic conditions from rapid (up to lm/min) and processive minus-end, to bidirectional, to slow plus-end motion. deletion of the uniquely large insert in loop of cin induced bias towards minus-end motility and strongly affected the directional switching of cin both in vivo and in vitro. the entirely unexpected in vivo and in vitro switching of cin directionality and speed demonstrate that kinesins are much more complex than thought. these results will force us to rethink molecular models of motor function and will move the regulation of motors into the limelight as pivotal for understanding cytoskeleton-based machineries. morphological and dynamical changes during tgf-b induced epithelial-to-mesenchymal transition david schneider , marco tarantola , and andreas janshoff institute of physical chemistry, georg-august-university, gö ttingen, germany, max planck institute for dynamics and self-organization, laboratory for fluid dynamics, pattern formation and nanobiocomplexity (lfpn), goettingen, germany the epithelial-to-mesenchymal transition (emt) is a program of cellular development associated with loss of cell-cell contacts, a decreased cell adhesion and substantial morphological changes. besides its importance for numerous developmental processes like embryogenesis, emt has also been held responsible for the development and progression of tumors and formation of metastases. the influence of the cytokine transforming growth factor (tgf-b ) induced emt on structure, migration, cytoskeletal dynamics and long-term correlations of the mammalian epithelial cell lines nmumg, a and mda-mb was investigated by time-resolved impedance analysis and atomic force microscopy (afm) performing force-indentation measurements. the three cell lines display important differences in cellular morphology mirrored in changes of their elastic response (young modulus), as well as their dynamics upon tgf-b treatment. impedance based measurements of micromotility reveal a complex dynamic response to tgf-b exposure which leads to a transient increase in fluctuation amplitude and long-term correlation. additionally, the investigation of cellular elasticity via afm depicts the different cytoskeletal alterations depending on the metastatic potential of the used cell type. physics of cellular mechanosensitivity studied with biomimetic actin-filled liposomes bjö rn stuhrmann, feng-ching tsai, guido mul, gijsje koenderink fom institute amolf, amsterdam, the netherlands biological cells actively probe the mechanical properties of their tissue environment by exerting contractile forces on it, and use this information to decide whether to grow, migrate, or proliferate. the physical basis for cell contractility is the actin cytoskeleton, which transmits motor generated stresses to mechanosensitive adhesions sites that anchor the cell to the tissue. the origins of mechanosensing are far from understood due to the complex interplay of mechanical effects and biochemical signaling that occurs far from equilibrium. we use a quantitative biophysical approach based on biomimetic constructs to elucidate physical principles that underlie active mechanosensing in biological cells. we have built realistic in vitro models of contractile cells by encapsulating cross-linked actin networks together with myosin motors in cell-sized membranous containers (liposomes). our method has several advantages over prior methods, including high liposome yield, compatibility with physiological buffers, and chemical control over protein/lipid coupling. i will show contour fluctuation spectra of constructs and first data on mechanical response obtained by laser tweezers microrheology. our work will yield novel insights into stress generation and stiffness sensing of cells. setting up a system to reconstitute cytoskeleton-based protein delivery and patterning in vitro nú ria taberner, liedewij laan, marileen dogterom fom institute amolf, amsterdam, the netherlands keywords: microtubules, fission yeast, cell polarity, protein patterning, plus end binding proteins. many different cell types, from mobile fibroblasts [ ] to fission yeast cells [ ] , display non-homogenous protein patterns on their cell cortex. in fission yeast the cell-end marker protein tea that among others is responsible for recruiting the actin dependent cell-growth machinery, is specifically located at the growing cell ends. tea travels at the tips of growing microtubules and is delivered to the cell ends [ ] . we aim to in vitro reconstitute a minimal microtubule plus-end tracking system that leads to cortical protein patterning in functionalized microfabricated chambers. our model will allow us to perturb microtubule-based transport and diffusion independently and evaluate the resulting protein patterns. [ the tropomyosins (tm) are dimeric actin-binding proteins that form longitudinal polymers along the actin filament groove. there is a great variety of isoforms, but the division of labour between the individual tms and their significance is poorly understood. as in most cell types, also in the neurons several isoforms are present, whose spatio-temporal localisation is differentially regulated. the neuron-specific brain isoform (tmbr- ) can be found in the axon of the mature cells. we aimed to clone, express and charaterise this protein in terms of its effects on the kinetic parameters of the actin filament. using a pet a construct we purified native, tag-free protein, and examined if it influences the rate of actin polymerisation or the stability of the filaments in the presence of either gelsolin or latrunculin-a, two depolymerising agents. in cosedimentation experiments the affinity of tmbr- to actin was* lm, about six times that of skeletal muscle tropomyosin. the net rate of actin polymerisation was reduced by % in the presence of tmbr- . the depolymerisation induced by gelsolin or latrunculin-a was inhibited in a concentration-dependent manner. tmbr- seems to stabilise actin filaments against disassembly without significant effect on the net polymerisation. cell mobility and metastatic spreading: a study on human neoplastic cells using optical tweezers f. tavano the primary causes of death in cancer patients are local invasion and metastasis but their mechanisms are not yet completely understood. metastatization is accompanied by alterations of the cytoskeleton and membrane structure leading to changes in their biomechanical properties [ ] . in this study we analyzed by means of optical tweezers the mechanical properties of two different breast adenocarcinoma cell lines corresponding to different metastatic potential. ot were used to grab the plasma membrane by a , um silica bead and form a plasma membrane tether. we measured the force exerted by the cell membrane on the bead and drew the force-elongation curves. fitting data in the kelvin body model [ ] we found out the values for the viscoelastic parameters influencing the pulling of the membrane tethers. the first cell line analyzed, mcf- , associated to a low metastatic potential showed tether stiffness of pn/um in average. the second cell line, mda-mb , poorly differentiated with a high metastatic potential had a tether stiffness of pn/um in average, that is a four times lower value. these results seems to confirm the hypotesis that metastasis prone cells are softer than less aggressive cancer cells, and support the use of ot for these measurements for its sub-pn force resolution and because cells are manipulated without damage. [ tubulin polymerization promoting protein (tppp/p ) is a brain-specific protein that primary targets the microtubule network modulating its dynamics and stability. tppp/p is a disordered protein with extended unstructured segments localized at the n-and c-terminals straddling a flexible region. tppp/p is primarily expressed in oligodendrocytes where its multifunctional features such as tubulin polymerization promoting and microtubule bundling activities are crucial for the development of the projections in the course of oligodendrocyte differentiation enabling the ensheathment of axons with a myelin sheath that is indispensable for the normal function of the central nervous system. microtubule network, a major constituent of the cytoskeleton, displays multiple physiological and pathological functions in eukaryotic cells. the distinct functions of the microtubular structures are attained by static and dynamic associations of macromolecules and ligands as well as by post-translational modifications. tppp/p is actively involved in the regulation of microtubule dynamics not exclusively by its bundling activity, but also by its tubulin acetylation-promoting activity. atypical histone deacetylases, such as nad-dependent sirt and histone deacetylase- , function outside of the nucleus and control the acetylation level of cytosolic proteins, such as tubulin. acetylation-driven regulation of the microtubule network during cellular differentiation is an ambiguous issue. tppp/p has been recently identified as an interacting partner and inhibitor of these deacetylases and their interaction decreased the growth velocity of the microtubule plus ends and the motility of the cells. we have established cell models for the quantification of the acetylation degree of microtubule network in correlation with its dynamics and stability as well as in relation to aggresome formation, that mimics the pathological inclusion formation. the intracellular level of tppp/p is controlled at posttranscription level by microrna and at protein level by the proteosome machinery. under pathological circumstances this disordered protein displays additional moonlighting function that is independent of its association with microtubule system or deacetylases; it enters aberrant proteinprotein interaction with a-synuclein forming toxic aggregates within the neuronal and glial cells leading to the formation of inclusions characteristic for parkinson's disease and multiple system atrophy, respectively. the cell membrane separates the intracellular from the extracellular environment while intimately interacting with the cytoskeleton in numerous cellular functions, including cell division and motility. cell shape changes are for a large part mediated by the contractile actomyosin network forming the cortex underneath the cell membrane. to uncover molecular mechanisms of cell shape control based on actin-membrane interactions, we built a novel biomimetic model system: a cell-sized liposome encapsulating an actively contracting actin-myosin network. our fabrication method is inspired by a recent report of liposome preparation by swelling of lipid layers in agarose hydrogel films. we extensively characterize important liposomal properties, finding diameters between and lm, unilamellarity, and excellent and uniform encapsulation efficiency. we further demonstrate chemical control of actin network anchoring to the membrane. the resulting liposomes allow quantitative tests of physical models of cell shape generation and mechanics. in the cohesive structure of the cytoskeleton functionally distinct actin arrays orchestrate fundamental cell functions in a spatiotemporally controlled manner. emerging evidences emphasize that protein isoforms are essential for the functional polymorphism of the actin cytoskeleton. the generation of diverse actin networks is catalyzed by different nucleation factors, like formins and arp / complex. these actin arrays also exhibit qualitative and quantitative differences in the associated tropomyosin (tm) isoforms. how the molecular composition and the function of actin networks are coupled is not completely understood. we investigated the effects of different tm isoforms (skeletal muscle, cytoskeletal nm and br ) on the activity of mdia formin and arp / complex using fluorescence spectroscopic approaches. the results show that the studied tm isoforms have different effects on the mdia -, and arp / complexmediated actin assembly. the activity of the arp / complex is inhibited by sktm and tm nm , while tmbr does not have any effect. all three tm isoforms inhibited the activity of mdia . these results contribute to the understanding of the mechanisms by which tropomyosin isoforms regulate the functional diversity of the actin cytoskeleton. chronic thromboembolic pulmonary hypertension (cteph) is a dual pulmonary vascular disorder, which combines major vascular remodelling with small-vessel arteriopathy. the presence of fibroblasts in the clot, occluding the pulmonary arteries, and its composition create a microenvironment with increased collagen level, which might affect the local endothelial function. in this study, human pulmonary artery endothelial cells (hpaec) were exposed to collagen type i to address the effect of the thrombotic microenvironment on the vessel wall forming cells. the hpaecs, cultured under standard conditions were treated with , and lg/ml of collagen type i for h and h. the changes in the endothelial cell barrier function were investigated by performing permeability and migration test as well as ve-cadherin staining. collagen type i treatment led to a decrease in ve-cadherin signal in hpaec. the loosening of cell-cell contacts could be proven with a significant increase in permeability after h of collagen treatment with different concentrations. besides the loosening of the cell-cell contacts, the hpaec migration was also dose dependently retarded by collagen application over time. our data show that collagen-rich microenvironment leads to a disruption of the junctional proteins in hpaecs, indicating an environmental induced possible alteration in the function of endothelial cells in the clots of cteph patients. the implementation of miniaturisation and high throughput screening has quickened the pace of protein structure determination. however, for most proteins the process still requires milligram quantities of protein with purity [ %. these amounts are required as a result of unavoidable losses during purification and for the extensive screening of crystallisation space. for integral membrane proteins (imps), one of the initial steps in the structure determination procedure is still a major bottleneck -the over-expression of the target protein in the milligram quantity range. with a view of developing guidelines for over-expression of human imps, a systematic approach using the three most common laboratory expression systems (e. coli, s. cerevisiae, sf insect cells) was implemented. initial expression levels were determined by either partial purification using ni ? -nta (e. coli), green fluorescence protein (gfp) fluorescence using a c-terminal gfp tagged protein (s. cerevisiae) or flag tagged partial purification (sf cells). the results show that e. coli is suitable for the over-expression of human imps in the required quantity range however protein size and complexity is an important factor. the yeast system is fast and affordable but, for the group of human imps tested, the expression levels were borderline. finally for the insect cell system, the timelines are slower and it is in comparison costly to run, however, it can produce relatively large quantities of human imps. the cu?-atpase copb of enterococcus hirae is a bacterial p-type atpase involved in resistance to high levels of environmental copper by expelling excess copper. the membrane protein copb was purified from an over-expressing strain and solubilized in dodecyl-maltoside. by uv circular dichroism the secondary structure is predicted to contain - % a-helices and - % b-sheets in agreement with estimates based on homology with the ca atpase serca . we present cd-spectroscopic data on thermal unfolding of the protein to address the influence of the binding of the atp analogs atpcs and the fluorescent analog mant-atp on the protein stability. such analogs are used to mimic functional states of the atpase but undergo different interactions with the binding site that are not well characterized. we propose a competition-based assay for nucleotide binding using cdspectroscopy to deduce the occupancy of the nucleotidebinding site by non-fluorescent nucleotides. alternatively, the change of intrinsic fluorescence of mant-atp upon binding to the atpase is exploited in these assays. finally, we show how the simultaneous measurement of protein cd and nucleotide fluorescence in thermal denaturation experiments may help to determine the stability of several functional conformational states of copb. showing the steady-state distribution of electric potential, ionic concentrations are obtained efficiently. channel current, a summation of drift and diffusive currents, can be further computed from the flux of ionic concentrations. the influence of finite size effect will be also addressed. effect of cholesterol and cytoskeleton on k v . membrane distribution jimé nez-garduño am , , pardo la , ortega a , stü hmer w unam, mexico-city, mexico, mpi-em, gö ttingen, germany the potassium channel k v . is expressed nearly exclusively in the central nervous system. besides its function as an ion channel, k v . has also been associated with non-canonical signaling functions. various membrane proteins associated with cholesterol-sphingolipids enriched microdomains are involved in signaling pathways. in this work we studied the membrane distribution of k v . in highly purified brain-tissue plasma membranes as a function of cholesterol content versus cytoskeletal proteins. the results show that one fraction of kv . associates to cholesterol-rich domains or detergent resistant membranes (drm) and another fraction to non-drm domains. the kv . fraction inserted in drm is dependent on cholesterol as well as on cytoskeleton proteins. depletion of cholesterol leads to a doubling of k v . current density. we suggest that k v . coexists in two different populations: one where the transmembrane domain fits cholesterol enriched membranes and another able to fit into a less packed lipid bilayer. the importance of this distribution on signaling processes needs to be further investigated. we use the reduced model of an axis-symmetric water-filled channel whose protein wall has a single charged site. the channel length, radius and fixed charge are selected to match experimental data for gramicidin a. the ion current, occupancy and escape rate are simulated by the d self-consistent bd technique with account taken of the electrostatic ion-ion interaction. the bath with non-zero ion concentration on one side of the channel is modelled via the smoluchowski arrival rate. it is shown that: a) the occupancy saturates with michaelis-menten kinetics. b) the escape rate starts from the kramers value at small concentrations and then increases with concentration due to the electrostatic amplification of charge fluctuations. the resulting dynamics of the current can be described by modified reaction rate theory accounting for ionic escape over the fluctuating barrier [ ] . many membrane-protein functions are amenable to biophysical and biochemical investigation only after the protein of interest has been reconstituted from a detergent-solubilised state into artificial lipid bilayers. unfortunately, functional reconstitution has remained one of the main bottlenecks in the handling of numerous membrane proteins. in particular, gauging the success of reconstitution experiments has thus far been limited to trial-and-error approaches. to address this problem, we have established high-sensitivity isothermal titration calorimetry (itc) as a powerful method for monitoring the reconstitution of membrane proteins into liposomes. itc has previously been employed for characterising liposome solubilisation and reconstitution in the absence of protein. here we show that itc is also excellently suited for tracking the complex process of membrane-protein reconstitution in a non-invasive and fully automated manner. the approach is exemplified for the prokaryotic potassium channel kcsa, which we first purified in detergent micelles and then reconstituted into stable proteoliposomes at very high protein densities. electrophysiological experiments performed in planar lipid membranes confirmed that kcsa regained its functional activity upon itc-guided reconstitution. gating currents of low-voltage-activated t-type calcium channels family má ria karmažínová , Ľ ubica lacinová institute of molecular physiology and genetics, sav, bratislava, slovak republic t-type calcium channels are distinguished by relatively low voltage threshold for an activation and steep voltage dependence of activation and inactivation kinetics just above the activation threshold. kinetics and voltage dependence of macroscopic inward calcium current through ca v channels was described in a detail. in contrast, very little information is available on gating current of these channels. therefore we compared gating currents measured from all three ca v . , ca v . and ca v . channels. voltage dependencies of charge movement differ dramatically from those for macroscopic current. first, their slope factors are several-fold bigger that slope factors of macroscopic current activation. second, activation mid-point for ca v . channels on-gating is shifted to more positive membrane potentials by about mv compare to ca v . and ca v . channels, whose activation mid-points are similar. the same is truth for off-gating voltage dependences. kinetics of both onand off-gating is remarkably faster for ca v . and ca v . channels compare to ca v . channels. further, more charge is moved per unit of macroscopic current amplitude in ca v . channels compare to ca v . and ca v . channels. supported by apvv- - and vega / / . the local anaesthetic lidocaine (lid) is generally believed to reach its binding site in the intracellular vestibule of the voltage-gated sodium channel via the cell membrane. qx (qx) is a permanently charged, quaternary amine analogue of lid, that can access this binding site via a hydrophilic route across the channel protein. the mutation i e of the adult rat muscle-type sodium channel (rna v . ) opens such a hydrophilic pathway. when bound to the internal vestibule, lid stabilizes both fast and slow inactivated states. we wondered whether qx, once bound to the internal vestibule, exerts a similar modulatory action on inactivated states as lid. the construct i e was expressed in tsa cells and studied by means of the patch-clamp technique. when applied from the extracellular side lm qx stabilized the slow but not the fast inactivated state in i e. when applied internally, qx entered the channel, but stabilization of inactivated states could not be observed. these results suggest that binding site for use-dependent block is in the inner vestibule of the channel, fast inactivation is modulated only by the hydrophobic form of lid, and the binding site for modulation of slow inactivation by qx is only accessible from the extracellular side of the channel. we observed that lipophilicity (quantified by the logarithm of the calculated water-octanol partition coefficient, logp) is important in determining both kr and ki, but had a greater effect on ki. distribution coefficients (logd) discriminated better between kr and ki than partition coefficients (logp). the ratio of positively charged/neutral forms (quantified by the acidic dissociation constant, pka) was a significant determinant of resting affinity: predominantly charged compounds tended to be more potent against resting channels, while neutral compounds tended to be more state-dependent. aromaticity was more important for inactivated state affinity. the acidification of intracellular compartments is critical for a wide range of cellular processes. a recent candidate for ph regulation within early endosomes and tgn is the highly conserved intracellular na ? /h ? exchanger isoform (nhe ), whose mutation leads to neurological syndromes in human patients. however, due to its intracellular localization, nhe biochemical features are still poorly characterized and its biological function remains elusive. we have developed somatic cell genetic techniques that enable the selection of variant cells able to resist h ? killing through plasma membrane expression of h ? extruders. this enabled us to obtain stable cell lines with forced plasma membrane expression of nhe . we used them to measure its functional and pharmacological parameters with high accuracy, using fast transport kinetics. to summarize, this exchanger displays unique features within the nhe family, especially with respect to its affinity for its substrates, lithium, sodium and protons and for its guanidine-derived inhibitors. taken together with our results on the subcellular localization of the native nhe , these unique biochemical features provide new insights on the biological function and pathological implications of this intracellular na ? /h ? exchanger. analysis of the collective behaviour of ion channels j. miśkiewicz, z. trela, s. przestalski wrocław university of environmental and life sciences, physics and biophysics department, ul. norwida , - wrocław, poland a novel approach to the analysis of the ion current recordings is proposed. the main goal of the standard patch clamp technique is to measure single channel activity (however the whole cell configuration is also used in various researches). in the presented study the ion channels time series recordings were several (up to four) ion channels were present are analysed and the collective behaviour of ion channels is investigated. the time ion current time series are converted into dwelltime series and the channel activity is analysed. the hypothesis of collective ion channels behaviour is verified and the influence of organolead compounds (met pbcl) on collective ion channel activity is measured. the analysis is performed on the sv cation channels of vacuolar membrane of beta vulgaris. the aim of our computed study was to examine the possible binding site of primaquine (pq) using a combined homology protein modeling, automated docking and experimental approach. the target models of wild-type and mutant-types of the voltage-dependent sodium channel in rat skeletal muscle (rna v . ) were based on previous work by tikhonov and zhorov. docking was carried out on the p-loop into the structure model of rna v . channel, in the open state configuration, to identify those amino acidic residues important for primaquine binding. the threedimensional models of the p-loop segment of wild types and mutant types (w . w c, w c and w a at the outer tryptophan-rich lip, as well as d c, e c, k c and a c of the deka motif) helped us to identify residues playing a key role in aminoquinoline binding. in good agreement with experimental results, a -fold inhibition loss was observed, tryptophan is crucial for the reversible blocking effects of pq. as a result, w c abolished the blocking effect of primaquine in voltage-clamp assays. hydrogen bond formation accelerates channel opening of the bacterial mechanosensitive channel mscl yasuyuki sawada and masahiro sokabe department of physiology, nagoya university graduate school of medicine, nagoya, japan the bacterial mechanosensitive channel mscl is constituted of homopentamer of a subunit with two transmembrane inner and outer (tm , tm ) a-helices, and its d structure of the closed state has been resolved. the major issue of mscl is to understand the gating mechanism driven by tension in the membrane. to address this question, we performed molecular dynamics (md) simulations for the opening of mscl embedded in the lipid bilayer. in the closed state of mscl, neighboring tm inner helices are crossed each other near the cytoplasmic side and leu and val in the constricted part form a stable hydrophobic environment called gate. upon membrane stretch, phe in tm outer helices was dragged by lipids, leading to an opening of mscl. thus phe was concluded to be the major tension sensor. during opening, tm inner helices were also dragged and tilted, accompanied by the outward sliding of the crossings. this led to a slight expansion of the gate associated with an exposure of oxygen atoms of the backbone to the inner surface of the gate. this allows water penetration in the gate and formation of hydrogen bonds between water and the exposed oxygen, which in turn weakened the hydrophobic interaction at the crossings, causing a further opening of the gate and water permeation. mitochondrial bk ca , mitobk ca has been proposed to be cardioprotective and formed by proteins of * to * kda. thus, we investigated the molecular characteristics of this channel in isolated mitochondria from murine heart. labeling of adult mouse cardiomyocytes with plasmalemma bk ca antibodies, mitotracker, and wheat germ agglutinin yielded remarkable mitochondrial but not plasma membrane localization. nanoscale fluorescence microscopy (stimulated emission depletion) revealed to of * - nm bk ca clusters per mitochondria. further, western blot analysis of purified mitochondria showed the presence of a full length * kda protein. systematic rt-pcr exon scanning of isolated cardiomyocyte mrnas were consistent with a full length * kda alpha-subunit protein and revealed the expression of three splice inserts. insertless-bk ca robustly localized to the plasma membrane of cho cells but when a c-terminal splice insert was present bk ca was readily targeted to the mitochondria (protein proximity index was * . indicating % colocalization). hence, cardiac mitobk ca is composed by full-length bk ca protein but with splice inserts which may facilitate its targeting to mitochondria. supported by nih and aha. patch-clamp technique was used to examine effect of trimethyl-lead and -tin on the sv channel activity in the red beet (beta vulgaris l.) taproot vacuoles. it was found that the addition of both investigated compounds to the bath solution inhibit, in a concentration-dependent manner, sv currents. when single channel properties were analyzed, only little channel activity can be recorded in the presence of lm of organometal. compounds investigated decreased significantly (by about one order of magnitude) the open probability of single channels. the recordings of single channel activity obtained in the presence and in the absence of organometal showed that compounds only slightly (by ca. %) decreased the unitary conductance of single channels. it was also found that organometal diminished significantly the number of sv channel openings, whereas it did not change the opening times of the channels. taken together, these results suggest that organometal binding site is located outside the channel's selectivity filter and that the inhibitory effect of both compounds investigated on sv channel activity probably results from organometal-induced disorder in compatibility between membrane lipids and membrane proteins. the research was financed by polish ministry of science and higher education by grant no. nn . electrophysiological investigation of the hvdac ion channel in pore-spanning membranes conrad weichbrodt, claudia steinem georg-august-universitä t gö ttingen, iobc, tammannstr. , d- gö ttingen, germany, e-mail: cweichb@gwdg.de the human voltage-dependent anion channel (hvdac ) plays an important role in cell life and apoptosis since it is the main porin of the outer mitochondrial membrane. as hvdac is believed to play a pivotal role in apoptosis-related diseases such as stroke, alzheimer, parkinson and cancer, the alterations of its electrophysiological properties under different conditions are of great value. to perform different investigations, refolded hvdac is reconstituted in artificial membranes which typically consist of dphpc with a cholesterol fraction of - %. they are prepared via the mü ller-rudin-technique on a functionalized porous alumina substrate containing pores with a diameter of nm. the quality of these so-called nano-black-lipid-membranes (nano-blms) is verified via electrochemical impedance spectroscopy (eis), hvdac is reconstituted and single channel recordings are made. membranes are also prepared by spreading proteoliposomes on hydrophobized porous silicon nitride with pores of lm diameter. information about altered gating-characteristics and related conductivities is gained by application of holding potentials up to ± mv and evaluation of the resulting currents. the hvdac was a kind gift of prof. c. griesinger, mpibpc, gö ttingen. pharmacological inhibition of cardiac herg k ? channels is associated with increased risk of arrhythmias. many drugs bind directly to the channel, thereby blocking ion conduction. ala-scanning mutagenesis identified residues important for drug block. two aromatic residues y and f were found to be crucial for block of most compounds. surprisingly, some cavity blocking drugs are only weakly affected by mutation y a. in this study we provide a structural interpretation for this observation. md simulations on the y a mutant suggest side chain rearrangements of f located one helical turn below y . loss of p-p stacking induces reorientation of f from a cavity facing to a cavity lining conformation, thereby substantially altering the shape of the binding site. docking studies reveal that due to their rigid shape and compactness y insensitive drugs can still favorably interact with the reoriented f aryl groups, while molecules with more extended geometries cannot. the ankyrin transient receptor potential channel trpa is a transmembrane protein that plays a key role in the transduction of noxious chemical and thermal stimuli in nociceptors. in addition to chemical activation, trpa can be activated by highly depolarizing voltages but the molecular basis of this regulation is unclear. the transmembrane part of the tetrameric trpa is structurally related to the voltagegated k ? channels in which the conserved charged residues within the fourth transmembrane region (s ) constitute part of a voltage sensor. compared to these channels, the voltage-dependence of trpa is very weak (apparent number of gating charges * . versus in k ? channels) and its putative voltage-sensing domain most likely lies outside the s because trpa completely lacks positively charged residues in this region. in the present study we used homology modelling and molecular dynamics to create models of the transmembrane part and the proximal cytoplasmic c terminus of trpa . in combination with electrophysiological data obtained from whole cell patchclamp measurements we were able to point out several positively charged residues which mutation strongly alter the voltage sensitivity of trpa channel. these may be candidates for as yet unrecognized voltage sensor. photosynthesis p- action of double stress on photosystem aliyeva samira a., gasanov ralphreed a. institute of botany, azerbaijan national academy of sciences, baku, azerbaijan the simultaneous effect of photoinhibitory illumination and toxic action of heavy metals ions (cd ? and co ? ) on activity of ps in vitro measuring by millisecond delayed fluorescence (ms-df) of chlorophyll a was studied. during action on chloroplasts only of cd ? ( - m) the fast component of ms-df, which originates via radiative recombination of reaction center with the camn -cluster or y z on donor side of ps , is inhibited stronger than at action of only co ? . the steadystate level at cd ? treatment is remain stable, while at co ? action it is increased. simultaneous action of cd ? and photoinhibitory illumination ( lmol photons m - s - ) have shown that fast component of ms-df was inhibited faster with time than in case of action of co ? and excess light. result indicates that damage sites of action cd ? and co ? are donor and acceptor side of ps , accordingly. we assume that binding site of cd ? is y z or camn -cluster, one of the recombination partners with p ? on the donor side of ps . thereby, action of cd ions on donor side of ps leads mainly to development of mechanism of donor-side photoinhibition. field instrument for determination of the photosynthetic capacity of intact photosynthetic bacteria e. asztalos , z. gingl and p. maró ti department of medical physics and informatics, university of szeged, hungary, department of experimental physics, university of szeged, hungary a combined pump and probe fluorometer and spectrophotometer with high power laser diodes has been constructed to measure fast induction and relaxation of the bacteriochlorophyll fluorescence yield and light-induced absorption changes in intact cells of photosynthetic bacteria. the construction is the upgraded version of our previous set up with better time resolution ( ls). the compact design of the mechanics, optics, electronics and data processing makes the device easy to use as outdoor instrument or to integrate into larger measuring systems. the versatility and excellent performance of the apparatus will be demonstrated on different fields: ) organization and redox state of the photosynthetic apparatus of the whole cells under different growth conditions deduced from fluorescence characteristics including the lag phase, the amplitude and the rise time of the variable fluorescence, ) electron transfer in the reaction center, cytochrome bc complex and in between obtained from relaxation of the fluorescence and ) re-reduction kinetics of the oxidized primary donor of the reaction center and energetization and relaxation of the intracytoplasmic membrane tracked by absorption changes at and nm, respectively. previous work has established that the iron stress induced protein a (isia) synthesized by cyanobacteria under stress conditions, has at least two functions: light harvesting [ ] and photoprotection [ ] . under prolonged iron starvation isia becomes the main chlorophyll-binding protein in the cell and occurs without a photosystem association. these isia aggregates have a strong ability to dissipate light energy and there is evidence of carotenoid participation in the quenching mechanism via downhill energy transfer from chlorophyll to the s state of a carotenoid [ ] . in the present work we have measured the temperature dependence of the fluorescence of carotenoid depleted mutants (echinenone and/or zeaxanthin) and isia monomers in order to investigate the role of carotenoid and aggregation in the quenching process. pigment analysis confirms the absence of the carotenoid mutated in its biosynthesis but shows that it is mainly replaced. the monomers are lacking two carotenoids, echinenone and one of the two ß-carotenes found previously in isia aggregates. temperature dependent fluorescence shows that quenching properties are affected in the monomers and the mutants lacking zeaxanthin. soon exhausted oil resources and global climate change have stimulated research aiming at production of alternative fuels, ideally driven by solar energy. production of solar fuels needs to involve the splitting of water into protons, energized electrons and dioxygen. in photosynthetic organisms, solar-energy conversion and catalysis of water splitting (or water oxidation) proceed in an impressive cofactor-protein complex denoted as photosystem ii (psii). the heart of biological water-oxidation is a protein-bound manganese-calcium complex working at technically unmatched efficiency. in an attempt to learn from nature, the natural paragon is intensely studied using advanced biophysical methods. structural studies by x-ray spectroscopy with synchrotron radiation play a prominent role in this endeavor. time-resolved methods provide insights in the formation of intermediate states of the reaction cycle. an overview is presented focusing on (i) the efficiency of solar energy usage in psii, (ii) the interrelation between electron transfer and proton relocations, and (iii) the mechanism of water oxidation. as an outlook, new results on water oxidation by biomimetic manganese and cobalt oxides, which may become a key element in future solar-fuel systems, are presented. the peridinin-chlorophyll-protein (pcp) is a light-harvesting complex (lhc) that works as antenna in the photosynthetic process of dinoflagellates. the protein contains both chlorophylls and carotenoids molecules, the latter being responsible to extend the spectral range of captured light to regions where chlorophylls are transparent. pcp crystal structures [ ] reveal that each chlorophyll is surrounded by or molecules of the carotenoid peridinin, located in non-equivalent positions. the different protein environment of the sites might be responsible of a spectral shift of the pigments with the functional role to extend the absorption spectra of the complex and enhance its light harvesting capabilities. high resolution x-ray diffraction data on a reconstructed pcp, the refolded peridinin-chlorophyll a-protein (rfpcp) [ ] , and on the less common high salt-pcp (hspcp) opened the way to the mechanistic understanding of peridinin spectral tuning, peridinin chlorophyll energy transfer and photoprotective mechanism [ ] . the two pcp forms differ in various features: spectral properties, molar mass, amino acid sequence and, above all, pigment stoichiometry, the peridinin:chlorophyll ratio being : for the rfpcp and : for the hspcp [ ] . in the present work we perform classical molecular dynamics simulations of the rfpcp and the hspcp in explicit water solution. we analyse the structure and dynamics of the proteins and of their pigments to characterize the different peridinin sites in both pcp forms in terms of quantities that can affect the chromophore spectra, such as distorsion, fluctuations and nature of the protein environment. the comparison between the data suggests correspondences between the pigments of the two forms. quantum and mixed quantum/ classical molecular dynamics simulations are also under progress to investigate the effect of the protein environment on the electronic and optical properties of the pcp pigments. peculiar applications, like in optoelectronics, biosensors, photovoltaics. among the existing carrier matrices conductive metal oxides (e.g. indium tin oxide, ito), carbon nanotubes, graphenes, silicon (si) are the most frequently used materials because of their unique characteristics such as good conductivity, good optical properties and excellent adhesion to substrates. in our work we combined purified photosynthetic reaction center protein (rc) and porous silicon (psi) investigating the morphology and optoelectronic properties of the bio-nanocomposite material. ftir spectroscopy, scanning electron microscopy and energydispersive x-ray spectroscopy indicated the binding of the protein to the psi. specular reflectance spectra showed a red shift in the characteristic interference peak of the psi microcavity which was saturated at higher concentration of the protein. the binding was more effective if the functionalization was done by the si-specific oligopeptide compared to the classical covalent binding via aminopropyl-triethoxysilane (aptes). excitation by single saturation flashes indicated that the rc still exhibited photochemical turnover after the binding. the role of reactive oxygen species (ros) in plant stress, both as damaging agent and as potential signal molecule is often assessed in experiments using photosensitized elicitor dyes. for these studies, it is essential to know how efficiently these chemicals generate ros, whether they are specific ros sources, as well as their cellular localization and additional side effects. the present study addresses these issues using a variety of dyes known and traditionally applied as singlet oxygen ( o ) sources. rose bengal (rb), methylene violet (mvi), methylene blue (mb), neutral red (nr) and indigo carmine (ic) were studied as putative ros sources in tobacco leaves. ros products of photosensitized dyes were measured in vitro, using spin trapping epr spectroscopy. dye concentrations and irradiation concentration leading to equal absorbed excitation quanta were determined spectrophotometrically. in vivo studies were carried out using tobacco leaves infiltrated with water solutions of the putative o sources. cellular localizations were identified on the basis of the dyes' fluorescence. rb, nr and mvi reached into mesophyll cells and were used to study the effects of these dyes on photosynthesis. photochemical yields and quenching processes were compared before and after photosensitization of the elicitor dyes inside the leaf samples. chlorophyll-chlorophyll charge transfer quenching is the main mechanism of non-photochemical quenching in higher plants alfred r. holzwarth max-planck-institut fü r bioanorganischechemie, stiftstraße - , d- mü lheim a.d.ruhr, germany non-photochemical quenching (npq) in plants protects against photochemical destruction of the photosynthetic apparatus under excess light conditions.while one location of the npq process has been shown to be centered on the major light harvesting complex ii (lhcii) (q type or qequenching), an additional quenching center responsible for qi type (identical to q center) quenchinghas been suggested to be located on the minor light-harvesting complexes upon accumulation ofzeaxanthin (zx), in particularon cp and cp we have performed femtosecond transient absorption and time-resolved fluorescence measurements of npq quenching in intact leaves of higher plants, on isolated light harvesting complexes in the minor (non-aggregated)light harvesting complex cp reconstituted with violaxanthin (vx) or zx, and in the isolated major lhc ii complex in the aggregated state. in all of these situations we find the formation of chl-chl charge transfer (ct) states to be the dominant quenching mechanism. the yield of formation of carotenoid cation states and/or carotenoid s state is either extremely low or absent, thus excluding their involvement in npq quenching as a major quenching mechanism. single-molecule spectroscopy (sms) is a powerful technique that allows investigation of fluorescence properties from single fluorescing systems. this technique enabled us to investigate the dynamics of the fluorescence intensity and spectral profiles of single, isolated light harvesting complex (lhc) on timescales of milliseconds to seconds, during continuous laser illumination. we were able to observe how each complex can rapidly switch between different emission states [ , ] and to characterise the intensity and the spectral dynamics of major and minor antenna complexes from plants, in two different environments, mimicking the light harvesting and the light dissipating state, respectively. the results will be discussed with respect to the current models for nonphotochemical quenching (npq) mechanisms [ , , ] , a vital photoprotection mechanism during which the lhcs of plants switch between a state of very efficient light utilisation and one in which excess absorbed excitation energy is harmlessly dissipated as heat. phaeodactylum tricornutum is one of the most utilized model organisms in diatom photosynthesis research mainly due to availability of its genome sequence ( ). it's photosynthetic antennae are the fucoxanthin chlorophyll a/c binding proteins (fcps) which share a high degree of homology with lhcs of higher plants and green algae ( ) . for detailed investigation of the antenna system of p.tricornutum, a transgenic strain expressing recombinant his-tagged fcpa protein was created which simplified the purification of a specific stable trimeric fcp complex consisting of fcpa and fcpe proteins. excitation energy coupling between fucoxanthin and chlorophyll a was intact and the existence of a chlorophyll a/fucoxanthin excitonic dimer was demonstrated ( ). we investigated in detail the existence of specific antenna system for psi and psii in p.tricornutum as in case of higher plants. our studies indicated that at least the main light harvesting proteins fcpa and fcpe are most probably shared as a common antenna by both psi and psii. harvesting complex ii (lhciib) from spinach or in native thylakoid membranes by picosecond time-resolved fluorescence. the domain size was estimated by monitoring the efficiency of added exogeneous singlet excitation quenchers -phenyl-p-benzoquinone (ppq) and dinitrobenzene (dnb). the fluorescence decay kinetics of the systems under study were registered without quenchers and with quenchers added in a range of concentrations. stern-volmer constants, k sv and k sv -for aggregates (membranes) and detergentsolubilized complexes, respectively, were determined from the concentration dependence of the ratio of the mean fluorescence lifetimes without/with quencher (s , s). the ratio k k sv • s /s was suggested as a measure of the functional domain size. values in the range of - were found for lhcii macroaggregates and - for native thylakoid membranes, corresponding to domain sizes of - chlorophylls. although substantial, the determined functional domain size is still orders of magnitude smaller than the number of physically connected pigment-protein complexes; thus our results imply that the physical size of an antenna system beyond these numbers has little or no effect on improving the light-harvesting efficiency. the interaction between photosynthetic reaction center proteins (rcs) purified from purple bacterium rhodobacter sphaeroides r- and functionalized and non-functionalized (single (swnt) and multiple (mwnt) walled) carbon nanotubes (cnt-s) has been investigated. both structural (afm, tem and sem microscopy) and functional (flash photolysis and conductivity) techniques showed that rcs can be bound effectively to different cnts. both physical sorption and binding through -nh or -cooh groups gave similar results. however, it appeared that by physical sorption some sections of the cnts were covered by multiple layers of rcs. after the binding the rcs kept their photochemical activity for a long time (at least for three months, even in dried form) and there is a redox interaction between the cnt and rcs. the attachment of rc to cnts results in an accumulation of positive and negative charges followed by slow reorganization of the protein structure after excitation. in the absence of cnt the secondary quinone activity decays quickly as a function of time after drying the rc onto a glass surface. the special electronic properties of the swnt/protein complexes open the possibility for several applications, e.g. in microelectronics, analytics or energy conversion and storage. the decay of the high fluorescence state generated by actinic illumination of different durations was measured in whole cells of various strains and mutants of photosynthetic purple bacteria. although similar method is used in higher plants, its application in photosynthetic bacteria is novel and highly challanging. the available data are restricted and only the re-oxidation of the reduced primary quinone (q a -? q a ) is usually blamed for the decay kinetics. here, we will analyse the complexity of the kinetics over a very broad time range (from ls to s) and show that the dark relaxation of the bacteriochlorophyll fluorescence reflects the overlap of several processes attributed to the intra-and interproteinous electron transfer processes of the reaction center (rc) and cytochrome bc complex of the bacterium. in the shorter (\ ms) time scale, the dominating effect is the re-reduction of the oxidized primary donor (p ? ? p) that is followed by the re-oxidation of the acceptor complex of the rc by the cytochrome bc complex. as the life times and amplitudes of the components depend on the physiological state of the photosynthetic apparatus, the relaxation of the fluorescence can be used to monitor the photosynthetic capacity of the photosynthetic bacteria in vivo. circular dichroism (cd) spectroscopy is an indispensable tool to probe molecular architecture. at the molecular level chirality results in intrinsic cd, pigment-pigment interactions in protein complexes give rise to excitonic cd, whereas ''psitype'' cd originates from large densely packed chiral aggregates. it has been well established that the anisotropic cd (acd), measured on samples with defined orientation, carries specific information on the architecture of molecules. however, acd can easily be distorted by linear dichroism of the sample or instrumental imperfections -which might be the reason why it is rarely studied in photosynthesis research. here we present acd spectra of isolated intact and washed, unstacked thylakoid membranes, photosystem ii membranes (bby), and tightly-stacked lamellar macroaggregates of the main light-harvesting complex ii (lhcii). we show that the acd spectra of face-and edge-aligned stacked thylakoid membranes and lhcii lamellae exhibit profound differences in their psi-type cd bands. marked differences are also seen in the excitonic cd of bby and washed thylakoid membranes. thus acd provides an additional dimension to the light induced conformation changes of quinone depleted photosynthetic reaction centers (rcs) purified from the carotenoid-less rhodobacter sphaeroides r- were investigated by transient absorption (ta) and grating (tg) methods. surprisingly, the decay of the ta signal measured at nm was divided into a ns and a ls components. the latter coincides with the life time of the tg signal, which was assigned earlier [nagy et al. ( ) febs lett. , - ] to spectrally silent conformational changes. the nature of the ls phase was investigated further. although, the probability of chlorophyll triplet formation under our measuring conditions was small, possible contribution of the triplet states was also studied. the presence of carotenoid in the wild type rcs eliminated the ls component indicating the role of carotenoid in the energy transfer within the rcs. there was no significant effect of the molecular oxygen on the ta. this fact may be explained if the chlorophyll triplets inside the protein have reduced accessibility to molecular oxygen. a differential effect of osmotic potential and viscosity on conformation changes accompanying the primary charge separation was measured by the effect of ficoll, glucose and glycerol as compared the ta to the tg signals. variable chlorophyll fluorescence: in part a yield change due to light-induced conformational change gert schansker , szilvia z. tó th , lá szló ková cs , alfred r. holzwarth and gy} oz} o garab institute of plant biology, biological research center, hungarian academy of sciences, szeged, hungary, max-planck-institut fü r bioanorganische chemie, mü lheim an der ruhr, germany on a dark-to-light transition the chlorophyll fluorescence rises from a minimum intensity (f ) to a maximum intensity (f m ). conventionally, this rise is interpreted to arise from the reduction of the primary quinone acceptor, q a , of photosystem ii -although this cannot explain all presently available observations. in untreated leaves, at room temperature, the fluorescence rise follows the reduction of the electron transport chain (etc). once induced, * - % of the variable fluorescence intensity relaxes within ms in darkness and can be re-induced within ms as long as the etc remains reduced. analyzing the fluorescence relaxation kinetics, ?/-dcmu, * % of the amplitude cannot be explained by q a -re-oxidation. special properties of this phase determined on dcmu-inhibited samples: at cryogenic temperatures (below - °c), where the q a -/s recombination is blocked, it still relaxes and it exhibits a strong temperature dependence with an apparent e a & kj/mol, whereas the reduction of q a is nearly temperature insensitive. a fluorescence yield change, driven by light-induced conformational change in the reaction center complex, can explain all these observations. tuning function in bacterial light-harvesting complexes katia duquesne , edward o'reilly , cecile blanchard , alexandra olaya-castro and james n. sturgis lism, cnrs and aix-marseille university, marseille, france, department of physics, university college, london, uk purple photosynthetic bacteria are able to synthesize a variety of different light-harvesting complexes, sometimes referred to as lh , lh and lh . here we have investigated the structural origins of these different forms and the manner in which the sequence tunes the absorption spectrum of the light-harvesting system. we then consider the functional consequences of this tuning for the organization of the light harvesting system and on the ecology of the organisms. specifically by spectroscopic techniques, in particular circular dichroism and resonance raman spectroscopy, we have been able to obtain information on the organization of the bacteriochlorophyll binding sites in the unusual lh of roseobacter denitrificans. this provides a picture of how different peripheral light-harvesting complexes are able to modulate the absorption spectrum. the structure and organization of this complex is the put in the context of the the recently published variability of the light-harvesting complexes. in particular the observation of their ability to form mixed complexes containing different polypeptides. we examine quantitatively the possible reasons for maintaining such variability by considering the transport properties of membranes containing either pure or mixed complexes and show that mixed complexes can permit light-harvesting to continue during adaptation. we then consider the different constraints that may be behind this type of adaptation in different bacteria and the conditions under which different types of antenna system might be optimal finally we integrate this into the evolutionary context of adaptation to variable light intensity and the ecological niches where such organisms are found. interaction between photosynthetic reaction centers and ito t. szabo , g. bencsik , g. kozak , cs. visy , z. gingl , k. hernadi , k. nagy , gy. varo and l. nagy departments of medical physics and informatics, physical chemistry and materials science, technical informatics and of, applied and environmental chemistry, university of szeged, hungary, institute of biophysics, has biological research center, szeged, hungary photosynthetic reaction center proteins (rc) purified from rhodobacter sphaeroides purple bacterium were deposited on the surface of indium-tin-oxide (ito), a transparent conductive oxide, and the photochemical/-physical properties of the composite was investigated. the kinetics of the light induced absorption change indicated that the rc was still active in the composite and there was an interaction between the protein cofactors and the ito. the electrochromic response of the bacteriopheophytine absorption at nm showed an increased electric field perturbation around this chromophore on the surface of ito compared to the one measured in solution. this absorption change is associated with the chargecompensating relaxation events inside the protein. similar life time, but smaller magnitude of this absorption change was measured on the surface of borosilicate glass. the light induced change in the conductivity of the composite as a function of the concentration showed the typical sigmoid saturation characteristics unlike if the chlorophyll was layered on the ito which compound is photochemically inactive. in this later case the light induced change in the conductivity was oppositely proportional to the chlorophyll concentration due to the thermal dissipation of the excitation energy. the supramolecular organization of photosystem ii in vivo studied by circular dichroism spectroscopy tü nde tó th , , herbert van amerongen , , gy} oz} o garab , lá szló ková cs institute of plant biology, biological research center, hungarian academy of sciences, hungary, wageningen university, laboratory of biophysics, wageningen, the netherlands, microspectroscopy centre, wageningen university, wageningen, the netherlands the light reactions of photosynthesis in higher plants take place in granal chloroplast thylakoid membranes, which contain chirally organized macrodomains composed of photosystem ii (psii) supercomplexes associated with light harvesting antenna complexes (lhciis). the physiological relevance of this hierarchic organization, which often manifest itself in semicrystalline assemblies, has not been elucidated but the diversity of the supramolecular structures and their reorganizations under different conditions indicates its regulatory role. the present work focuses on the structural and functional roles of different components of lhcii-psii supercomplexes. we used various growth conditions, influencing the protein composition, and different arabidopsis mutants (kocp , kocp , kopsbw, kopsbx, dgd ), with altered organization of the membranes, and measured their circular dichroism (cd) spectra as well as their chlorophyll fluorescence kinetics to characterize the chiral macro-organization of the chromophores and the functional parameters of the membranes, respectively. we show that the formation of chiral macrodomains require the presence of supercomplexes. our data also reveal specific functions of some of the protein or lipid compounds in the light adaptation processes of plants. excitation energy transfer and non-photochemical quenching in photosynthesis rienk van grondelle department of physics, vu university, de boelelaan , hv, amsterdam, the netherlands the success of photosynthesis relies on two ultrafast processes: excitation energy transfer in the light-harvesting antenna followed by charge separation in the reaction center. lhcii, the peripheral light-harvesting complex of photosystem ii, plays a major role. at the same time, the same light-harvesting system can be 'switched' into a quenching state, which effectively protects the reaction center of photosystem ii from over-excitation and photodamage. in this talk i will demonstrate how lhcii collects and transfers excitation energy. using single molecule spectroscopy we have discovered how lhcii can switch between this light-harvesting state, a quenched state and a red-shifted state. we show that the switching properties between the light-harvesting state and the quenched state depend strongly on the environmental conditions, where the quenched state is favoured under 'npq-like' conditions. it is argued that this is the mechanism of non-photochemical quenching in plants. photobiology in the soil: arrested chlorophyll biosynthesis in pea epicotyl sections beá ta vitá nyi, katalin solymosi, annamá ria kó sa, bé la bö ddi eö tvö s university, institute of biology, department of plant anatomy, pá zmá ny p. s. /c, h- budapest, hungary the key regulatory step of chlorophyll (chl) biosynthesis is the nadph:protochlorophyllide oxidoreductase (por) catalyzed reduction of protochlorophyllide (pchlide)which is light activated in angiosperms. this process is usually described on artificially dark-grown plants. in this work, we studied epicotyl segments developed under the soil surface, which were dissected from pea plants grown under natural light conditions. using k fluorescence spectroscopy, pigment analyses, electron microscopy and fluorescence microscopy, we found that upper segments showed transitional developmental stages, i.e. chl appeared besides pchl(ide) and etio-chloroplasts were typical. in regions under cm depth, however, the characteristics of the segments were similar to those of plants germinated artificially in complete darkness, i.e. only pchl(ide) and etioplasts were present. the results of this work prove that these latter symptoms may occur in shaded tissues of fully developed, photosynthetically active plants grown under natural conditions. in this overview talk it will be shown how atomistic computations can complement experimental measurements in our quest to understand biological electron and proton transfer reactions. at first the molecular simulation methods for calculation of important electron transfer parameter such as reorganization free energy, electronic coupling matrix elements and reduction potentials will be explained. then three applications will be discussed where such computations help interpret experimental data on a molecular level. the first example concerns electron tunneling between heme a and heme a in the proton pump cytochrome c oxidase. this reaction is very fast, occurring on the nano-second time scale, and it is unclear if this is due to an unusually low reorganization free energy or due to high electronic coupling. carrying out large-scale all-atom molecular dynamics simulation of oxidase embedded in a membrane, we do not find evidence for unusually small values of reorganization energy as proposed previously, implying that the nanosecond tunneling rate between heme a and a is supported by very efficient electronic coupling. the second example is on electron transport in a deca-heme 'wire'-like protein, used by certain anaerobic bacteria to transport electrons from the inside of the cell to extracellular substrates. the crystal structure of such a protein has been solved recently for the first time. however, it is unclear if and in which direction the wire structure supports electron transport. here we present results of heme reduction potential calculations that help us reveal the possible electron flow in this protein. in a third example we explain how quantum mechanical/molecular mechanical methods (qm/mm) recently helped us understand why the catalase from h. pylori is prone to undergo an undesired protein radical migration reaction during catalysis. proton pumping activity of purple and brown membranes regenerated with retinal analogues k. bryl , k.yoshihara university of warmia and mazury, department of physics and biophysics, olsztyn, poland, suntory institute for bioorganic research, wakayamadai, osaka , japan the retinal protein bacteriorhodopsin (br) acts as a light-driven proton pump in the purple membrane (pm) of halobacterium salinarium (h.s.). the aim of these studies was to clarify whether the specific crystalline structure of protein and protein-substrate interactions are significant for h ? transfer into the aqueous bulk phase. two membrane systems were prepared: purple membranes (br arranged in a two-dimensional hexagonal lattice) and brown membranes (br not arranged in a crystalline lattice) were regenerated with -fuororetinals. light-induced proton release and reuptake as well as surface potential changes inherent in the regenerated systems reaction cycles were measured. signals of optical ph indicators residing in the aqueous bulk phase were compared with signals of ph indicator covalently linked to the extracellular surface of proteins and with surface potential changes detected by the potentiometric probe. the energies of activation of proton transfer have been calculated. experimental results and thermodynamic parameters (energies of activation) suggest the different mechanism of proton transfer into the aqueous bulk phase in these two systems. the implications for models of localized-delocalized energy coupling by proton gradients will be discussed. iron regulation is a vital process in organisms and in most of them it is accomplished through the metal solubilisation and storage by ferroxidase enzymes of the ferritin family, which have the ability of sequester, oxidize and mineralize ferrous ions using oxygen or hydrogen peroxide as substrate. dnabinding proteins from starved cells (dps) belong to this ferritin's family. dps belongs to the sub-type designated as miniferritins and, besides iron storage and release capability, is responsible for hydrogen peroxide resistance showing the ability to form stable complexes with dna. the preferable cosubstrate of this enzyme is h o although the reaction can occur in the presence of oxygen with lower rate [ , ] . in this work, the electrochemical behaviour of the recombinant dps from pseudomonas nautica, was assessed as a function of metal content in anaerobic environment with h o as co-substrate. the obtained electrochemical results together with spectroscopic studies allowed inferring some new hypothesis on the dps iron uptake mechanism. for myoglobin electrostatically immobilized on au-deposited mixed self-assembled monolayers (sams) of the composition: -s-(ch ) -cooh/-s-(ch ) -oh. our approach allows for a soft switching of the haem group charge state and accurate probing of the accompanying reorganizational dynamics of conformational (quasi-diffusional) and quantum (e.g. protonrelated) modes. the electron transfer rate constants were determined with h o or d o as solvent, under the variable temperature ( - k) or pressure ( - mpa) conditions, revealing the overall reorganization free energy of . ± . ev, activation volume of - . ± . cm mol - and inverse solvent kinetic isotope effect of . ± . ( °c). on the grounds of an extended charge-transfer theory, we propose specific protoncoupled et mechanism additionally coupled to the slow conformational dynamics of the protein matrix accompanied by translocation(s) of haem-adjacent water molecule(s). proton gradients across pore spanning membranes: towards on-chip energy conversion daniel frese, claudia steinem institute for organic and biomolecular chemistry, georg-august-university goettingen, germany in cell organelles, chemiosmotic potentials resulting from proton gradients across membranes are widely used to fix chemical energy in forms of atp. the high efficiency of this protein-mediated energy conversion raises interest for artificial proton gradient setups. to investigate proton transport across artificial membranes, we prepared pore spanning membranes (psms) on porous silicon substrates via painting technique. this allowed us to trap aqueous content of well-defined composition and volume inside the substrates microcavities. nigericin, a peptide that acts as an h ? -k ? -antiporter and bacteriorhodopsin, a transmembrane protein which is well known to be a light driven proton pump, were reconstituted into the pre-formed psms to achieve proton transport from one aqueous compartment to the other. changes of proton concentrations inside the pores were monitored by means of confocal laser scanning microscopy (clsm). therefore, pores were filled with pyranine, a ph-sensitive fluorescence dye and variations in intensity were measured to analyze proton translocation. we were able to show that both, nigericin and bacteriorhodopsin are capable of building up a proton gradient across psms and plan to co-reconstitute atp synthases for on-chip energy conversion by formation of atp. application of the gibbs free energy profiles to sequential biochemical reactions pé ter farkas, tamá s kiss and eugene hamori department of biological physics, eö tvö s ló rá nd tudomá ny egyetem, budapest, hungary, and department of biochemistry, tulane university, new orleans, la., usa the full understanding of the energetic details of complex metabolic reaction sequences requires a step-by-step analysis of the gibbs free energy (g) changes of the ''parasystem'' (i.e., a collection of atoms comprising all the molecules participating in a given reaction) as it gradually changes from its initialreactants state to its final-products state along the reaction pathway. knowing the respective equilibrium constants of each of the participating reaction steps and also the actual in vivo concentrations of the metabolites involved, a free-energy profile can be constructed that will reveal important information about the progress of the reaction as driven by thermodynamic forces. this approach will be illustrated on some biochemical reactions including the glycolytic/gluconeogenetic pathways. furthermore, the often misleading text-book representation of enzymatic catalysis will be reexamined and explained in thermodynamic terms using the free-energy profiles of both the non-catalyzed and the enzyme-catalyzed reactions. redox-active proteins can be diversely functionalized at metaldeposited self-assembled monolayers (sams) of a widely variable composition and thickness. the voltammetric methodology in combination with the advanced data processing procedures allow for comprehensive kinetic data within the congruent series of nano-devices and the subsequent calculation of the key physical parameters, such as the medium reorganization energy of et, the donor-acceptor electronic coupling, effective relaxation time (related to the protein's and environment's fluctuational dynamics), etc. in our studies the ''model'' redox protein, cytochrome c (cytc), was either freely diffusing to sam terminal groups (mode ), or attached to sams through the electrostatic interaction (mode ), or specific ''wiring'' (mode ). another redox-active protein, azurin, was confined at terminal sam groups through the hydrophobic interaction (mode ). diverse experimental strategies including the variation of sam thickness, solution viscosity temperature and hydrostatic pressure, allowed for a severe demonstration of the full adiabatic and nonadiabatic control (thinner and thicker sams, respectively), and the intermediary regime, in a nice agreement with the major theoretical predictions. proton transfers in a light-driven proton pump j.k. lanyi dept. physiology & biophysics, university of california, irvine, usa illumination of bacteriorhodopsin causes isomerization of alltrans retinal to -cis, -anti and a cyclic reaction ensues, in which the protein and the chromophore undergo conformational changes with an overall ten millisecond turn-over time and a proton is transported from one membrane side to the other. with crystal structures of six trapped intermediate states and plausible structural models for the remaining two intermediates, structures are now available for the initial bacteriorhodopsin state and all intermediates. they reveal the molecular events that underlie the light-induced transport: protonation of the retinal schiff base by asp , proton release to the extracellular membrane surface, a switch event that allows reprotonation of the schiff base from the cytoplasmic side, side-chain and main-chain motions initiated in the cytoplasmic region, formation of a single-file chain of hydrogen-bonded water molecules that conducts the proton of asp to the schiff base, and reprotonation of asp from the cytoplasmic surface. the observed changes can be summarized as a detailed atomic-level movie in which gradual relaxation of the distorted retinal causes a cascade of displacements of water and protein atoms results in vectorial proton transfers to and from the schiff base. electron transfer (et) processes are fundamental in photosynthesis, respiration and enzyme catalysis. the relative importance of superexchange and sequential mechanisms in biological et is still a matter of debate. the identification of any ''stepping stones'' necessary for electron hopping is a key point in the understanding of long range et. hence, the study of a single event in the sequence of reactions occurring in these phenomena is a fundamental but formidable task. muon spin relaxation (lsr) has been shown to be sensitive to charge transport on a molecular lengthscale. the muon is a very sensitive probe of electron transport, as any changes to the electronic density sampled by the muon can change its spin polarization, which can easily be measured. in this context, a very useful tool is the detection of the so-called avoided level crossing (alc) resonances [ ] . the enhancement in the loss of polarization of the muon's spin on these resonances dramatically increases sensitivity. we show that a laser pump -lsr probe technique can measure et processes at particular, and most importantly known, sites within the amino acids chain, and therefore track the time evolution of the electron over the molecule. keywords: photosynthesis, reaction center, electron transfer, proton transfer, fourier transform infrared, l dn, isotopic labeling, band assignment, histidine, mechanism in photosynthesis, the central step in transforming light energy into chemical energy is the coupling of light-induced electron transfer to proton uptake. in the photosynthetic reaction center (rc) of rhodobacter sphaeroides, fast formation of the charge separated state p ? q a is followed by a slower electron transfer from the primary quinone q a to the secondary quinone q b and the uptake of a proton from the cytoplasm to q b . previous fourier transform infrared (ftir) measurements on rc suggested an intermediate x in the q a -q b to q a q b transition. mutation of the amino acid aspl to asn (l dn mutant) slows down proton uptake and oxidation of q a -. using time-resolved ftir spectroscopy we characterized this rc mutant and proposed specific ir bands that belong to the intermediate x. to study the role of the iron-histidine complex located between q a and q b , we performed fast-scan ftir experiments on the l dn mutant marked with isotopically labelled histidine. we assigned ir bands of the intermediate x between cm - and cm - to histidine vibrations. these bands show the protonation of a histidine, most likely hisl , during the q a -q b to q a q b transition. based on these results we propose a new mechanism of the coupling of electron and proton transfer in photosynthesis. complex i of respiratory chains is an energy transducing enzyme present in most bacteria and in all mitochondria. it is the least understood complex of the aerobic respiratory chain, even though the crystallographic a-helical structures of bacterial and mitochondrial complexes have been recently determined [ ] [ ] . this complex catalyses the oxidation of nadh and the reduction of quinone, coupled to cation translocation across the membrane. rhodothermus marinus complex i, our main model system, is a nadh:menaquinone oxidoreductase and has been extensively characterized. we have made an exhaustive study in order to identify all the subunits present in the complex [ ] . the nature of the coupling charge of r. marinus complex i was investigated using inside-out membrane vesicles, which were active with respect to nadh oxidation and capable of creating and maintain an nadh-driven membrane potential (dw) positive inside. it was observed that this bacterial complex i is able of h ? and na ? transport, although to opposite directions. the coupling ion of the system was shown to be the h ? being transported to the periplasm, contributing in this way to the establishment of the electrochemical potential difference, while na ? is translocated to the cytoplasm [ ] . the sodium ion extrusion from the membrane vesicles was due to the activity of complex i, since it was sensitive to its inhibitor rotenone, and it was still observed when the complex i segment of the respiratory chain was isolated by the simultaneous presence of cyanide and external quinones. additional studies have shown that although neither the catalytic reaction nor the establishment of the dph requires the presence of na ? , the presence of this ion increased the proton transport. combining all these results, a model for the coupling mechanism of complex i was proposed, suggesting the presence of two different energy coupling sites, one that works only as a proton pump (na ? independent), and the other functioning as a na ? /h ? antiporter (na ? dependent) [ ] . this model was reinforced by further studies performed in the presence of the na ? /h ? antiporter inhibitor, -(n-ethyl-n-isopropyl)-amiloride (eipa) [ ] . a deeper inside into the coupling mechanism of this enzyme was provided by studying the influence of sodium ions on energy transduction by complexes i from escherichia coli and paracoccus denitrificans. it was observed that the na ? / h ? antiporter activity is not exclusive of r. marinus complex i, since the e. coli enzyme is also capable of such a transport, but is not a general property given that the p. denitrificans enzyme does not performed sodium translocation [ ] . due to the fact that r. marinus and e. coli enzymes reduce menaquinone while p. denitrificans complex i reduce ubiquinone, it is suggested that the na ? /h ? antiporter activity may be correlated with the type of quinone used as substrate. under anaerobic conditions some bacteria can use nitrate instead of oxygen in a process called denitrification. during denitrification, the reduction of no to n o is catalyzed by a membrane-bound enzyme nitric oxide reductase (nor). this enzyme is an important step in the evolution of a respiratory system: nor belongs to the superfamily of o -reducing heme-copper oxidases and is assumed to be the evolutionary ancestor of cytochrome c oxidase. the understanding of nor functioning was limited by the lack of structural information, but recently the first structures (cnor type from ps. aerug. and qnor type from g. stearoth.) were solved [ ] [ ] . we will present results of the first computational studies of nor (both cnor and qnor types) [ ] [ ] . the studies include: (i) large-scale all-atom md simulations of proteins in their natural environment (i.e. embedded in membrane and solvent), which were performed to describe water dynamics inside the protein and to identify potential proton transfer pathways, and (ii) free-energy calculations by the empirical valence bond (evb) method [ ] for the explicit proton translocations along the pathways established by md. among important findings are new proton pathways, which were not predicted from the x-ray structure and could be identified only by means of computer simulations. simulations also reveal that, despite a high structural similarity between cnor and qnor, these enzymes utilize strikingly different proton uptake mechanisms. our results provide insights into the functional conversion between no and o reductases, and into evolution proton transfer mechanisms and respiratory enzymes in general. the genome of the bacterium geobacter sulfurreducens (gs) encodes for c-type cytochromes ( ). genetic studies using cytochrome deficient gs strains and proteomic studies identified cytochromes that were produced under specific growth conditions ( ) ( ) ( ) . a putative outer-membrane cytochrome, omcf, is crucial for fe ? and u ? reduction and also for microbial electricity production ( ). omcf is a monoheme c-type cytochrome with sequence similarity to soluble cytochromes c of photosynthetic algae and cyanobacteria ( ). the structure of oxidized omcf was determined ( ) being the first example of a cytochrome c -like structure from a nonphotosynthetic organism. the structural features of omcf hinted a different function compared to cytochromes c from photosynthetic organisms, being an excellent example of how structurally related proteins are specifically design by nature to perform different physiological functions. in order to elucidate omcf structural-functional mechanism, isotopic labeled protein ( n and c) was produced and its structure in the reduced form determined by nmr. single point-mutations at key residues were produced by site-directed mutagenesis and their impact on the structural and functional properties of omcf will be presented. in the early s, the search for the source of nitrogen monoxide (no) production in mammals led to the discovery of three major isoforms of no-synthases (nos): the neuronal nos (nnos), the inducible nos (inos), and the endothelial nos (enos) ( ( )). years later, based on genomic analysis, numerous nos-like proteins have been identified in the genome other organism and in particular of several bacteria (bacillus anthrax, staphylococcus aureus… ( )). in spite of superimposable d structures and the ability to catalyse no production, all these enzymes are carrying different (if not opposite) physiological activities including cgmp signalling, cytotoxic activities, anti-oxidant defence, metabolism… moreover, noss become increasingly associated to oxidative-stress related pathologies ranging from neurodegenerative disorders, cardiovascular and inflammatory diseases, diabetes, cancers ( )…. this apparent paradox seems related to the belief that the strong similarity of sequence and structure of no-synthases must lead to a unique and identical functioning (no production) for all isoforms. this is blatant for bacterial nos-like proteins that are lacking the essential components required for no biosynthesis but remain considered as genuine no synthases. this approach might remain an obstacle to the understanding of nos actual biological role and could prevent the design of efficient nos-targeted therapeutic strategies. to elucidate this ''nos paradox'' our group has initiated a multidisciplinary approach that aims to relate the wide diversity of nos biological activities to variations in the catalytic mechanism of noss, to modifications of their regulation patterns and to adaptations to their physiological environment. in this context we have been investigating the mechanism of bacillus subtilis nos-like proteins with a special focus on the features that are specific to nos mechanism: i) electron and proton transfers and the role of the substrate and the pterin cofactor ii) oxygen activation and the role of the proximal ligand iii) the very molecular mechanism and the variations in the nature of reaction intermediates. for that matter we have been using a combination of radiolytical techniques (cryoreduction with co y-irradiation, pulse-radiolysis with the elyse electron accelerator), stateof-the-art spectroscopies (epr, atr-ftir and resonance raman, and picoseconds uv-visible absorption spectrosocopies), organic synthesis (synthesized substrates and cofactors analogues) biochemistry and molecular biology (site-directed mutagenesis). we will present our results on the coupling of electron and proton transfers, on the tuning of the proximal ''push effect'' and we will discuss the conditions that favour for each nos isoform no production versus other reactive nitrogen and oxygen species. photosynthetic iron oxidation (pio) is an ancient form of photosynthesis with relevant consequences in the shaping of the planet. this form of metabolism may have been involved in the deposition of geological structures known as banded iron formations, which hold key information regarding the co-evolution of photosynthesis and earth. rhodopseudomonas palustris tie- and rhodobacter ferroxidans sw both use ferrous iron as an electron donor to support photosynthetic growth (i.e. photoferrotrophy). the sw foxeyz operon can stimulate light-dependent iron oxidation by other bacteria. it codes a two-heme cytochrome, a pyrroloquinoline quinone protein and an inner membrane transporter, respectively. in tie- the pioabc operon is required for photoferrotrophy. it codes for a ten-heme cytochrome, an outer membrane beta-barrel and a high potential iron-sulfur protein (hipip) respectively. here we present functional and structural characterization of proteins involved in pio. this molecular characterization is essential for understanding this mode of bioenergetic metabolism, and may one day aid the development of biotechnological applications like microbial fuel cells and bioremediation. alongside classical, cytochrome respiratory pathway, phycomyces blakesleeanus possess alternative, cyanideresistant respiration (crr) facilitated by alternative oxidase (aox). in order to study role of oxygen in regulation of crr, the effects of cyanide on respiration of h old mycelia in aerated (control), hypoxic and anoxic conditions were measured. mycelium was incubated in these conditions for . h, h and h. after . h, aox activity was increased only in specimens incubated in anoxic conditions ( . %). after h, increase in aox activity was significant in both hypoxic and anoxic specimens ( . % and . %, respectively), with even greater increase after h, . % for hypoxic and . % for specimens in anoxic conditions. mycelia treated for h was then oxygenated for minutes. this induced decrease in aox activity of % in anoxic and even . % in hypoxic mycelia. aox is recognized as one of the mechanisms for maintaining low levels of reduced ubiquione which can function in conditions in which cytochrome chain is disabled, such as anoxia. this is in concordance with results obtained on p. blakesleeanus, where aox levels rise in hypoxic and anoxic conditions and decrease close to control level shortly after introduction of oxygen into the system. influence of escherichia coli f f -atpase on hydrogenase activity during glycerol fermentation k. trchounian , , g. sawers , a. trchounian department of biophysics, yerevan state university, yerevan, armenia, institute for microbiology, martin-luther university halle-wittenberg, haale, germany e. coli encodes four hydrogenases (hyd); only three of these, hyd- , hyd- and hyd- have been well characterized. hyd- has been shown recently to reversibly evolve hydrogen during glycerol fermentation at ph . [ ] . proton reduction was inhibited by n,n'dicyclohexylcarbodiimide suggesting a link with the proton-translocating f f -atpase. indeed, at ph . in an e. coli mutant (dk ) lacking f f overall hyd-activity was reduced to approximately % of the wild type activity; hyd- , but not hyd- , was detected in an in-gel activity assay. f f is therefore suggested to be required for hyd- activity. at ph . in glycerol medium hyd-activity in dk was * % of wild type activity and hyd- and hyd- exhibited only weak activity. this indicated a significant f f contribution towards hyd-activity as ph decreased. furthermore, at ph . hydactivity was negligible and only a very weak activity band corresponding to hyd- could be observed. these results suggest that f f -atpase is essential for hydrogenase activity during glycerol fermentation at ph . . taken together, the results suggest an interdependence between hyd- , hyd- and f f -atpase activity. [ ion channels and transporters control many facets of cancer cell biology and blocking the activity of these impairs tumor cell growth in vitro and in vivo. this new paradigm has opened new opportunities for pharmaceutical research in oncology , . we have contributed to this field showing that k v . (herg ) channels are aberrantly expressed in several human cancers where they control different aspects of the neoplastic cell biology such as proliferation and apoptosis, invasiveness and angiogenesis, the latter through the regulation of vegf secretion (reviewed in ). the herg dependent effects were shown in vitro and, more recently, in vivo. in preclinical models of both leukemia and colorectal cancer , herg overexpression confers a higher malignancy to neoplastic cells. moreover, herg blockers have therapeutic potential, since preclinical tests showed that treatment with specific herg blockers overcame chemoresistance in acute leukemias as well as reduced gi cancer growth, angiogenesis and metastatic spread . the overall message emerging from our data is that the herg protein represents a novel biomarker and drug target in oncology. up to now, herg was considered an ''antitarget'' due to the cardiac side effects that many (but not all) herg blockers produce and that result in lengthening the electrocardiographic qt interval. we report here recent studies on known and newly developed herg blockers that exhibit no cardiotoxicity and are more specific for the herg channels expressed in cancer cells. we reported previously that the increase of the cholesterol content of the cell membrane (in vitro) modified the biophysical parameters of the gating of kvl. k ? ion channels in human t-lymphocytes. in our present study we aimed to determine the effect of hypercholesterolemia on the biophysical parameters of kv . gating and the proliferation of t cells. t-lymphocytes were isolated from the peripheral blood of patients with cholesterol level considered normal (\ . mmol/l,control group) and patients with hypercholesterinaemia (hc). whole-cell k? currents were measured in patchclamped t cells and the kinetic (activation and inactivation kinetics) and equilibrium parameters (voltage-dependence of steady-state activation) of kv . gating were determined. lymphocyte proliferation was measured using cfse staining with and without anti-cd and anti-cd stimulation. our results indicate that the biophysical parameters of kv . gating are similar in the control group and in the 'hc' samples. the cfse-based assay showed that hypercholesterolemic t cells had higher spontenaous activation rate compared to control group. however, t cells from high cholesterol level patients challenged by anti-cd and anti-cd exhibited lower proliferation rate than control cells. generalized epilepsy with febrile seizures plus (gefs?, omim ) is a childhood genetic epilepsy syndrome correlated to mutations in the ancillary b-subunit of neuronal voltage-gated sodium channels (nachs). b -subunit is non-covalently associated with nach a-subunits, serving as modulator of channel activity, regulator of channel cell surface expression and as cell adhesion molecule. the first and best characterized gefs? mutation is the c w. this mutation changed a conserved cysteine residue into a tryptophan, disrupting a putative disulphide bridge that should normally maintain an extracellular immunoglobulinlike fold. in this study, we investigated the presence of this putative disulphide bond using -d-diagonal-sds-page, where the proteins were separated in the first dimension in absence of a reduction agent and in presence of it in the second dimension. this method allows to visualize the protein above the diagonal experimentally confirming that the disulphide bond is intramolecular. duchenne muscular dystrophy (dmd) is associated with severe cardiac complications. recent research suggests that impaired voltage-gated ion channels in dystrophic cardiomyocytes accompany cardiac pathology. it is, however, unknown if ion channel defects are primary effects of dystrophic gene mutations, or secondary effects of the developing cardiomyopathy. here, we studied na and ca channel impairments in dystrophic neonatal cardiomyocytes, derived from dmd mouse models, prior to cardiomyopathy development. dystrophin-deficiency reduced na current density. in addition, extra utrophin-deficiency altered na channel gating. moreover, also ca channel inactivation was reduced, suggesting that ion channel abnormalities are universal primary effects of dystrophic gene mutations. to assess developmental changes, we also studied na channel impairments in dystrophic adult cardiomyocytes, and found a stronger na current reduction than in neonatal ones. the described na channel impairments slowed the action potential upstroke in adult cardiomyocytes, and only in dystrophic adult mice, the qrs interval of the ecg was prolonged. ion channel impairments precede pathology development in the dystrophic heart, and may be considered cardiomyopathy triggers. supported by austrian fwf (p ). it has been over years since sensory neuron-specific sodium channel na v . was identified. since then na v . has been shown to play a crucial role in pain pathways, and it became a prominent drug target for novel pain killers. in contrast to myelinated neurons, mechanisms that target voltagegated sodium channels to the unmyelinated c-fibre axons are largely unknown. we investigated the localisation of na v . in unmyelinated primary sensory neurons. na v . was found to be clustered in lipid rafts on unmyelinated axons. when the lipid rafts are disrupted, remarkable reduction in both the conduction velocity and the number of cells responsive to mechanical stimuli to the unmyelinated axons were seen. using a compartment culture system, we also found that disruption of rafts in the middle region of the sensory axons caused a significant reduction in the responsiveness of the neurons to chemical stimuli to nerve endings. this is due to the failure of action potential propagation through the axons. these data suggest that clustering of na v . in lipid rafts of unmyelinated fibres is a key factor for the functional properties of the channel, which may due to a change in the voltage threshold. disruption of the na v . cluster and modifying lipid rafts in primary sensory neurons may be a useful new approach to control the excitability of nociceptive neurons. ion currents in are crucially important for activation of t-lymphocytes. our aim was to investigate how the blockage of various ion channels in isolation or in combination affects mitogen-dependent activation and proliferation of t-cells. we activated human peripheral blood lymphocytes using monoclonal antibodies against the tcr-cd complex and cd . we applied specific channel blockers inhibiting the major ion channels of the t-cell: either the kv . (tea or anuroctoxin), ikca (tram- ), or the crac channel ( -apb) alone or in combination. five days after the stimulus we measured the change in cell size and cellular granulation with flow cytometry along with the proportion of dividing cells, using cfse (carboxyfluorescein succinimidyl ester) dilution assay. our measurements indicated that the ion channel blockers suppressed the proportion of dividing cells in a dosedependent manner. increasing the strength of the stimulation reduced the potency of the blockers to inhibit cell proliferation and eventually the blockers were ineffective in decreasing lymphocyte proliferation. we experienced the greatest amount of inhibition using the combination of the blockers, which indicates synergy in the regulation pathway of the various ion channels. recently, sodium dependent phosphate transporter napi b was revealed as potential marker for breast, thyroid and ovarian cancer. in vivo, napi b is involved in maintenance of phosphate homeostasis and mutations or aberrant expressions of its gene (slc a ) are associated with several diseases including cancer. however, data about napi b mrna expression in different types of cancer and correspondent normal tissues are controversial and restricted. we investigated slc a gene expression level in normal ovarian tissues and different histomorphological types of ovarian tumors using real-time pcr analysis. it was found that slc a gene was highly expressed in well-differentiated endometrioid and papillary serous tumors, but was not expressed in low-differentiated tumors, benign tumors and in most normal tissues. mrna expression of slc a in serouse and endometrioid ovarian tumors accurately correlated with protein expression that was detected in these tumor samples by western blot analysis and immunohistochemistry in our previous investigation. upregulation of slc a gene expression in well-differentiated tumors may reflect cell differentiation processes during ovarian cancerogenesis and could serve as potential marker for ovarian cancer diagnosis and prognosis. in the present contribution a procedure for molecular motion characterization based on the evaluation of the mean square displacement (msd), through the self-distribution function (sdf), is presented. it is shown how msd, which represents an important observable for the characterization of dynamical properties, can be decomposed into different partial contributions associated to system dynamical processes within a specific spatial scale. it is shown how the sdf procedure allows us to evaluate both total msd and partial msds through total and partial sdfs. as a result, total msd is the weighed sum of partial msds in which the weights are obtained by the fitting procedure of measured elastic incoherent neutron scattering (eins) intensity. we apply sdf procedure to data collected, by in , in and in spectrometers (institute laue langevin), on aqueous mixtures of two homologous disaccharides (sucrose and trehalose) and on dry and hydrated (h o and d o) lysozyme with and without disaccharides. the nature of the dynamical transition is highlighted and it is shown that it occurs when the system relaxation time becomes shorter than the instrumental energy time. finally, the bioprotectants effect on protein dynamics and the amplitude of vibrations in lysozyme are presented. we evaluated q for genotoxicity in mcf- breast cancer cells in the presence or absence of doxorubicin (dox), docetaxel (dtx) and paclitaxel (ptx), anticancer drugs commonly used in chemotherapy of different solid tumors. damage to dna was determinated by comet assay. the cells, after treatment with investigated compounds, were washed up and cultured in fresh medium for , , , and hours. we have found that q by itself caused significant dna damage. moreover flavonol enhanced genotoxic effect of anticancer drugs. the highest amount of dna in the comet tail was observed h after treatment with combination of q with dox. similar changes were found in cells incubated with combination of q with taxanes -ptx or dtx. however, damage to dna in this case was considerably lower than damage caused by combination of q with dtx. our results confirmed anticancer and genotoxic activity of quercetin, which makes it a promising candidate for a potential use as a modulator of cytotoxicity and anticancer activity of anthracycline and taxane chemotherapeutics. although the health effects of low-frequency and intensity electromagnetic fields (lfi-emfs) are controversial, increasing evidence suggests that lfi-emfs are capable for initiating various healing processes. many (bio)physical ideas were suggested to explain the influence of lfi-emfs in living systems but the main effect of lfi-emfs on cell functions remains vague. however, some effects of lfi-emfs may be explained by redox and membrane processes. during diseases, cells not only demonstrate altered biochemical processes but also produce altered non-linear bioelectromagnetic complex patterns. thus, it is reasonable to use non-linear bioelectric and bioelectromagnetic signals from cells of the body for potential therapeutic applications that may be more effective than the artificial lfi-emfs signals. our novel emost (electromagnetic-own-signal-treatment) method is based on the utilization of the non-linear, bioelectric and bioelectromagnetic signals of the patients without any electromagnetic wave modulation and inversion of recorded output signals of subjects. here, we report our some restorative results after emost application. we also suggest that the possible effects of the emost may be achieved via redox-related processes. background or leak potassium conductances are a major determinant of resting potential and input resistance, two key components of cell excitability. these currents are not passive but finely tuned and adapted to cell specific functions. k p channels producing these currents are tightly regulated by a variety of chemical and physical stimuli including temperature, membrane stretch, free fatty acids, ph, ca ? , neurotransmitters and hormones as well as protein partners. these different stimuli converge on gating mechanisms that show remarkable conservation between intracellular k p channels (twik channels) and k p channels located at the plasma membrane (trek / channels). living at the edge: volume the conduction system of interfacial forces into the alveolar type ii cell in previous investigations, using new microscopic approaches, we found that the presence of an a li leads to a paradoxical situation: it is a potential threat that may cause cell injury, but also a important stimulus: at ii cells respond promptly, and show sustained ca ?signals that activate exocytosis. exocytosed surfactant, in turn, clearly prolonged the time to irreversible cell damage, and may be an adaptive and evolutionary defense mechanism against the harmful nature of surface forces. recently we published that at ii cells are sensing the a li but how this stimulus is conducted and converted into the cell, is still obscure. currently we are searching for potential calcium sources and it seems that the cells signal ca ? by extracellular ca ? entry probably through mechanosensitive channels. specialdesigned gene chips allowed a whole genome profiling of a li exposed single cells. these cells react with rapid changes on the transcriptional level: cellular pathways that are involved include e.g. defense response and lipid metabolism, and we identified genes associated with several lung diseases and injuries. we summarize, interface forces are strong, they are acting on the cells and triggering cellular events that are closely related with classical concepts in mechanotransduction, it is very plausible that those forces play a crucial role in the lung surfactant homeostasis. calorimetric method and instrumentation were worked out and applied for investigation aqueous solutions of proteins. thermal effects were analyzed by own construction heat-fluxtype dsc cell designed for temperature range from the boiling point of water down to k. the achieved sensitivity of heat flow rate (hf) of the instrument is better than %w tested using ll of mm aqueous solution of nacl. from the integral value of hf -the total enthalpy change dh total , the enthalpies of transitions were separated from the heat capacities. using the method, several types of proteins (bsa, erd , ubq, a-, b-casein, and wt, a t, a-synuclein mutants) were investigated in that temperature range. the results are shown in detail as an illustrative example. potential applications are outlined, which include (i) the distinction between the solvent accessible surfaces of globular and intrinsically disordered proteins, (ii) the distinction between protein mutants, and (iii) the identification of monomer and polymer protein states. this method provides a possibility to study the polymerization process (amyloid formation) and to investigate in-situ the reason and circumstances of that. morphometry due to self gravity in living organism is an integral part in understanding self gravitation bio. computational studies of biological system, especially in ab-initio embryo as self gravitating mass, floating over amniotic fluid, as if maintaining anti -gravitation (extrinsic) mechanism, would be an interesting one to explore biomechanics of intrinsic gravity. since the work would be of exploratory nature, both from the point of view of identifying appropriate stage of development of embryological morulla and available computational logics, it was contemplated to initiate functional study with a few reported ultrasound evidential works on small animals like mice, grasshopper including compact human embryo as mathematical structure that may allow the formal definition of concepts such as convergence, mapping and continuity. as many of the finite-dimensional function analysis in topological vector spaces are available, we initiated our simulation and studies on concentrating locally compact banach spaces. institute for x-ray physics, university of gö ttingen, germany we report on hard x-ray phase contrast imaging of black lipid membranes (blms), which are freely suspended over a micro machined aperture in an aqueous solution. this new way of membrane structure analysis allows investigating bio molecular and organic substances in aqueous environments by parallel and divergent beam propagation imaging, using partially coherent multi-kev x-ray radiation. the width of the thinning film is significantly smaller than the detector pixel size, but can be resolved from quantitative analysis of the intensity fringes in the fresnel diffraction regime down to its native thickness of about nm. to our knowledge this is the first time that such small features of a very weak phase object have been visualized by direct x-ray imaging techniques. we have put forward a simplified but extendable model, which enables the theoretical description of image formation and characterization of membrane thickness and its decrease during the thinning process from a bulk to a bimolecular film. on the basis of recent experiments, future investigations will be performed to study the interactions of membranes, as they are for example known from synaptic fusion, with high spatial resolution. shown that the acid incorporates mainly in the exterior part of the erythrocyte membrane, inducing creation of echinocytes. this suggests that it interacts predominantly with the outer part of the lipid layer of erythrocytes and liposomes. it was also shown that the cga decreases the packing order of the hydrophobic part of the membranes, without changing the anisotropic fluorescence of the hydrophobic part. one of the unique features of single molecule absorption/ emission is their anisotropy due to the well-defined transition dipoles for both processes allowing the determination of the molecule's d orientation. therefore, several techniques have been proposed in order to determine the full d orientation of dipole emitters on a single molecule level. we recently demonstrated a technique that combines emission distribution and polarization detection [ , , ] . as the method is an intensity distribution technique and based on single photon detection in principle, one can extend the d orientation determination to fluorescence correlation spectroscopy (fcs) as well as dynamical anisotropy measurements. this allows for the determination of the dynamics in d orientation of single molecules down to a nanosecond timescales. the d orientation is particularly interesting in non-isotropic environments. a lipid membrane is such a non-isotropic environment of enormous importance in biological systems. we therefore use giant unilamellar vesicle (guv) labeled with dyes like dio as a model system. due to the defined curvature of such vesicles all possible dipole orientations can be achieved. this allows us to show the capabilities of our method on different timescales and to quantify the error in determination of d orientation dynamics in lipid membranes. [ the aim of the studies was to determine the changes that occur in a biological membrane and the model lipid membrane as a result of interaction with strawberry leaf extract. numerous studies conducted all over the world have documented a beneficial effect of polyphenolic compounds on the human organism. however, the mechanism of the interaction on the molecular and cell level is not yet known. in the work presented, the effect of strawberry leaf extract on the erythrocyte and black lipid membranes has been investigated. the applied methods -spectroscopic, fluorimetric and electric -allowed to determine the hemolytic and antioxidant activity, and the packing order in the erythrocyte membrane as well as the electric capacity of blms. the results obtained indicate that the extract is efficient in protecting membrane lipids against oxidation, does not induce hemolysis, increases osmotic resistance and decreases packing order in the hydrophilic region of the erythrocyte membrane. moreover, it increases stability and life-time of flat lipid membranes, without altering their specific capacity. supported lipid bilayers are an abundant research platform for understanding the behavior of cell membranes as they allow for additional mechanical stability and enable characterization techniques not reachable otherwise. however, in computer simulations these systems have been studied only rarely up to now. we present systematic studies on different length scales of the changes that a support inflicts on a phospholipid bilayer using molecular modeling. we characterize the density and pressure profiles as well as the density imbalance induced by the support. it turns out that the changes in pressure profile are strong enough that protein function should be impacted leading to a previously neglected mechanism of transmembrane protein malfunction in supported bilayers. we determine the diffusion coefficients and characterize the influence of corrugation of the support. we also measure the free energy of transfer of phospholipids between leaflets using the coarsegrained martini model. it turns out that there is at equilibrium about a - % higher density in the proximal leaflet. these results are in agreement with data obtained by very large scale modeling using a water free model where flip-flop can be observed directly. we are additionally characterizing the intermediate states which determine the barrier height and therefore the rate of translocation. we also study the influence of surface roughness and curvature on the behavior. simulations in atomistic detail are performed for selected systems in order to confirm the findings. [ the inverse bar (i-bar) domain is part of the superfamily of the membrane-deforming protein is bin-amphiphysin-rvs (bar) proteins which induce either positive or negative membrane curvature both in vitro and in cells. generation of membrane curvature by these membrane deforming proteins often works together with actin dynamics. i-bar shares its function between actin bundling and membrane binding but it is still obscured what molecular mechanisms are responsible for these functions. the aim of our project is to investigate the detailed membrane binding properties of the i-bar of irsp and its relations to the actin cytoskeleton. in vitro fret experiments and fluorescence quenching studies were carried out between the i-bar and liposomes made up from different lipid constructs. we have found that the i-bar has preference to bind to the negatively charged lipids however it can also bind to the uncharged lipids. the fluorescence quenching studies reflected that the accessibility of the i-bar surface was higher toward the negatively charged lipids than for the uncharged ones. tns fluorescence assay reflected that the i-bar domain binds to the surface of the micells rather than penetrating into its core. lipid bilayers present a well-known order-disorder chain transition at ambient temperatures. this transition may become anomalous if the lipid head-group presents ionic dissociation at low ionic strength, as detected by several experimental techniques: between the gel and the liquid phases an intermediate phase appears as a shoulder in the specific heat, a deep in turbulence or a maximum in conductivity. we propose a statistical model which allows ionic dissociation of the polar group on the membrane surface and thus introduces competition between the hydrophobic interaction of hydrocarbonic chains, which favours the gel phase, at low temperatures, and the electrostatic interaction of charged head-groups, which favours the fluid phase, at higher temperatures. the model presents an intermediate fluid phase with higher dissociation and charge ordering on the membrane surface, beyond a sharp gel-fluid transition. the model presents increasing temperature of the main transition by addition of salt, as well as the shrinking of the anomalous region as chain length increases. model thermodynamic behavior is compared to results for pgs, phospholipids with a glycerol head-group. the well-programmed membrane fusion systems, operating in a weakly acidic environment, have attracted attention in the fields of biochemistry, biophysics, and pharmacy because these acidic conditions are generally observed in endosomal membranes or tumor tissues. we have reported a selective liposomal membrane fusion system toward a sugar-like cyclic cis-diol structure on the target liposome. this system consists of a lapidated phenyl boronic acid derivative as membrane -bound fusogen and phosphatidylinositol as target. here we report the preparation of a boronic acid / ph-responsive polypeptide conjugate as a novel membrane fusion device and the development of a target selective liposomal membrane fusion system with endosomal ph-responsiveness. during the course of lipid-mixing, inner-leaflet lipid-mixing, and contents-mixing assays to characterize membrane fusion behavior, we clearly observed a liposomal membrane fusion phenomenon when the ph of the experimental system was changed from . (physiological) to . (endosomal). our highly effective methods, which include a target selective liposomal membrane fusion, can be useful in the area of nanomedicine such as hybridoma technology and liposomebased drug or gene delivery. complete and reversible chemical denaturation of an a-helical membrane protein jana broecker, sebastian fiedler, sandro keller molecular biophysics, university of kaiserslautern, erwin-schrö dinger-str. , kaiserslautern, germany the question of how an unordered polypeptide chain assumes its native, biologically active conformation is one of the greatest challenges in molecular biophysics and cell biology. this is particularly true for membrane proteins. chemical denaturants such as urea have been used successfully for in vitro un-and refolding studies of soluble proteins and b-barrel membrane proteins. in stark contrast with these two protein classes, in vitro unfolding of a-helical membrane proteins by urea is often irreversible, and alternative denaturation assays using the harsh detergent sodium dodecyl sulphate suffer from a lack of a common reference state. here we present the complete and reversible chemical denaturation of the bacterial a-helical membrane protein mistic out of different micellar environments by urea. we applied multidimensional spectroscopy and techniques typically used in b-barrel membrane protein unfolding. mistic unfolds reversibly following a two-state equilibrium that exhibits the same unfolded reference state. this allows for a direct comparison of the folding energetics in different membrane-mimetic systems and contributes to our understanding of how a-helical membrane proteins fold as compared with both b-barrel membrane proteins and water-soluble proteins. in recent years, buckwheat has been of great interest in the world markets of healthy food, due to its high energy value, the content of unsaturated fatty acids, mineral constituents and vitamins. its seeds contain flavonoids which are natural, efficient antioxidants. the aim of the present studies was to investigate the effect of buckwheat extracts on the properties of biological membrane, which is the main site of the interaction between the substances buckwheat contains and the organism. the research was conducted on red blood cells and their isolated membranes, using the spectrophotometric, microscopic and fluorimetric methods. from the results obtained it follows that the compounds contained in buckwheat extracts increase the osmotic resistance of erythrocytes, making them less sensitive to the medium's osmotic pressure, induce changes in cell shape, producing increased number of echinocytes, and decrease the packing order of the polar heads of membrane lipids. it can thus be inferred that the compounds contained in the extracts penetrate the hydrophobic region of the erythrocyte membrane and alter its properties. .due to its small size, symmetric structure, amphipathicity, proteolytic stability and testable mode of activity, the gs backbone is a convenient model system to examine the structure-activity relationship of individual amino acid substitutions. we have previously reported the structure analysis of two gs analogues in which either the val or leu residues on the hydrophobic face of the molecule were substituted by the aliphatic f-labeled amino acid f-phg. using f-ssnmr in oriented lipid bilayers, we observed a re-alignment of the peptide that is compatible with the formation of a putative pore [top.curr.chem. : ]. here, we present novel analogs of gs with different f-prolines in the b-turn region, and with cf -bpg in place of leu. based on these f-ssnmr results and supported by cd, dsc and activity tests, we could demonstrate that all analogues are structurally intact and antimicrobially active. we observe, however, differences in the re-alignment propensity when comparing these gs analogues in dlpc and dmpc bilayers. these differences can be rationalized in terms of molecular shape being changed upon incorporation of unnatural amino acids at various sites of the molecule. the beta-propiolactone (bpl) is an inactivating reagent commonly used to produce viral vaccine preparations (whole virions or split-virions). although bpl has been reported to inactivate nucleic acids, its mechanism of action on proteins and the outcome on viral infection remains ill-defined. in this work, h n /victoria/ / influenza virus strain has been submitted to various bpl inactivation conditions (from lm to mm). cell infection ability was progressively reduced and entirely abolished at mm bpl. to clarify the bpl effect, we focused on membrane fusion infection steps using kinetic fluorescence molecule leakage from liposome and lipid fret assays combined with cryo electron microscopy. membrane fusion measured at ph on gm liposomes was reduced in a dose-dependent manner. interestingly the fusion activity was partially restored using the proton-ionophore monensin as confirmed by cryoem images. in addition, a decrease of molecule leakage irrespective to bpl concentration was measured suggesting that the hemagglutinin affinity for gm was slightly modified even at low bpl concentration. altogether these results strongly suggest that bpl treatment impairs m protein activity likely by preventing proton transport and bring new light on the mechanism of action of bpl. cellular membranes have a heterogeneous lipid composition, potentially forming nano-domains or membrane rafts, believed to be platforms of altered fluidity involved in protein sorting and trafficking . an alternative mechanism, potentially leading to protein sorting, has recently been proposed, suggesting that the curvature of membranes can also actively regulate protein localization . recently we showed that a variety of protein anchoring motifs are membrane-curvature sensors and thus up concentrate in regions of high membrane curvature . furthermore the curvature sensing ability of the anchoring motifs persisted independently of their structural characteristics. this leads us to speculate that curvature sensing might be an inherent property of any curved membrane and as a consequence, the lipid composition of the bilayer could potentially regulate this recruitment by membrane curvature. thus there might be an intimate, yet unrecognized, link between the way raft-like membrane domains and membrane-curvature promotes the localization of membrane-anchored proteins. we examined how changing the lipid composition of liposomes influenced the recruitment by membrane curvature of a model amphiphilic protein-anchoring motif. employing our single liposome curvature assay, we tested lipid mixtures with different ratios of dopc, sphingomyelin and cholesterol, giving rise to liposome populations of different phase-states. we found an amplified recruitment by membrane curvature for all raft-like l o phase-state mixtures when compared to the l d phase-state counterparts. based on these findings we suggest a synergetic effect when combining a raft-like lipid phase-state and high membrane curvature, resulting in a highly potent mechanism for selective localization of membrane-anchored proteins. keywords: non-lamellar lipid structure, phase transition, minerval, -hydroxylated fatty acid. minerval ( -hydroxyoleic acid), a potent antitumoral drug, is known to modulate the lipid membrane structure by decreasing the lamellar-to-non-lamellar phase transition temperature (t h ). a series of -hydroxy fatty acid derivatives, varying in acyl chain length and degree of unsaturation, have been analyzed in terms of their ability to stabilize the inverted hexagonal (h ii ) phase in palmitoyl-oleoyl-phosphatidylethanolamine membranes. differential scanning calorimetry and p-nuclear magnetic resonance showed that mono-and polyunsaturated, but not saturated, -hydroxylated fatty acid molecules were able to decrease the t h . lipid vesicles mimicking the lipid composition of a cell membrane were solubilized at °c in the presence of triton x- . the results demonstrated that the amount of detergent-resistant membranes, which are related to liquid ordered (lo) structures, decreased in the presence of -hydroxylated fatty acids. the so-called lipid membrane therapy focuses on the reversion of cell disfunction through the modulation of the membrane structure, thus altering the activity of membrane-associated proteins. the ability to modify the biophysical properties of a lipid membrane makes the studied -hydroxylated fatty acid molecules be prospective candidates for the use in the lipid membrane therapy. ]. however, a significant downward divergence occurs above mol % of probe content, which might indicate deviations to ideal mixing in fluid phase. results for b-py-c -hpc, in mixtures of popc with and mol % pops were indistinguishable from those obtained with pure popc vesicles; however excimer formation in pure pops bilayers appears to be appreciably higher. we also compared the excimer formation findings with quenching of the same probes by low concentrations of doxyl quencher groups labeled acyl phospholipid chain at the same depth of the pyrenyl group. the results are also scrutinized by the same two-dimensional kinetic formalism and good correlation was also found. derek marsh max-planck-institut fü r biophysikalische chemie, gö ttingen, germany the amassing of comprehensive data on the lipid composition of biological membranes by lipidomics initiatives provides a potent challenge to the membrane biophysicist interested in lipid structure. this resolves itself essentially into two aspects. the first systematises the dependence of membrane biophysical parameters on lipid molecular structure. lipid volumes, membrane dimensions, chain-melting temperatures and enthalpies, nonlamellar phase formation and structure, critical micelle concentrations and thermodynamics of membrane formation, membrane-membrane interactions and lipid transfer are amongst the properties of central biophysical interest. the relevant structural parameters are lipid chain length, degree of unsaturation, chain branching and headgroup configuration. the second, more complex and less well developed, aspect concerns the lipidlipid interactions that determine the membrane properties of lipid mixtures. in part, these can be obtained from binary phase diagrams, and the more limited number of ternary phase diagrams -notably with cholesterol -that are available. extrapolation to higher order mixtures lies in the future. i shall attempt to summarise some of the progress in these directions. the immediate aim is a second edition of my handbook of lipid bilayers, which, in addition to a vastly expanded database, will include interpretative features and will be available in the early part of next year. lateral diffusion dynamics in phosphatidylcholine/cholesterol bilayers has been mostly accessed by means of epr, nmr and fcs spectroscopic techniques. reliable stady-state florescence quenching analysis of diffusion-controlled processes has been ampered by the lack of a self-consistent kinetic formalism for the two-dimensional ( d) counterpart of the classical stern-volmer analysis for three-dimensional ( d) solvents. we studied the excimer formation of phospholipid-labeled pyrenyl probes (proportion of mol %) in mixed popc/cholestrol mlv liposomes by combined steady-state and timeresolved fluorescence the findings are in very good agreement with the theoretical predictions of the kinetic formalism specific for fluorescence quenching processes occurring in the small hydrophobic head group, the closely packed acyl chains, and the capability of interfacial hydrogen bonding have been suggested to govern the characteristic membrane behavior of long chain saturated ceramides: the self-segregation, and the formation of hexagonal phases and highly ordered gel phases. while it has been shown that structural alterations of the ceramide acyl chains induce position dependent effects on their behavior, we wanted to study the effect of interfacial properties, including hydrogen bonding, on ceramide membrane properties. the h-bond donor functions of nh and oh in the sphingosine backbone of palmitoylceramide were disrupted either separately or simultaneously by replacing the hydrogen with a methyl-group. when the lateral phase behavior of mixed bilayers containing cholesterol/sphingomyelin-rich domains was studied in the presence of the ceramide analogs, the o-methylated ceramide appeared to form a thermally stable, sterol-excluding gel phase with sphingomyelin, whereas the o-methylated ceramide failed in both thermal stabilization and sterol displacement. the doubly methylated analog was the poorest ceramide mimic. together with the possible steric effects induced by the methylations, the lack of nh h-bond donor function impaired ceramide membrane behavior to a greater degree than the lack of oh h-bond donor function. sugar-based surfactants are made from renewable resources using the ''green chemistry'' methods, are easily biodegradable and used in washing agents, cosmetics, and drug carriers. besides, there are attempts to use them as nonviral vectors in gene therapy. we studied the influence of new x-(alkyldimethylamonium)alkylaldonamide bromides (c n gab) with different chain lengths (n = , , , ) on the thermotropic phase behavior of dppc, and dppc/chol bilayers by means of differential scanning calorimetry. the surfactants were added either to the water phase or directly to the lipid phase (a mixed film was formed). we analyzed the changes in the temperatures, enthalpies and shapes of the main phase transitions as a function of concentration. molecular modeling methods were also used. cytotoxicicity of the c n gabs was determined in the cell line l and a . for cytotoxicity test, the cells were seeded in -well plates ml of Á cells/ml in the culture medium eagle'a or dulbecco with % calf serum, penicillin and streptomycin was deposited into each plates. the cells were treated with various doses of surfactants and incubated. the minimal concentration which was toxic to approximately % of cells was taken as tccd . this work was supported by grant n n . membrane fusion is ubiquitous in life requiring remodeling of two phospholipid bilayers. as supported by many experimental results and theoretical analyses, merging of membranes seems to proceed via similar sequential intermediates. contacting membranes form a stalk between the proximal leaflets which expand radially into a hemifusion diaphragm (hd) and subsequently open to a fusion pore. direct experimental verification of the hd is difficult due to its transient nature. using confocal fluorescence microscopy we have investigated the fusion of giant unilamellar vesicles (guvs) containing fluorescent membrane protein anchors and fluorescent lipid analogues in the presence of divalent cations. time resolved imaging revealed that fusion was preceded by displacement of peptides and lipid analogues from the guv-guv contact region being of several lm in size. a detailed analysis showed that this structure is consistent with the formation of an hd. a quantitative model of the hemifusion equilibrium and kinetics of the growing hd was developed. bilayer tension could be shown to drive hd expansion and interleaflet tension was found to act as a counterforce, because the outer leaflets are compressed upon hd growth. the model and its predictions fit nicely with observations above. concentration effects of trehalose in the equivalent polarity of fluid popc bilayers c. nobre , d. arrais , j. martins , ibb -cbme, faro, portugal, dcbb -fct, universidade do algarve, faro, portugal trehalose is an important disaccharide, formed by two units of glucose linked by a a- , glicosidic bond. it is capable of replacing water molecules in the hydration shell of the phospholipid headgroups, in cases of extreme dehydration, by establishing hydrogen bonds with their -co and -po groups, preserving this way the membrane structure. the polarity gradient is a significant feature of lipid bilayers and is influenced by the amounts of water within this medium. it is therefore important to understand the effects of different concentrations of trehalose in simple model membranes. using the pyrene empirical polarity scale, we monitorized changes in the polarity values when varying trehalose concentration in the bounding aqueous phase. for lower concentrations (until . m), we observed a decrease in polarity, comparing with popc bilayers in pure water. for higher trehalose concentrations (above . m), the polarity values are indistinguishable from those popc in water. using the freeze and thaw technique we obtained the same results, except for the lower trehalose concentrations. general anesthetics are indispensible tools of daily surgery. yet, their molecular mode of action remains elusive. while one school favors specific (direct) interactions with proteins of the central nervous system, another school adheres to a nonspecific modulation of biophysical membrane properties. one of the strongest arguments against lipid theories is the absence of stereo-specific effects in model membranes, as opposed to their detection by electrophysiological measurements on ionchannels. we have combined x-ray scattering and molecular dynamics simulations on palmitoyl-oleoyl-phosphatidylcholine bilayers with fluorescence microscopy on live cells to study the effects of the stereoisomers of ketamine on membrane properties. we find significant effects of both enantiomers on the distribution of lateral pressures at clinically relevant concentrations, being more pronounced for s-(?)-ketamine. we further calculated the effect of the lateral pressure profile changes on the opening probability of an ion-channel using crystallographic information. the observed channel inhibition compares remarkably well with clinically observed effects of the enantiomers. we thus provide first evidence for a stereo-specific, but indirect effect of general anesthetics on ion-channels. dependence of gramicidin a channel lifetime on membrane structure obtained from x-ray scattering measurements horia i. petrache department of physics, indiana university purdue university indianapolis, in , usa the activity of ion channels, in particular the lifetime of their conducting (open) state depends on the physical properties of lipid bilayers [ , ] which in turn depend on lipid headgroup and acyl chain composition. in order to investigate this dependence, we have performed measurements of gramicidin a (ga) channel lifetimes in three different lipid series. in each series, the lipid headgroups were phosphatidylcholine (pc), phosphatidylethanolamine (pe), and phosphatidylserine (ps), while the acyl chains consisted of symmetric monounsaturated di( : ), mixed ( : )( : ), and methylated di( : - me). in order to minimize the effect of headgroup electrostatics, measurements where performed in m kcl salts. we show how ga lifetimes depend on headgroup and acyl chain composition and on structural parameters determined by x-ray scattering. for the lipids considered, ga lifetimes cover a range from . seconds in the dope lipid to seconds in dphps. in this range, we find a gaussian dependence of ga lifetime on bilayer thickness, consistent with hydrophobic matching models. we discuss different aspects of channel-lipid interactions and to what extent measurements of ga lifetime in binary mixtures are consistent with measurements in pure lipid systems. [ the aim of the studies was to determine the effect of chlorogenic acid (cga), which is the main constituent of plant extracts, on properties of the model membranes. its effect was studied on temperature of the main phase transition of various lipids with and without presence of cholesterol, using the differential scanning calorimetry (dsc) method and the fluorimetric method. in particular, the degree of packing order of the hydrophilic phase of liposomes was determined using the laurdan and prodan probes, and fluorescence anisotropy of the hydrophobic phase with the probes dph and tma-dph. it had also been studied the effect of chlorogenic acid on the structure and capacity of black lipid membranes (blms), formed of egg lecithin and lipids extracted from erythrocytes. the results obtained indicate that cga lowers the main phase transition temperature slightly, without changing the fluorescence anisotropy in the hydrophobic part of the bilyer, and causes a decease in the packing order of the hydrophilic phase. by monitoring the capacity during blm formation we have found that the presence of chlorogenic acid accelerates the process of lipid self-organization into a bilayer, and increases stability and life of the blms. however, the was no effect of cga on specific capacity of the membranes, and thus on thickness of the liposome membrane hydrophobic layer. this work was sponsored by the ministry of science and education, scientific project no. n n and n n . many examples have recently been found where biological processes in the lipid bilayer are affected by the changes in the physicochemical properties of the membrane, e.g. the local curvature, the membrane tension and certainly the membrane structure. it has been shown that the activity of polyene antibiotics is strongly correlated to the phase diagram in a membrane composed of a mixture of popc and ergosterol or cholesterol (j membrane biol, : - , ( )). it is known that polyene action is quite sensitive to the type of sterol in the membrane, which enables its medical use, mainly as antifungals. it has been proposed that this selectivity of the drug to fungi is related to structure modulation by the sterols (see for example, biophys. j., , , ( )) and therefore the correlation found could be due to structural differences between popc/ergosterol and popc/ cholesterol along the corresponding phase diagrams. to investigate this, molecular dynamics simulations of the above mixtures along their phase diagrams were performed. it was found that there are indeed marked differences in structure along the phase diagrams, but for the sterol-sterol distribution function. an analysis of the behavior of this observable and the implications on polyene action is discussed. acyl transfer from lipids without enzyme catalysis: a new paradigm for membrane protein ageing? john sanderson, catherine pridmore, jackie mosely, paul yeo durham university, department of chemistry, durham, uk membrane proteins are recycled in cellulo with half-lives ranging from minutes to days. in other systems, such as enveloped viruses, proteins may equally remain membranebound for periods of days. it is therefore of interest to examine the behaviour of proteins in model membranes over extended periods in order to determine the long-term stability of the mixed systems, both in kinetic terms (attainment of equilibrium states) and chemical terms (reactivity). the reactivity of proteins towards membranes has been examined using the peptide melittin as a model for membrane proteins. acyl transfer from phospholipids to the peptide was found to occur over a period of several days, in the absence of any enzyme catalysis. transfer was detectable after days and reached % conversion in days. using tandem mass spectrometry approaches, the sites of melittin modification were localised. these sites included the side chain of lysine, opening the possibility that this residue may be modified in any membrane protein where this residue has an appropriate disposition. these observations challenge preconceptions concerning the membrane as an inert medium and highlight potential new mechanisms for membrane protein ageing. interaction of poly(l-arginine) with negatively charged bilayers studied by ft-ir spectroscopy christian schwieger, alfred blume martin-luther-university, halle-wittenberg, institute of chemistry, van-dankelman-platz , halle / saale, germany e-mail: christian.schwieger@chemie.uni-halle.de oligoarginine residues attached to macromolecules are known to facilitate the transport through lipid membranes. since the mechanism of this transport is still unclear, the effect is often called ''arginine magic''. we studied the interaction of poly(larginine) (pla) of different molecular weight with negatively charged lipid bilayers. we have shown by calorimetric and monolayer techniques that the interaction is due to a combination of electrostatic and hydrophobic forces. now we present an ft-ir spectroscopic study to reveal the effect of pla binding on membrane organisation and peptide conformation. we will show that pla binding reduces the lipid miscibility of negatively charged (pg or pa) and zwitterionic (pc) lipids within the bilayer. from the shift of the c=o stretching vibration we deduce that arginine side chains penetrate into the hydrophobic/ hydrophilic interface and replace hydration water molecules. the binding reduces the rotational freedom of the lipid molecules, as could be shown by an analysis of the ch -streching vibrations. pla binds in a b-sheet conformation to pg or pa gel phase membranes whereas its structure in bulk is random coil. the shift of the guanidyl vibration frequencies shows that also hydrogen bonds contribute to the pla -lipid interactions. neutron scattering studies of model membrane as a function of hydration and temperature federica sebastiani , , alessandra filabozzi and giovanna fragneto dipartimento di fisica, università degli studi di roma ''tor vergata'', roma, italy, institut laue-langevin, grenoble, france cell membranes carry out highly specialised functions in living materials. the composition of bacterial membranes is essential to understand the mechanism of action of antimicrobial peptides. in order to understand the role of the various components contributing to the overall behaviour, we have reproduced the membrane of bacillus subtilis and carried out neutron diffraction studies on d (small momentum-transfer diffractometer) and d (reflectometer used as a diffractometer), at ill. an ordered and homogeneous sample has been obtained by using the widely studied dmpc. the measured d-spacing of dmpc as a function of the relative humidity (rh) is related to the physical and chemical conditions affecting the sample. consequently the reliability of the humidity chamber, which has been previously upgraded, has been stated. moreover, the most suitable preparation technique has been set up. in order to investigate the component roles within bacillus subtilis membrane, three samples of phospholipids were prepared (with pope, popg and cardiolipin). neutron diffraction measurements, performed at controlled rh and temperature, suggested the presence of interesting phase transitions or coexistence of phases. the rupture of membrane vesicles near solid surfaces annamá ria taká ts-nyeste, imre deré nyi department of biological physics, eö tvö s university, h- budapest pazmany p. stny. /a, hungary the behavior of lipid membranes near solid surfaces has a great significance both in medicine and in technology. in spite of the widespread use and study of such membrane phenomena, their theoretical analysis is rather scarce. our main goal here is to understand the process during which membrane vesicles first adhere to solid surfaces, then rupture (or go through a series of transient ruptures) due to the mechanical tension induced by the adhesion, and finally spread along the surface forming a supported lipid bilayer. in our theoretical description we simultaneously consider the dynamics of spontaneous pore opening and closing; volume loss via leakage through the pores; and the advancement of the adhesion front. all these processes are supposed to follow an overdamped dynamics and coupled to each other through membrane tension. our numerical simulations reveal that the rupture process consists of three well distinguishable phases: a fast initial volume loss; followed by a slow volume loss; ending with a final burst and surface spreading. the second phase can be skipped if either the first phase advances far enough or the third phase sets in early enough. the smaller the vesicle, the further the first phase can advance. the third phase can start earlier if either the surface is smooth enough, or the adhesion energy is large enough, or the line tension is small enough. when the second phase is not skipped the time needed for the rupture process can take very long with a large variance. in the realistic range of the material properties (line tension, bending rigidity) the process is qualitatively always the same, so the most decisive parameter remains the size of the vesicle: the smaller the vesicle the faster and easier it ruptures. ( )). we chose four plant-derived polyphenols (flavonoids and stilbenes) of documented biological activity to study their influence on lipid domain number, area, shape, and borderlength. we found that resveratrol elevated the number of domains per vesicle, decreased their area and markedly increased the total length of domain border without affecting domains' circular shape. surprisingly, no such effect was observed for piceatannol differing from resveratrol by one hydroxyl group only. neither genistein nor -prenylnaringenin changed the morphology of lipid domains significantly. the possible mechanism of resveratrol-induced effect on lipid domains' morphology could be its selective accumulation in the interfacial regions between liquid ordered and liquid disordered domains. putative cholesterol recognition amino acid consensus (crac) motif in hiv coreceptors cxcr and ccr mikhail a. zhukovsky, albrecht ott biological experimental physics department, saarland university, saarbruecken, germany we identified a cholesterol recognition amino acid consensus (crac) motif in transmembrane domain (tmd ) of two g protein-coupled receptors (gpcrs), human chemokine receptors cxcr and ccr , coreceptors of human immunodeficiency virus (hiv). we suggest that residues belonging to this crac motif are involved in cholesterol binding to cxcr and ccr that is responsible for cholesterol requirement for cxcr and ccr conformation and function and for the role that cell cholesterol plays in the cell entry of cxcr -using and ccr -using hiv strains. putative crac sequences involve residues v /l -y -k in cxcr and l /v /v -y -k in ccr . in cxcr , crac motif is highly conserved across chordata species, whereas in ccr , crac motif is less conserved. t curve describe quantitatively the interfacial landscape around the protein molecules and can be used for the distinction between the globular and idp states. the behavior of the t and t data showed that there are two reorientation types present for every protein solutions below °c, irrespective for the nature of the protein or the solvent composition. local field fluctuation and the bpp models were applied, which failed for the buffered protein solutions and for the idps dissolved in water. a main cause of the failure is the changing h in the analyzed temperature range. this case is valid for the solutions of idps and for buffered solutions of both protein types. another cause can be the active relaxation channels other than dipolar when ions of quadrupolar nuclei are present. ligand-induced disorder-to-order transition plays a key role in the biological functions of many proteins that contain intrinsically disordered regions. this trait is exhibited by rtx (repeat in toxin) motifs found in more than virulence factors secreted by gram-negative pathogenic bacteria. we investigated several cyaa rtx polypeptides of different lengths ranging from to residues. we showed that the rtx proteins exhibit the hallmarks of intrinsically disordered proteins in the absence of calcium: they adopt premolten globule conformations and exhibit a strong timeaveraged apparent hydration, due in part to the internal electrostatic repulsions between negatively charged residues, as revealed by the high mean net charge. calcium binding triggers a strong reduction of the mean net charge, dehydration and compaction, folding and stabilization of secondary and tertiary structures of the rtx proteins. we propose that the intrinsically disordered character of the rtx proteins may facilitate the uptake and secretion of virulence factors through the bacterial secretion machinery. these results support the hypothesis that the folding reaction is achieved upon protein secretion and, in the case of proteins containing rtx motifs, could be finely regulated by the calcium gradient across bacterial cell wall. occupational exposure to heavy metals has been recognized to be a risk factor for parkinson's disease via metaltriggered deposition of alpha-synuclein (as) , . in the present work, al ? induced conformational change and instant oligomerization of as have been studied using fret and fcs as main techniques. donor and acceptor were labeled in the c-terminal at positions a c and a c. the average lifetime of donor in the presence of acceptor increases with the increase of al ? concentration, indicating as adopts a more extended conformation upon al ? binding. the intrinsic tyr fluorescence rises sharply within the mixing dead time, reflecting an enhanced hydrophobicity of the tyr environment and a fast conformational change of as. al ? also induces an immediate oligomerization of as as monitored by fcs. the diffusion coefficient of as changes from ± lm /s as monomer state to ± lm /s as oligomer state. the oligomerization is supposed to be induced by the ligand bridging of trivalent al ions. nearly % of human genes encode protein-kinases (pk), enzymes involved in cellular signaling and several other vital biochemical functions, which transfer phosphate groups from atp to specific target molecules, modifying their activity. [ ] deregulated pk have been linked to numerous diseases including cancer and diabetes, making them attractive targets for drug design. [ ] conformational transitions play a central role in regulating the phosphorylation activity. pk adopt an on state that is maximally active and one or more inactive states that shows minimal activity. [ ] the similarity of the relatively rigid and largely conserved atp binding site makes the design of selective inhibitors binding to the active state very difficult. indeed some of the best cancer therapies available are based on inhibitors, as imatinib, that bind to inactive states peculiar to a small subset of pk (abl, c-kit and pdgfr in the case on imatinib). thus, understanding the atomic details of the active to inactive transitions in kinases has a great importance. here we study a particular active-toinactive transition of c-src, a fundamental proto-oncogene involved in cancer and metastasis, by using multi-microsecond long fully solvated molecular dynamics simulations, metadynamics and ptmetad calculations [ , ] . the results, validated by mutagenesis, x-ray crystallography and binding kinetics, are suggestive of a functional role for the conformational transition. moreover, we were able to single out the most important residues affecting the conformational transition and to show that even a very conservative amino-acid substitution can have a dramatic effect on the conformational free energy landscape. the time-scales of protein folding events range over many orders of magnitude. in order to understand the complex folding mechanisms, peptides with well-defined secondary structure are often used as model systems as they may be regarded as smallest folding units of proteins. the formation of secondary structure elements occur on the nanosecond to low microsecond time scale. thus, stopped-flow techniques are too slow whereas pulsed laser techniques are capable to trigger folding processes in nanoseconds and to analyze faster folding events. we study ns-to-ls peptide dynamics by temperature-jump infrared spectroscopy. after initiation of a nanosecond temperature jump, the spectral response is monitored at single wavelengths in the amide i region reflecting the dynamics of the peptide backbone. relaxation rates are obtained. the helix-to-coil relaxation of polyglutamic acid is a multi-step process and requires more complex models than two-state kinetics. however, there are kinetic steps that are well described by single-exponential behavior and a two-state model. we demonstrate how equilibrium and time-resolved infrared spectroscopic data can be combined to deduce folding rates. unfolding and refolding studies using chemical denaturants have contributed tremendously to our understanding of the thermodynamics and kinetics of protein folding and stability. however, a major limitation of this approach lies in the large uncertainty inherent in the extrapolation of the free energy of unfolding in the absence of denaturant from free energy values measured at finite denaturant concentrations. here we show that this limitation can be overcome by combining multiple spectroscopic signals-including fluorescence, circular dichroism, and absorbance-recorded in a quasi-simultaneous and fully automated way at different wavelengths. we have optimised the number of wavelength values used, the integration time per data point, the increment in the denaturant concentration, and the weighting scheme applied for global data fitting. compared with the traditional approach based on the use of a single or a few wavelengths, we could thus improve the precision of the free energy value by an order of magnitude. we exemplify and validate this novel approach using representative, well-studied globular proteins and explain how it can be exploited to quantify subtle changes in membrane-protein stability which have thus far remained elusive. the rates of protein conformational changes are usually not only limited by external but also internal friction, however, the origin and significance of this latter phenomenon is poorly understood. it is often found experimentally that a linear fit to the reciprocal of the reaction rate as a function of the viscosity of the external medium has a non-zero , the physical basis of pressure unfolding is still largely unknown. we report here a specific study of cavities contributions to the volume difference between unfolded and folded states (dv u ), using four single point mutants of staphylococcus nuclease (snase). each mutation is localised in a strategic position on the protein structure and was designed to change a large buried hydrophobic side chain into alanine, thus opening tunable cavities in the snase d structure. measuring hsqcs peaks intensities up to bar monitored the equilibrium high pressure unfolding and leads us to precise estimations of dv u for more than two-thirds of the residues of each mutant. so-fast hmqc experiments were also performed to measure folding and unfolding rates from bar pressure jumps. high-pressure fluorescence experiments were done on six additional alanine mutants to complement the nmr study, allowing a more complete exploration of the local pressure sensitivity along the protein d structure. all these highly reliable measurements shed light on the real signification of the thermodynamic parameter dvu, and bring an unprecedented complex and heterogeneous picture at a residue level of the apparent two-state folding process of snase. determination of contributing factors to the volume change magnitude between unfolded and folded states (dv u ) is a longstanding question in the high-pressure field. we provide here new experimental and computational data using two wellcharacterized model proteins: notch ankyrin repeat domain (nank) and staphylococcal nuclease (snase). the repetitive nature of the nank protein was used to study influence of the protein size on dv u in a systematic way with a set of deletion mutants. high-pressure fluorescence data provided new evidences that neither peptide bonds hydration nor side chains differential hydration could be considered as major contributor to the measured dv u value. additional molecular dynamics (md) simulations rather suggested that the heterogeneous distribution of void volume in the folded states structures could explain the dv u variations among the nank deletion mutants. the specific issue of the void volume contribution to dv u values was studied using cavity mutants in snase, allowing a large structural mapping of the alanine mutations on this globular protein. combination of x-ray crystallography, highpressure fluorescence, high-pressure nmr and md simulations provided a first clear determination of the void volume contribution to the dv u values. these results also bring an unprecedented complex and heterogeneous picture at a residue level of the apparent two-state folding process of snase. we expressed an ig domain (i ) and a -residue-long fragment of the pevk domain in order to investigate the effect of temperature and pressure on their conformation. ftir spectroscopy is a useful method for investigating the secondary structure of proteins. we analyzed the amide i band to obtain information on protein structure. fluorescence labeling was also used in some experiments. to generate high pressures, a diamond anvil cell was employed. the ftir and fluorescence spectra of the protein fragments were recorded across the pressure and temperature ranges of - gpa and - °c, respectively. moderate changes were observed in the conformation of the pevk fragments in the explored range of the t-p plane, suggesting that the domain is a highly flexible, random-coil across the entire studied t-p range. by contrast, the i domain showed quite stable secondary structure. intrinsically disordered proteins participate in important regulatory functions in the cell, including regulation of transcription, translation, the cell cycle, and numerous signal transduction events. disordered proteins often undergo coupled folding and binding transitions upon interaction with their cellular targets. the lack of stable globular structure can confer numerous functional advantages, including, paradoxically, both binding promiscuity and high specificity in target interactions. nmr is unique in being able to provide detailed insights into the intrinsic conformational preferences and dynamics of unfolded and partly folded proteins, and into the mechanism of coupled folding and binding. the function of intrinsically disordered protein domains in transcriptional regulation and signaling will be described, with particular reference to the general transcriptional coactivators cbp and p , the tumor suppressor p , and the adenovirus e a oncoprotein. the globular domains of cbp/p are targets for coupled folding and binding of disordered transactivation motifs of numerous transcription factors and viral oncogenes, which compete for binding to limiting amounts of cbp/p . many intrinsically disordered proteins contain multipartite interaction motifs that perform an essential function in the integration of complex signaling networks. the role of multipartite binding motifs and post translational modifications in regulation of p -mediated signaling pathways will be discussed. the early vascular network is one of the simplest functioning organs in the embryo. its formation involves only one cell type and it can be readily observed and manipulated in avian embryos or in vitro explants. the early vascular network of warm-blooded vertebrates self-organizes by the collective motility of cell streams, or multicellular ''sprouts''. the elongation of these future vascular network segments depends on a continuous supply of cells, moving along the sprout towards its tip. to understand the observed self-organization process, we investigate computational models containing interactions between adherent, polarized and self-propelled cells. by comparing the simulations with data from in vivo or simplistic in vitro experiments, we explore the role of active migration, leader cells, invasion of the ecm, and cell guidance by micromechanical properties of adjacent cell surfaces. boron neutron capture therapy (bnct) is a promising method for treating the highly fatal brain tumor; glioblastoma multiform. it is a binary modality; in which use is made of two components simultaneously; viz. thermal neutrons and boron- . the biophysics of bnct is very complicated; primarily due to the complexity of element composition of the brain. moreover; numerous components contributes to the over all radiation dose both to normal brain and to tumor. simple algebraic summation cannot be applied to these dose components, since each component should at first be weighed by its relative biological effectiveness (rbe) value. unfortunately, there is no worldwide agreement on these rbe values. thermal neutrons were formerly employed for bnct, but they failed to prove therapeutic efficacy. later on; epithermal neutrons were suggested proposing that they would be enough thermalized while transporting in the brain tissues. however; debate aroused regarding the optimum source neutrons energy for treating brain tumors located at different depths in brain. insufficient knowledge regarding the rbe values of different bnct dose components was a major obstacle. a new concept was adopted for estimating the optimum source neutrons energy appropriate for different circumstances of bnct. four postulations on the optimum source neutrons energy were worked out, almost entirely independent of the rbe values of the different dose components. four corresponding condition on the optimum source neutrons energy were deduced. an energy escalation study was carried out investigating different source neutron energies, between . ev and . mev. mcnp b monte_carlo neutron transport code was utilized to study the behavior of these neutrons in the brain. the deduced four conditions were applied to the results. a source neutron energy range of few electron volts (ev) to about kev was estimated to be optimum for bnct of brain tumors located at different depths in brain. simulation of mutation induction by inhaled radon progenies in the bronchial epithelium balá zs g. madas, Á rpá d farkas and imre balá shá zy hungarian academy of sciences kfki atomic energy research institute, konkoly-thege mikló s ú t - ., budapest, h- , hungary radon is considered as the second most important cause of lung cancer after smoking. to understand the mechanisms leading from radon exposure to cancer formation is of crucial importance. this study focuses on the description of mutation induction by radon progenies in the bronchial epithelium. computational fluid and particle dynamics approach was applied to determine the radio-aerosol deposition distribution in the central airways. a numerical replica of a small fragment of the bronchial epithelium was prepared based on experimental data. microdosimetric computations were performed to quantify the cellular radiation burdens at the very site of deposition accumulation. a mutagenesis model was applied supposing that radiation induces dna damages and enhances the cell turnover rate. the results show that both considered mutagenic effects of densely ionising radiation contribute significantly to mutation induction and mutation rate depends non-linearly on exposure rate. furthermore, simulations suggest that the local maintenance capacity of the bronchial epithelium can be exhausted by chronic exposure to radon progenies with activity concentration characteristic of some uranium mines. the present work demonstrates possible applications of numerical modelling in radon related carcinogenesis studies. the neural crest is a group of cells found in all vertebrate embryos. it forms in the neural folds at the border of the neural plate and gives rise to a huge variety of cells, tissues and organs. one of the astonishing characteristic of neural crest cells is that they are able to migrate very long distances in the embryo. the neural crest has been called the ''explorer of the embryo'' as it is one of the embryonic cell types that migrate most during development, eventually colonizing almost every tissue. in this talk i will discuss our recent finding about neural crest migration. we have shown that neural crest cells, classically described as mesenchymal cells, migrate in large clusters cytokinesis relies on tight regulation of the mechanical properties of the cell cortex, a thin acto-myosin network lying under the plasma membrane. although most studies of cytokinetic mechanics focus on force generation at the equatorial acto-myosin ring, a contractile cortex remains at the poles of dividing cells throughout cytokinesis. whether polar forces influence cytokinetic cell shape is poorly understood. combining cell biology and biophysics, we demonstrate that the polar cortex makes cytokinesis inherently unstable and that any imbalance in contractile forces between the poles compromises furrow positioning. we show that limited asymmetric polar contractions occur during normal cytokinesis, and that perturbing the polar cortex leads to cell shape oscillations and division failure. a theoretical model based on a competition between cortex turnover and contraction dynamics accurately accounts for the oscillations. we further propose that blebs, membrane protrusions that commonly form at the poles of dividing cells, stabilise the position of the cleavage furrow by acting as valves releasing cortical contractility. taken together, our findings show that the physical properties of the entire cell are integrated into a finetuned mechanical system ensuring successful cytokinesis. collective motion of individual cells marks the onset of the transition to multicellularity in many microorganisms. this transition is often mediated by intercellular communication signals between cells. here, we show, in contrast, that the transition from single cell to collective motion in an ensemble of gliding bacterial cells can be understood as a dynamical selfassembly process of self-propelled rods. experiments were carried out with a mutant of the bacterium myxococcus xanthus moving by means of the a-motility system only and without undergoing reversals. the collective motion phase is confined to a monolayer and is characterized by the organization of cells into larger moving clusters. a transition to collective motion is detected in experiments by image analysis, that reveals a qualitative change of the cluster-size distribution at a critical cell packing fraction around %. this transition is characterized by a scale-free power-law cluster size distribution with an exponent . . we provide a theoretical model for cluster formation of self-propelled rods that reproduces the experimental findings for the cluster size distribution. our findings suggest that the interplay of selfpropulsion of bacteria and volume exclusion effects of the rodshaped cell bodies is sufficient to explain the onset of collective motion and the related changes in the cluster statistics. despite much speculation on the existence of structurally distinct oligomeric species associated with the conversion of certain monomeric proteins into amyloid fibrils, it has not previously been possible to observe them directly or to relate them to any key mechanistic steps involved in the interconversion process. we have developed a novel application of singlemolecule intermolecular fret to investigate in unprecedented detail the aggregation and disaggregation of alpha-synuclein, the protein whose pathogenic deposition as intracellular lewy bodies is a characteristic feature of parkinson's disease. our study reveals that a range of oligomers of different size and structure are formed, even at physiologically relevant concentrations. interestingly, the resistance to degradation of the aggregated state of alpha-synuclein, which is a well- we focused on the structure-dynamics interplay and showed how the fractal-like properties of proteins lead to such anomalous dynamics. we used diffusion, a method sensitive to the structural features of the protein fold and them alone, in order to probe protein structure. conducting a large scale study of diffusion on over pdb structures we found it to be anomalous, an indication of a fractal-like structure. taking advantage of known and newly derived relations between vibrational dynamics and diffusion, we demonstrated the equivalence of our findings to the existence of structurally originated anomalies in the vibrational dynamics of proteins. more specifically, the time dependent vibrational mean square displacement (msd) of an amino acid is predicted to be subdiffusive. the thermal variance in the instantaneous distance between amino acids is shown to grow as a power law of the equilibrium distance. the autocorrelation function in time of the instantaneous distance between amino acids is shown to decay anomalously. our analysis offers a practical tool that may aid in the identification of amino acid pairs involved in large conformational changes. more recently, we studied the effect of the hydrodynamic interaction between amino acids using a zimm-type model. we computed the time-dependent msd of an amino acid and the time-dependent autocorrelation function of the distance between two amino acids, and showed that these dynamic quantities evolve anomalously, similar to the rouse-type behavior, yet with modified dynamic exponents. we also studied the dynamic structure factor s(k,t) of proteins at large wavenumbers k, kr g [ [ , with r g the gyration radius, that are sensitive to the protein internal dynamics. we showed that the decay of s(k,t) is dominated by the spatially averaged msd of an amino acid. as a result, s(k,t) effectively decays as a stretched exponential. we compared our theory with recent neutron spin-echo studies of myoglobin and hemoglobin for the rouse and zimm models of hydrodynamic friction. in addition, i will mention two other projects currently underway: (i) a new elastic network model that accounts for the tensorial aspects of protein elasticity and is a combination of stretch-compress springs and bond-bending energies. (ii) the unfolding of a protein under the exertion of a large pulling force. allosteric regulation of enzymatic activity is crucial for controlling a multitude of fundamental cellular processes. yet the molecular level details underlying regulation often remain poorly understood. here we employed single molecule activity studies to dissect the mechanistic origin of enzymatic activity regulation. as a model system we employed a lipase and measured its activity as a function of accessibility to surface tethered liposomes ( ), which are known regulators of its activity. our results surprisingly revealed that the lipase oscillates between states of different activity. we accurately quantified for the first time both the interconversion rates between activity states and the inherent activity of these states. based on these we calculated the energetic landscape of the entire reaction pathway and identified that regulatory interactions redistributed the probability to reside on preexisting enzymatic activity states but did not alter the activity of these states. our findings provide the missing link between conformational and activity substates supporting and represent the first direct validation of the textbook hypothesis of conformational selection for regulation of enzymatic activity to identify the potential targets of cgmp in arabidopsis plants we adopted a proteomic approach to isolate possible cgmp-binding proteins. purification of soluble cgmp-binding proteins was performed using cgmp-agarose-based affinity chromatography procedure. next eluted proteins were analyzed by sds-page which revealed ten bands. we focused the subsequent analysis on low-molecular peptides of , and kda which were bound cgmp more intensively. after d-ief-page of the proteins isolated by cgmp-agaroseaffinity chromatography eight most abundant protein spots in the low-molecular area were visualized. these spots of interest were excised from the gel and in gel digested by trypsin. then tryptic peptides were analyzed by maldi-tof mass spectrometry and identified as isoforms of nucleoside diphosphate kinase (ndpk) from arabidopsis. thus, our data suggest that ndpk is a potential target of cgmp signaling in arabidopsis. dual-color fluorescence-burst analysis (dcfba) was applied to measure the quaternary structure and high affinity binding of the bacterial motor protein seca to the protein-conducting channel secyeg reconstituted into lipid vesicles. dcfba is an equilibrium technique that enables the direct observation and quantification of protein-protein interactions at the single molecule level. seca binds to secyeg as a dimer with a nucleotideand preprotein-dependent dissociation constant. one of the seca protomers binds secyeg in a salt-resistant manner, while binding of the second protomer is salt-sensitive. since protein translocation is salt-sensitive we conclude that the dimeric state of seca is required for protein translocation. a structural model for the dimeric assembly of seca while bound to secyeg is proposed based on the crystal structures of the thermatoga maritima seca-secyeg and the escherichia coli seca dimer. • dcfba is a flurorescence based single molecule technique that allows assessment of the stoichiometry of ligands bound to membrane receptors • dimeric seca binds asymmetrically to the protein-conducting membrane channel secyeg • monomeric seca binds secyeg but dimeric seca is required for protein translocation • protein translocation depends on receptor cycling of the dimeric seca if the dna charge is sufficiently neutralized by counter-ions, electrostatic interactions between helical charge patterns can cause attraction [ ] . helix specific interactions also cause tilt, in one direction, between two dna fragments [ ] . in braids and supercoils, this impetus to tilt breaks positive-negative supercoil symmetry. we show that these effects may cause spontaneous braiding of two molecules, lowering the dna pairing energy [ ] . the pairing is more energetically favourable for homologues (same base pair text) than for nonhomologous pairs. this might explain pairing between only homologues observed in nacl solution [ ] . also, we construct a simple model for a closed loop supercoil, including chiral electrostatic interactions. there are very interesting effects, for sufficient charge neutralization and groove localization of counter-ions. i.) positive super-coils are more energetically favourable than negative ones. ii.) a transition between loosely and tightly wound supercoils as one moves from negative to positive values of the supercoiling density. iii.) in positive super-coils the chiral interaction underwinds dna. [ von willebrand factor (vwf) is a large multimeric protein that is crucial for the force sensing cascade triggering primary hemostasis. it mediates binding of activated thrombocytes to injured epithelial tissue and serves as a transporter for coagulation factor viii. while it was shown that the hemostatic activity of vwf is affected by shear stress [ ] , the exact impact that shear forces have on the inflammatory cascade remains unclear. it is assumed that hydrodynamic forces lead to partial unfolding of vwf, which in consequence exposes more binding sites. in order to observe shear-induced changes of the protein's functionality, we measure conformational changes of vwf under flow with fluorescence correlation spectroscopy (fcs). we aim to measure the degree of uncoiling of vwf multimers under various buffer conditions, e.g. in the presence of colloids, vesicles or platelets. as only large multimers show significant hemostatic activity we intend to monitor the molecular weight distribution of vwf. shifts in this distribution indicate various pathological conditions making our multimer analysis a fast diagnostical tool for vwf-related diseases. this will serve as a basis for studies of vwf binding to collagen, fviii, gpib, vesicles and membranecoated nanoparticles under shear flow. [ data on mechanical properties of medically important proteins located in neural junctions are very limited. contactins (cntn) and paranodin proteins, located in extracellular part of ranvier nodes, are important for proper brain wiring. here we study a new series of fniii modules from human cntn- and - using a single molecule afm force spectroscopy and advanced, all-atom steered molecular dynamics (smd) computer simulations. mutations in cntns are responsible for numerous brain disorders including autism or pathological development of odor maps. perhaps mechanical properties of individual fniii mutated protein modules are compromised, thus we address this problem. a comparison of our afm force spectra with those of reference proteins will be presented [ ] [ ] , and the molecular level interpretation fniii nanomechanics, based on our smd data will be given. we believe that these data should help to understand a role of cntn in regulation of sodium ion channels in both normal and autistic subjects. supported in part by polish ministry of education and science, grant no. n , the computational center task in gdansk and license for accelrys software. recent achievements in rational dna-motors engineering demonstrate the possibility to design nano-motors and nanorobots capable of performing externally controlled or programmed tasks. a major obstacle in developing such a complex molecular machine is the difficulty in characterizing the intermediates, the final products and their activity. typically, non in-situ gel and afm and in-situ bulk fluorescence methods are used. i will present two dna-motors recently developed and studied using in-situ single-molecule fluorescence resonance energy transfer (smfret), alternating laser excitation (alex) and total internal reflection fluorescence spectroscopy (tirf), and will demonstrate that these methods can improve the way we design, construct, measure and understand highly complex dna-based machines. a motor made of bipedal dna-walker, which walks on a dna track embedded on a dna-origami, capable of long walking distance and maintaining structural stability, will be presented. the motor is non-autonomous; it receives ss-dna fuel/anti-fuel commands from outside (as in shin & pierce, jacs, ). the motors assembly stages and singlemotor's walking steps are monitored using smfret. the second motor is based on published bipedal autonomous dna-motor (seeman, science ). it is characterized by coordinated activity between the different motor domains leading to processive, linear and synchronized movement along a directionally polar track. to prove that the motor indeed walks, the authors chemically froze the motor at each step and use a complicated radioactive gel assay. i will demonstrate that using single-molecule approach, we are able to directly and in-situ measure single-motor's movements in few simple experimental steps, and measure its structural dynamics and kinetics. translation by a single eukaryotic ribosome using single molecule total internal reflection fluorescence microscopy, we observed translation of a short messenger rna (mrna) strand by single eukaryotic ribosomes. the ribosome-mrnas complexes are fixed to a microscope coverslip through the mrna, and mrnas are located through fluorescently labelled oligonucleotides hybridized to it downstream start codon. because of the ribosome helicase activity, the double strand formed by the oligonucleotide and the mrna is opened while the ribosome translates this region of the mrna. thus, the loss of the fluorescence signal allows us to measure the distribution of translation speed of single ribosomes. careful attention was given to photobleaching for the data analysis. this experiment opens the door to the study of eukaryotic translation at the single molecule level. erythrocyte hyperaggregation, a cardiovascular risk factor, has been associated to high plasma concentrations of fibrinogen. using atomic force microscopy (afm)-based force spectroscopy measurements, we have recently identified the erythrocyte membrane receptor for fibrinogen, an integrin with a a or a -like subunit [ ] . after this, we extended the study to the influence of erythrocyte aging on fibrinogen binding [ ] . force spectroscopy measurements showed that upon erythrocyte aging, there is a decrease of the binding to fibrinogen by decreasing the frequency of its occurrence (from . % to . %) but not its force. this observation is reinforced by zeta-potential and fluorescence spectroscopy measurements. knowing that younger erythrocytes bind more to fibrinogen, we could presume that this population is the main contributor to the cardiovascular diseases associated with increased fibrinogen blood content, which disturbs the blood flow. our data also show that sialic acid residues on the erythrocyte membrane contribute for the interaction with fibrinogen, possibly by facilitating the binding to its receptor. antimicrobial peptides are usually polycationic and amphiphilic with high affinity for bacterial membranes. in order to characterize their therapeutic potential it is crucial to disclose which properties of the peptide/lipids are important for target selectivity, and to examine the peptide structure and its association with lipid bilayers. in this work, first experiments have been carried out on a promising peptide called sb , which might represent the basis for developing a novel class of antibiotics. with the goal of enhancing the activity of a new semi-synthetic sequence, two identical peptides (wkkirvrlsa) were assembled via a lysine-linker, carrying also an octanoyl-lipid anchor. a highly active compound was obtained, but its structure and mode-of-action remain unexplored. this dendrimeric peptide and its linear deca-peptide counterpart are being studied in parallel to highlight the relevant properties and differences between dendrimeric structure and the sequence. monolayer intercalation is investigated with microtensiometry, fluorescence spectroscopy is applied to study thermodynamics and kinetics of the binding process. circular dichroism, nmr and md simulations are employed with the aim of elucidating the d structure in the membrane-bound state. the capability of proteins to build structures via self-organization is fascinating biophysicists since decades. with the advent of single-molecule methods, namely fluorescence correlation spectroscopy (fcs) and fluorescence resonance energy transfer (fret), the process of complex formation is becoming accessible to direct observation. coronaviruses (cov) are enveloped positive-stranded rna viruses. for sars-cov, it was shown that coronaviruses encode a rna-dependent rna-polymerase (rdrp) build from non-structural protein (nsp ) and non-structural protein (nsp ). this hexadecameric nsp -nsp complex is a hollow, cylinder-like structure assembled from eight copies of nsp and held together by eight nsp molecules [ , ] . we are aiming at understanding the assembly process and conformational changes of the complex for the related feline coronavirus. first results implicate that nsp alone forms a dimer, where interchain fret is more efficient than intrachain fret. for the complex the results indicate that nsp -nsp form a heterodimer which is different from sars-cov. our experiments highlight the potential of single-molecule fret for the study of protein complex formation. diffracted x-ray tracking (dxt) has been considered as a powerful technique for detecting subtle dynamic motion of the target protein at single molecular level. in dxt, the dynamics of a single protein can be monitored through trajectory of the laue spot from the nanocrystal which was labeled on the objective protein. in this study, dxt was applied to the group ii chaperonin, a protein machinery that captures an unfolded protein and refolds it to the correct conformation in an atp dependent manner. a mutant group ii chaperonin from thermococcus strain ks- with a cys residue at the tip of the helical protrusion was immobilized on the gold substrate surface and was labeled with a gold nanocrystal. we monitored diffracted spots from the nanocrystal as dynamic motion of the chaperonin, and found that the torsional motion of the chaperonin in the presence of atp condition was times larger than that in the absence of atp condition. and uv-light triggered dxt study using caged atp revealed that the chaperonin twisted counterclockwisely (from the top view of chaperonin) when the chaperonin closed its chamber, and the angular velocity from open to closed state was % faster than that from closed to open state. peptides or proteins may convert (under some conditions) from their soluble forms into highly ordered fibrillar aggregates. in vivo such transitions can lead to neurodegenerative disorders such as alzheimer's disease. alzheimer's disease is characterised by the extracellular deposition of abeta peptide in amyloid plaques, and the intracellular formation of neurofibrillary tangle (nft) deposits within neurons, the latter correlating well with disease severity. the major constituent of nft deposits are paired helical filaments (phf) composed of a microtubule-associated protein known as tau. studying the process by which tau forms these large aggregates may be an essential step in understanding the molecular basis of alzheimer's disease and other tauopathies. we have applied a two-colour single molecule fluorescence technique, and single molecule intermolecular fret measurements to study the soluble oligomers of tau which are formed during the aggregation and disaggregation of phf's. the neuronal protein alpha-synuclein is considered to play a critical role in the onset and progression of parkinson's disease. fibrillar aggregates of alpha-synuclein are the main constituents of the lewy bodies that are found in the brains of parkinson patients. however, there is growing evidence suggesting that oligomeric aggregates are significantly more toxic to cells than fibrillar aggregates. very little is known about the structure and composition of these oligomeric aggregates. we present results using single-molecule photobleaching approaches to determine the number of monomeric subunits constituting the oligomers. our results show that the oligomers have a narrow size distribution, consisting of * - monomers per oligomer. fluorescence correlation spectroscopy data confirm the narrow size distribution and additionally indicate a very loose packing of the oligomers. in combination with bulk fluorescence spectroscopy results of tryptophan containing mutants of alpha-synuclein, we present a structural model for the alpha-synuclein oligomer. gold colloids are widely used for in vitro and in vivo imaging. compared to the traditional optical tags sers-coded nanoparticles show a narrow emission bandwidth with structured spectra typical of the molecule used, a wider excitation bandwidth, higher emission intensity, a better photo-stability, and a lower toxicity. this is why in cancer therapy, besides being considered good tools for the delivery of anti-tumor drugs, aunp can be also good optical tags for the analyses of both np localization by laser scanning microscopy and the process of drug release inside the cells by raman. in our work we used nm diameter aunp loaded with rhodamine g, a molecule with a high raman and fluorescence efficiency, and with a chemical structure similar to doxorubicin, the antitumoral drug used in our system. the data showed that aunp are internalized by cells and sers can be performed. nm and nm diameter aunp loaded with doxorubicin were incubated at different time points with a cell line (human adenocarcinomic alveolar basal epithelial cells). only nm aunp showed intense raman emission typical of the doxorubicin phonon transitions. in recent years biomedical applications of diamond nanoparticles have become of significant interest, which rises questions of their biocompatibility and mechanisms of interactions with cells. the aim of this study was to compare the effect of nonmodified diamond nanoparticles (dnps) and dnps modified by the fenton reaction on human endothelial cells. dnps (\ nm particle size, sigma) were modified by the fenton reaction introducing surface -oh groups. immortalized human endothelial cells (huvec-st) were incubated with - lg/ml dnps in the optimem medium. diamond nanoparticles modified by the fenton reaction had smaller hydrodynamic diameter estimated by dynamic light scattering and the surface potential (zeta potential) measured using laser-doppler electrophoresis. they were more cytotoxic as evaluated by the mtt reduction assay. dnps augmented generation of reactive oxygen species in the cells, estimated by oxidation of ', '-dichlorofluorescin, the effect being higher for the fenton-modified dnps after -h incubation. cellular production of nitric oxide, estimated with daf-fm, was also affected by dnps; after h, fentonmodified oh, in contrast to non-modified diamond, decreased no production. diamond nanoparticles affected also the cellular level of glutathione and activities of main antioxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and glutathione s-transferase). we aim at investigating how photoreactions of proteins can be controlled by means of intense thz radiation tuned in resonance to specific vibrational modes, much in analogy to coherent control experiments conducted by fs nir laser pulses [ ] . for this we will combine a time-resolved ir difference spectroscopic setup with uniquely intense, tunable narrow bandwidth thz radiation ( - lm) at the ps beamline of the thz free electron laser felbe. these experiments will be performed on bacteriorhodopsin (br) which is the sole protein of the purple membrane of the archaebacterium halobacterium salinarum [ ] . upon illumination, the chromophore retinal isomerizes around the c -c double bond [ ] and br pumps a proton from the cytoplasmic to the extracellular side. this proton gradient is used by the bacterium to drive photosynthetic atp production under low oxygen tension [ ] . in our experiment, the photoreaction is initiated by a visible laser pulse as in standard experiments, but then the sample will be irradiated by a thz pulse from the free electron laser tuned into resonance with low-energy vibrational modes which is supposed to influence the photoreaction [ ] . such vibrational control will be monitored by time-resolved ftir spectroscopy using the step-scan technique [ ] . liposomes are increasingly studied as nanoscale drug delivery systems and biomembrane models. however the exact structure dynamics and mechanical behavior of liposomes is little known. atomic force microscopy (afm) is a powerful tool to characterize nanoscale morphology and enables the mechanical manipulation of submicron-sized vesicles. a drawback of afm, however, is that liposomes may flatten and rupture on substrates to form patches or supported planar bilayers (spb). our aim was to obtain better understanding of factors affecting liposomes on substrates and find experimental conditions at which liposomes preserve their structural integrity. in the presence of divalent cations dppc liposomes formed spb on mica. vesicles sedimented subsequently preserved their integrity and showed stronger attachment to spb. in addition to cross-bridging lipid head groups, divalent cations influence the surface charge of liposomes, thereby modulating liposome-substrate and liposome-liposome interfacial interactions. preserved vesicles stabilized by divalent cations may provide a unique experimental system for studying membrane-protein interactions. the influence of e.g. ph and ionic strength on various chromatographic bead -biomass combinations. to analyze the force curves, possible elastic contributions, e.g. from deforming cellular membranes, have to be decoupled from the interaction forces. then, bead-biomass interactions will be modeled using (extended) dlvo theory and resulting data can also be compared to real-life eba processes. the project aims for a better understanding of the interaction forces in chromatography and might help to improve the process quality of eba. long-term non-invasive in vivo monitoring of the survival, migration, homing and fate of transplanted cells is of key importance for the success of cell therapy and regenerative medicine. tools for in vivo magnetic resonance (mr) imaging of labeled cells are therefore being developed. we have prepared superparamagnetic iron oxide nanoparticles by the coprecipitation of fe(ii) and fe(iii) salts and oxidation. to stabilize the particles and to facilitate their internalization by the cells, the nanoparticles were coated with several novel low-and highmolecular weight compounds including d-mannose, poly(llysin), poly(n,n-dimethylacrylamide) and dopamine-hyaluronate conjugate. the surface-modified magnetic nanoparticles were thoroughly characterized by a range of physico-chemical methods, which proved the presence of the coating on the particles. the particles were then investigated in stem cell experiments in terms of real time cell proliferation analysis, viability, labeling efficiency and differentiation. the iron oxide concentration of the labeled cells was assessed using mr relaxometry. the advantages/disadvantages of particular iron oxide coatings will be discussed and the optimal coating suggested. excellent contrast was achieved by labeling the cells with dopamine-hyaluronate-coated nanoparticles. support of the as cr (no. kan ) is acknowledged. in the last decades the interest towards the fabrication of innovative bio-sensors with improved sensitivity and reliability for medical-diagnostics applications has been constantly risen. among the different techniques, microfluidic systems are playing a major role. in order to detect extremely low concentrations of biomolecules (pm and fm), attention should be placed on the controlled, selective functionalization of micro-and nano-channels. in this work we propose a new approach to functionalize gold patches inside fluidic channels. we start from self-assembled monolayers (sams) of thiolated molecules on a gold electrode deposited inside the channel. then, by using an electrochemical approach [ , ] we remove molecules from the sam at selected locations, by applying a negative voltage to the electrode. the newly exposed gold surface can be re-functionalized by using a thiolated biomolecule (i.e an antibody) capable to bind specific proteins flowing inside the channel. the cycle can be applied to other electrodes in the microfluidic system, creating a multiplexing device which, as we will show, can differentially measure ionic current flows in different channels. optically actuated micromanipulation and micro-probing of biological samples are increasingly important methods in the today's laboratory. microbeads as probes are the most commonly used tools in this field, although the only manipulative motion they allow is translation. we present polymerized d microstructures which can also be used for optical micromanipulation with more degree of freedom than microbeads. the two-photon polymerization (tpp), based on focused fs laser beam into appropriate photopolymers is a powerful method to build structures of arbitrary complexity with submicrometer resolution. the presented tools have the advantage of being capable of twisting and rotational manipulative motion, and also that the position of biological manipulation and optical trapping is spatially separated. different manipulative interfaces, the positioning stability and surface activation of the manipulators will be discussed. the potential application of multiplexed quantum dot labeling, mqdl, in clinical detection, prognosis and monitoring therapeutic response has attracted high interests from bioengineers, pathologists and cancer biologists. mqdl is superior comparing with conventional organic dye staining in its narrow emission bandwidths, wide signal dynamic ranges, high detection sensitivity, and low noise to signal ratios. however, the majority of the mqdl application has been limited to identification of specific cell type or cancer subtype and the improvement in labeling methodology. in this study, we focused on simultaneous detection and analysis of proteins in the c-met activation pathway, i.e. rankl, vegf, nrpln- , p-c-met and mcl- , which are known to be associated with human prostate cancer progression and metastasis. two experimental systems were analyzed: ) fixed xenograft tissues from an established ltl castration resistance human prostate cancer or crpc model; and ) clinical prostate tissue specimens from localized cancer and bone metastasis. in the presentation we will report our experience in ) the mqdl protocol optimization for the sequential reactions of individual primary antibody, the biotinylated secondary antibody and streptavidin-coated qd conjugate with nuclear dapi staining; and ) the multiplexed image catching, image unmixing, and subsequent per cell base quantification. for future multi-specimen analyses and validation, we will introduce a high throughput vectra image analysis system. carbon nanotubes (cnts) are already quite popular among many scientific and technological disciplines . in recent years they have been targeted for biotechnological and medical applications. in this work we have investigated the nanostructural self assemblies of biological lipid molecules in presence of cnts. advantage of using highly aligned cnts for this purpose being the possibility of studying the interactions of lipid molecules on the macromolecular surface as well as in the confinement of aligned cnts. we have observed various lyotropic nanostructures that are found for corresponding lipids in the bulk under dry and hydrated conditions. nanostructural studies were mainly performed using small and wide angle x-ray scattering techniques. this work is crucial for designing the nano-micro-fluidic architectures and supported model membranes where both -functionalization of cnts and nanostructural assembling of lipids, could be employed simultaneously. alternative of the commonly used measuring tools in protein research and medical diagnostics. thazolidone derivatives are novel synthetic compounds possessing various biological activities. we selected three such compounds les- , , which passed national cancer institute in vitro tests. annexin v/pi and dapi staining, dna electrophoresis in agarose gel, and western-blot analysis using specific antibodies against cellular proteins involved in apoptosis were applied to study molecular mechanisms of tumor cell death induced by these compounds. it was found that molecular targets of thiazolidones in target cells strongly depend on structure of their side groups: les- containing isatine fragment, activated caspase- involved in receptor-mediated apoptosis, while les- possessing benzthiazol residue, induced mitochondrial apoptosis mediated by caspase- , and les- which has a unique chlorine atom in side chain, also led to mitochondrial apoptosis mediated by aif (apoptosis-inducing factor). to increase anticancer potential of these molecules, in silico study was performed and most active groups of les- and les- were combined into one molecule. in vitro studies showed that such hybrid molecule called les- possessed times higher anticancer potential (ic = lm) comparing with initial compounds. we report on the synthesis and characterization of vaterite microcontainers for controlled drug release. moreover, we present experiments on possible release strategies of encapsulated substances via recrystallization, ph controlled, or by desorption methods. vaterite spherical particles were fabricated with controllable average sizes from ± nm till ± hm. we considered two ways of functionalization of the containers: encapsulation of the substances during the vaterite synthesis or their adsorption onto the prepared particles. as model experiments, vaterite containers, encapsulating rhodamine g, were imaged by two-photon microscopy, showing dye release into the aqueous medium due to recrystallization to calcite within days. differently, in ethanol only small amounts of the encapsulated markers were diffusion released after one week. the release mechanisms can be further controlled by covering the microcontainers with additional polymer layers to increase diffusion and recrystallization time. a change of the ph from neutral to acid conditions leads to the destruction of the vaterite matrix followed by a quick release of the encapsulated materials. these flexible control mechanisms make this system an interesting candidate for pharmaceutical applications. magnetic nanoparticles (np) in combination with therapeutic molecules represent one of most promising methods for targeted drug delivery. one of major current limitations of magnetic drug targeting is to achieve efficient concentration of magnetic carrier-drug complexes at the targeted sites due to poor mobility of nanoparticles in tissue structures. interstitial delivery is hindered by microscopic extracellular matrix, which represents a major barrier for nanoparticles motilities. in order to achieve efficient magnetic drug targeting it is crucial to know particle mobility in a given in vivo environment as well as to apply magnetic field having appropriate field gradient which drags magnetic nps. we used gel magnetophoresis in order to measure motilities of different magnetic nps (co-ferrite, c-fe o ) in agarose gel. numerical modeling using fem method was used to determine appropriate settings of magnets, which generate sufficient magnetic field gradient. further, we used the numerical modeling to evaluate the magnetic force on the nps for different geometries. we obtained that one of crucial factors which determines final mobility in tissue is formation of larger aggregates of nanoparticles under physiological conditions and interaction of nanoparticles with surrounding matrix. defining the forces required to gate mechanosensitive channels in mammalian sensory neurons kate poole and gary lewin department of neuroscience, max delbrueck center for molecular medicine, robert-roessle str , , berlin-buch, germany our sense of touch and mechanical pain is based on mechano-electrical transduction (met) at the terminal endings of subsets of dorsal root ganglion (drg) neurons innervating the skin. to quantify the stimulus strengths required to gate mechanosensitive channels in these subsets of neurons, we developed an approach using microstructured surfaces. the drg neurons are grown on laminin-coated pdms pillar arrays, mechanical stimuli are applied by deflecting individual pili and the deflection is monitored using light microscopy. as the pili behave as light-guides, the center of each pilus can be determined from a fit of the intensity values, allowing detection of movements of a few nanometers. the response to such stimuli is monitored using whole-cell patch-clamp. pili deflections of nm can gate the rapidly adapting-current in mechanoreceptor cells, while deflections above nm are required for gating of slowly adapting-currents in nociceptors. smaller stimuli are required to generate currents via pili deflection ( nm) vs neurite indentation ( - nm), suggesting that gating occurs at the cell-substrate interface. we have also characterized the met currents present in n a cells which we show are modulated by the substrate to which the cells are attached. enhanced stimulation of toll-like receptor via immunostimulatory nanoparticles jan rother, anna pietuch, andreas janshoff georg-august university gö ttingen, institute of physical chemistry, tammanstr. , d- gö ttingen, germany e-mail: jrother@gwdg.de among the toll-like receptor family (tlrs), the tlr has been subject of intensive research because of its predominant localization in the lysosomes of immune cells and its ligand rendering it a potential candidate for immunotherapy of autoimmune diseases and cancer. additionally, a use as an adjuvant in vaccination is aimed using synthetic cpg-oligodeoxynucleotides (cpg-odn's). albeit, immunostimulatory cpg-odns already showed promising results in animal experiments and clinical trials, several groups found that tlr is also expressed by tumor cells. first experiments show that activation of tlr displayed by cancerous cells leads to a decreased apoptosis rate and proliferation posing unpredictable threat to tumor patients exposed to cpg-odns. therefore, detailed knowledge about the impact of cpg-odns on cancer cells is inevitable for a save use in pharmaceutics. herein, we describe a sophisticated way to address tlr in cancer cells using cpg-odn functionalized ''superparamagnetic'' mno-and c-fe o -nanoparticles (nps) to stimulate tlr in a cells. analysis of impedimetric measurements revealed a cytotoxic effect of the mno-nps. cells treated with immunostimulatory fe o -nps showed an increased micromotility as well as a higher long-term correleation of the impedance signal. biomedical diagnostics like high-sensitivity, single-molecule study, easy sample preparation. furthermore, sers allows to conduct non-invasive studies of conformations of the molecules without destruction of living cells, i.e. in vivo [ ] . this work presents a sers study of cytosolic hemoglobin (hb c ) using silver nanoparticles (agnps). the hb c was isolated from cytoplasm of red blood cells taken from rat erythrocytes and diluted. agnps were prepared by developing leopold and lendl method [ ] . three types of colloids were prepared at various temperatures ( , and °c). the resulted agnps were characterized by uv-vis-, ftirspectroscopy, dls and tem. reduction of ag ions leads to the formation of predominantly spherical agnps but also silver nanorods, faceted and aggregated agnps in small quantities with a surface plasmon resonance band in the range of - nm. for agnps synthesized at °c, for example, a bimodal size distribution was observed (about and nm medium sizes, respectively). sers measurements were optimized for each type of agnps. it was demonstrated that agnps gave strong raman enhancement from hb c and types of sers spectra differ from each other. nano-zno is characterized by unique properties, low toxicity and high biocompatibility instead of a lot of others nanomaterials. for this fact nanoparticles of zno have great potential for applications in biosystems, for example biolabeling, biosensoring, delivery systems and others, which can be used in genetics, pathology, criminology, safety of food and many other industries. for these bioapplications are necessary surface modifications, which can made to the nanostructures to better suit their integration with biological systems, leading to such interesting properties as enhanced aqueous solubility, bio-recognition or applicability for biological systems. for synthesis of zno nanoparticles in aqueous solution we used -mercapto-undecanoic acid (mua) as stabilizing agent. the coating of nanoparticles with mua could allow their solubility in the water and the binding through carboxyl groups present in its structure. we defined the optimal ph for mua modificated nano-zno solubility and their ability interaction with positive charges. we studied the optical properties of pure and surface modificated nanoparticles and their conjugates with cytochrome c and also the effect of ph on the interaction between nano-mua and horse cytochrome c. the permeation of water soluble molecules across cell membranes is controlled by channel forming proteins and particularly the channel surface determines the selectivity. an adequate method to study properties of these channels is electrophysiology and in particular analyzing the ion current fluctuation in the presence of permeating solutes provides information on possible interactions with the channel surface. as the binding of antibiotic molecules in the channels of interest is significantly weaker than that of preferentially diffusing nutrients in substrate-specific pores, the resolution of conductance measurements has to be significantly increased to be able to resolve the events in all cases. due to the limited time resolution, fast permeation events are not visible. here we demonstrate that miniaturization of the lipid bilayer; varying the temperature or changing the solvent may enhance the resolution. although electrophysiology is considered as a single molecule technique, it does not provide atomic resolution. molecular details of solute permeation can be revealed by combining electrophysiology and all atom computer modeling. [ novel functionalized nanocomposites (nc) were designed and synthesized on the basis of polymeric surface-active oligoelectrolytes. the developed technology permits controlling: ) quality and quantity of structural blocks of nc, and size unimodality; ) branching at specific sites in polymer chain of nc; ) providing nc with reactive chemical groups; ) covalent conjugation of specific bio-targeting molecules. provided bioactive elements were: a) specific anticancer drugs, antibiotics, alkaloids; b) dna and sirna; c) immunoglobulins and lectins; d) lipids and amino acids; e) polyethylene glycol. fluorescent, luminescent, super-paramagnetic, or x-ray detectable compounds were also incorporated in nc to make them detectable and measurable. biocompatible nc possessing low toxicity towards mammalian cells in vitro and in vivo (mice) were created. they were effective in delivery of: ) drugs (doxorubicine and antibiotics) for chemotherapy in vitro and in vivo; ) dna for transfection of mammalian, yeast and bacterial cells; ) protein antigens for animal immunization and specific lectins for targeting apoptotic cells. these and other approaches in application of developed nc and nanobiotechnologies are considered. this work was supported by stcu grants # , # , # . in the anti-cancer drug delivery domain, nanotechnologies are a promising tool, providing a good tissue distribution and a low toxicity. drug delivery vehicles relying on solid nanoparticles have been proposed, among which diamond nanoparticle (size\ nm) is a very promising candidate [ ] . we have investigated the delivery of sirna by nanodiamonds (nd) into cells in culture, in the context of the treatment of a rare child bone cancer (ewing sarcoma), by such a gene therapy. sirna was bound to nds after nds coating by cationic polymers, so that the interaction is strong enough to pass the cell membrane without loss of the drug and does not prevent its subsequent release. the cellular studies showed a specific inhibition of the gene expression at the mrna and protein level by the nd vectorized sirna. we also uses the fluorescence of color center created in the nanodiamonds [ ] to monitor the release of fluorescently-labeled sirna in the intracellular medium. this technique brings a quantitative insight in the efficiency of sirna to stop cell proliferation. considering the success of the cell model we recently started the drug delivery in tumor xenografted on nude mice. silica nanoparticles are stable aqueous suspension of condensed siloxane nanocomposites, having an average diameter between and nm. particles containing organic functional groups on their surface are called organically modified silica nanoparticles (ormosil). due to the various chemical and physical properties of the surface groups, ormosil nanoparticles may have an enormous variety of biological applications, such as in vivo bioimaging, non-viral gene delivery or targeted drug delivery. our aim was to synthesize both void and fluorescent dye doped amino functionalized ormosil nanoparticles through the microemulsion method and use them for gene delivery. the obtained nanoparticles have been characterized by transmission electron microscopy and dynamic light scattering. furthermore, the nanoparticles have been investigated to exploit their transfection efficiency and the possible toxicity caused by surfactants used in the synthesis. the transfection efficiency was tested on various cell cultures. our further aim is the in vivo transfection of salivary glands using ormosil nanoparticles. our work has shown that the nanomedicine approach, with nanoparticles acting as a dna-delivery tool is a promising direction for targeted gene therapy. in vivo amperoetric cells for detection of fast diffusing, physiologically important small molecules lívia nagy, bernadett bareith, tü nde angyal, erika pinté r, gé za nagy university of pé cs, pé cs, hungary h s is a naturally occurring gas that is toxic in high concentration. it exists also in different tissues of living animals sometimes in concentrations as high as lm. it is generally accepted, that h s has important roles in modulating different, physiologically important biochemical processes similarly to other, fast diffusing molecules like no, co and h o . for investigation of the physiological effects of these species their local concentration in the studied biological media is important to know. this means methods needed for measuring the instantaneous concentration with high spatial resolution in living tissues without major invasion. electrometric micro, and ultramicro sensors are often gain application in experimental life sciences for measurement of local ion concentration or following neurotransmitter species in vivo measurements. in our work efforts are being carried out to improve the applicability of selective electrometric sensors in life science experiments. as a result of these work an improved h s measuring cell and improved electrode and method was developed for measurement of electroactive small molecules like no or h o . in the poster to be presented the structure, the working principles and the performances of the different sensors mentioned will be described. bacteriorhodopsin (br) is the only protein in the purple membrane of the halophilic organism halobacterium salinarium. it is a light-driven proton pump converting light into a transmembrane proton gradient through isomerization of the covently bound retinal chromophore. its stability, as well as its photoactivity in dried films, has made br an attractive material for biomolecular devices. such studies, however, have used br within the membrane, on relatively large surfaces. here, conducting-probe atomic force microscopy (c-afm) analysis was performed after isolating the protein from its native membrane environment while keeping its basic trimeric structure, and demonstrated that the molecular conductance of br can be reversibly photoswitched with predictable wavelength sensitivity. intimate and robust coupling to gold electrodes was achieved by using a strategically engineered cysteine mutant located on the intracellular side of the protein which, combined with a % delipidation, generated protein trimers homogenously orientated on the surface. c-afm proximal probe analysis showed a reproducible fold drop of br mean resistance over * cycles of interspersed illuminations at the same gold-br-gold junction when k[ nm, while no shift was observed with other wavelengths. capture of circulating tumor cells with a highly efficient nanostructured silicon substrates with integrated chaotic micromixers shutao wang ). this core technology shows significantly improved sensitivity in detecting rare ctcs from whole blood, thus provides an alternative for monitoring cancer progression. by assembling a capture-agent-coated nanostructured substrate with a microfluidic chaotic mixer, this integrated microchip can be applied to isolate ctcs from whole blood with superb efficiency. ultimately, the application of this approach will open up opportunities for early detection of cancer metastasis and for isolation of rare populations of cells that cannot feasibly be done using existing technologies. this technology helped to find a needle in a haystack and will open up the opportunity for single cell genomic and epigenetic sequencing and gene expression profiling. results from further development of this technology will assist the physicians in follow-up patients and testing vigorously the concept of personalized oncology with individualized therapy. this novel technology has recently been reviewed and highlighted by nature medicine the growing crisis in organ transplantation and the aging population have driven a search for new and alternative therapies by using advanced bioengineering methods. the formation of organized and functional tissues is a very complex task: the cellular environment requires suitable physiological conditions that, presently, can be achieved and maintained by using properly-designed bioreactors reproducing all specific functions and bioactive factors that assure viability/regeneration of cells cultured in an appropriate scaffold. the creation of biomimetic environment requires the use of biomaterials such as membranes with specific physico-chemical, morphological and transport properties on the basis of the targeted tissue or organ. tailor-made membranes (organic, functionalized with specific biomolecules, in hollow-fiber configuration), designed and operated according to well-defined engineering criteria are able to sustain specific biotransformations, to provide adequate transport of oxygen, nutrients and catabolites throughout the cellular compartment, and to supply appropriate biomechanical stimuli of the developing tissue. in this talk the author will show the development of membrane engineered constructs focusing on liver and neuronal systems. the role of membrane surface and transport properties in providing instructive signals to the cells for the guiding of proliferation and differentiation will be discussed. membrane bioreactors, which through the fluid dynamics modulation may simulate the in vivo complex physiological environment ensuring an adequate mass transfer of nutrients and metabolites and the molecular and mechanical regulatory signals, will be presented. here we present a novel but simple system for cell-based assays enabling simultaneous testing of multiple samples on a same tissue without cross-contamination between neighbouring assays, as well as sequenced or repeated assays at the same tissue location. the principle of this method lies in the spatially-controlled diffusion of test compounds through a porous matrix to the target cells. a simple microfabrication technology was used to define areas where diffusion processes are allowed or inhibited. we performed proof-of-principle experiments on madin-darby canine kidney (mdck) epithelial cells using hoechst nuclear staining and calcein-am cell viability assay. fluorescent staining superimposed properly on membrane pattern with a dose-dependent response, indicating that both compounds specifically and selectively diffused to the target cells. mdck cells similarly treated with cytochalasin b showed their actin network rapidly altered, thus demonstrating the suitability of this system for drug screening applications. such a well-less cell-based screening system enabling multiple compounds testing on a same tissue and requiring very small volumes of test samples appears interesting for studying potential combined effects of different biochemicals applied separately or sequentially. it is generally believed that all-optical data processing is the most promising direction to achieve serious improvements both in capacity and speed of internet data traffic. one of the bottlenecks of the state-of-the-art photonic integration technology is to find the proper nonlinear optical (nlo) materials that are supposed to serve as cladding media in waveguidebased integrated optical circuits performing light-controlled active functions. recently, the unique chromoprotein bacteriorhodopsin (br) has been proposed to be used as an active, programmable nlo material in all-optical integrated circuits. in integrated optical applications of br, its light-induced refractive index change is utilized. in this paper we exploit the refractive index changes of a dried br film accompanying the ultrafast transitions to intermediates i and k, which allows even sub-ps switching, leading beyond tbit/s communication rate. in the experiments direct pulses of a femtosecond laser system at nm were used along with synchronized ultrafast laser pulses at nm. we believe that the results may be the basis for the future realization of a protein-based integrated optical device, and represent the first steps to a conceptual paradigm change in optical communication technologies. last years, such autoantibodies attract an increasing attention of researchers as potential cancer biomarkers. since the sera of cancer patients typically contain a unique set of antibodies that reflect the tumor-associated antigens expressed in a particular malignant tissue, diagnosing and predicting the outcome of disease such as breast cancer based on serum autoantibody profiling is an attractive concept. to create a representative panel of antigens for detecting of breast cancer autoantibody profile we selected breast cancer associated antigens. these antigens were identified by screening of tumor cdna libraries with autologous sera using serex (serological investigation of recombinantly expressed clones) approach. all antigens were cloned, expressed, purified in bacteria and tested with sera of breast cancer patients and healthy donors in large-scale allogenic screening using elisa. the utility of selected tumor associated antigens for detecting of autoantibody profile in different types of breast cancer was evaluated. (controlled by architectural software) is carried out according to a design template, consistent with the geometry and composition of the desired organ module. structure formation occurs by the post-printing fusion of the discrete bio-ink units. when the bio-ink units contain more than one cell type, fusion is accompanied by sorting of the cells into the physiologically relevant pattern. thus structure formation takes place through self-assembly processes akin to those utilized in early embryonic morphogenesis. we demonstrate the technology by detailing the construction of vascular and nerve grafts. spherical and cylindrical bio-ink units have been employed to build fully biological linear and branching vascular tubular conduits and multiluminal nerve grafts. upon perfusion in a bioreactor the constructs achieved desirable biomechanical and biochemical properties that allowed implantation into animal models. our results show that the printing of conveniently prepared cellular units is feasible and may represent a promising tissue and organ engineering technology. femtosecond lasers have become important tools for noncontact microprocessing of biological specimens. due to the short pulse length and intensity-dependent nature of the multiphoton ionization process, fs-laser pulses affect only a small volume of a treated cell, providing a high degree of spatial localization. we employed fs-laser to address topical bioengineering and biomedical problems such as cell fusion and embryo biopsy respectively. a tightly focused laser beam (cr:f seed oscillator and a regenerative amplifier, nm, fs, hz) was used for a fusion of blastomeres of two-cell mouse embryos and for a polar body (pb) biopsy. in order to fuse blastomeres the contact border of cells was perforated by a single laser pulse. the fusion process usually completed within * min. in order to perform a noncontact laser based pb biopsy we initially drilled an opening in the zona pellucida with a set of laser pulses, and then extracted the pb out of zygote by means of optical tweezer (cw laser, nm). the energy of laser pulses was thoroughly optimized to prevent cell damage and increase the fusion and biopsy rates. the proposed techniques demonstrate high efficiency and selectivity and show a great potential for using fs lasers as a microsurgical tool. new insights into mechanisms of electric field mediated gene delivery maš a kanduš er and mojca pavlin university of ljubljana, faculty of electrical engineering, si- ljubljana, slovenia gene electrotransfer is widely used for transfer of genetic material in biological cells by local application of electric pulses and is currently the most promising non-viral delivery method for gene therapy for a series of diseases as well as for dna vaccination. current description of the process defines several steps: electropermeabilization, dna-membrane interaction, translocation, trafficking to nucleus and into nucleus. but the mechanisms of electrotransfer are still not fully understood. we present results of the systematic in vitro analysis using pegfp of all steps involved in electrotransfection from electropermeabilization, analysis of different pulsing protocols, theoretical analysis of plasmid mobility to visualization of the processes of dna-membrane interaction. we demonstrate that in order to translate in vitro results to tissue level sub-optimal plasmid concentrations have to be used. furthermore, sofar the method of dna entry into cytoplasm was only speculated. our results suggest that it is crucial that first, membrane is electropermeabilized, then sufficient electrophoretic force is crucial for insertion of dna into destabilized lipid bilayer followed by dna translocation into cytoplasm via a slow process. efficiency of electrotransfer depends also on the stage of of cell culture -cells in dividing phase are easer to electrotransfect. gentamicin interaction with b f cell membrane studied by dielectrophoresis dielectrophoresis (dep) is the translational motion of polarizable particles due to an electric field gradient. positive-dep and negative-dep correspond to particle movement forward or backward the region of high field intensity, respectively. our study reveals some of the cell membrane modifications induced by gentamicin (gt), as they are reflected in the crossover frequency f co of b f murine cells incubated with gt for different concentrations and durations. f co is the ac frequency when cells turn from positive-dep to negative-dep. gentamicin is a positively charged aminoglycosidic antibiotic, with concentration-dependent killing action; it is widely used because of its low cost and reliable bactericidal activity. gt drawbacks consist in high toxicity for renal and hearing cells; the molecular mechanisms of this toxicity are still unclear. for low external medium conductivities (& . s/m), f co of control and gt-cells was found to range from to khz. f co shifts to higher frequencies with the increase of gt concentration and incubation time. cells dielectrophoretic behavior is discussed using the cell singleshell based model. extracellular matrix (ecm) is a major obstacle for succesful delivery of genes. chitosan is a versatile and biocompatible polysaccharide derived from chitin and is a promising gene carrier. chitosan-dna interactions, and hence dna polyplexation and release can be controlled through chitosan de-acetylation degree, molecular weight and functionalization of chitosan cationic groups. grafting of poly(ethylene glycol) peg to gene delivery vectors increases circulation time of gene delivery systems in blood vessels and reduces polyplexes charge. diffusion and unpacking of pegylated and non-pegylated chitosan-dna polyplexes through articial ecms based on collagen and collagen-hyaluronic acid (ha) gels were compared using fluorescence correlation microscopy, confocal microscopy and colocalization analysis. non-pegylated polyplexes were immobilized in the gels whereas pegylated polyplexes were diffusing. the smaller charge of pegylated polyplexes seems to decrease interactions between polyplexes and ecm components. furthermore, ha might also screen collagen fibers-pegylated polyplexes interactions. pegylated polyplexes also showed a higher degree of unpacking in gels, probably due to a looser compaction of dna by pegylated chitosan compared to non-pegylated chitosan. fabrication of vesicles, a close membrane made of an amphiphile bilayer, has great potentiality for encapsulation and controlled release in chemical, food or biomedical industries but also from a more fundamental point of view for the design of biomimetic objects. methods based on lipid film hydratation , inverse emulsion techniques and more recently microfluidic techniques such as double emulsion or jetting method are limited either by a low yield, a low reproducibility, a poor control on the size, or by the presence of remaining solvent or defects. we propose a fast and robust method easy to implement: continuous droplet interface crossing encapsulation (cdice), that allows the production of defect-free vesicles at high-yield with a control in size and content. the vesicles have controlled bilayer composition with a polydispersity in size lower than %. we have shown that solutions as diverse as actin, cells, micrometric colloids, protein and high ionic strength solutions can easily be encapsulated using this process. by adjusting the parameters of our set-up, we are able to produce vesicles in the range - lm in diameter, stable for weeks. we believe this method open new perspectives for the design of biomimetic systems and even artificial tissues. under appropriate conditions. the extremely variable d domain of flagellin subunits, comprising residues - , protrudes at the outer surface of flagellar filaments. the d domain has no significant role in the construction of the filament structure. thus, replacement of d may offer a promising approach for insertion of heterologous proteins or domains without disturbing the self-assembly of flagellin subunits. our work aims at the construction of flagellinbased fusion proteins which preserve the polymerization ability of flagellin and maintain the functional properties of the fusion partner as well. in this work a fusion construct of flagellin and the superfolder mutant of green fluorescent protein (gfp) was created. the obtained gfp variant was highly fluorescent and capable of forming filamentous assemblies. our results imply that other proteins (enzymes, binding domains etc.) can also be endowed by polymerization ability in a similar way. this approach opens up the way for construction of multifunctional filamentous nanostructures. generation polyamidoamine (pamam) dendrimer has been shown to be highly efficient nonviral carriers in gene delivery. however, their toxicity limits their applications. in this study, to improve their characteristics as gene delivery carriers, g pamam dendrimer was modified with anti-tag nanobody through hetrobifunctional peg, then complexed with t-bid coding pdna, yielding pamam-peg-anti-tag nanobody/pdna nanoparticles (nps). nuclear magnetic resonance (nmr) spectroscopy, zeta sizing and gel retardation assay results provided evidence that the nanovector was successfully constructed. the transfection efficiency of vector/pdna complexes were evaluated in vitro. real time pcr results also demonstrated that anti-tag nanobody modified nps are more efficient in t-bid killer gene expressing in colon cancer cell line than the unmodified nps. in conclusion, pamam-peg-anti-tag nanobody showed great potential to be applied in designing tumour-targeting gene delivery system. dept of chemistry, faculty of science, national university of singapore, singapore, dept of biochemistry, yong loo lin school of medicine, national university of singapore, singapore, division of bioengineering, faculty of engineering, national university of singapore, singapore macromolecular crowding (mmc) is a biophysical tool which has been used extensively to enhance chemical reactions and biological processes by means of the excluded volume effect (eve). the in vivo stem cell microenvironment contains macromolecules which are crucial for stem cell selfrenewal and cell fate determination. in order to mimic this physiological microenvironment, crowders are included in cell culture medium. we have observed that the ex vivo differentiation of human mesenchymal stem cells (hmscs) into the adipogenic lineage is significantly amplified when a crowder mixture comprising ficoll and ficoll is added to the culture medium. stem cell differentiation is modulated by soluble chemical substances as well as interactions between cells and the extracellular matrix (ecm), and both these external influences may be affected by mmc. measurements we have performed by fluorescence correlation spectroscopy (fcs) show that ficoll additives cause anomalous subdiffusion within a crowder concentration range of to mg/ml. the diffusion of fluorophorelabelled molecules in artificial lipid bilayers and membranes of living cells is not changed by crowders, suggesting that these crowders do not directly alter membrane properties and cell surface signalling. however, we have data to suggest that crowders increase actin polymerization reaction rates in vitro. we have also observed that crowders are taken up by stem cells and that they localize to specific compartments. based upon our observations, we hypothesize that crowders can influence stem cell differentiation by influencing molecular kinetics. lignocellulose-based composites are becoming extremely important and perspective sustainable and renewable natural materials. fibre modification enhancing their existing properties can be obtained to broaden the application areas. in response to shortcomings of traditional chemical and physical methods, enzymes and chemo-enzymatic methods have emerged as eco-friendly catalysts working under mild conditions and enable tailoring of the material surface properties by substrate specificity and regional selectivity. recently, binding of different functional molecules to lignin-rich fibres by using an oxidative enzyme (e.g. laccase) has been reported leading to their functionalisation through free radical reactions. by the application of electron paramagnetic resonance spectroscopy (epr) laccase action was inspected. consumption of substrates was investigated and their polymerization traced. stable radical intermediates were detected with epr when substrate molecules were in contact with active enzymes. secondly, oxidation of mediators like nitroxides was determined via epr spectroscopy of stable water-soluble nitroxide radicals. finally, the generation of short-lived radicals as well as their reduction was measured via epr spin trapping using dmpo as sensitive water soluble spin trap. mammalian ovary hormone stimulation (ohs) is known to be an inalienable stage of reproductive biotechnology as well as human infertility treatment. the basic aim of the ohs is to receive a stock of valuable oocytes and early embryos for subsequent utilization in the reproductive technology, experimental work et al. however, it is known that ohs itself affects the character of ovulation and oocyte quality, which in its turn affects the development of embryos and even has distant consequences. the wideness of cell parameters and appropriate methods for investigation of gamete/embryo quality are very important. the aim of this study is determination of specific electric conductivity of mouse oocytes and early embryos which have been received after ohs in comparison with the ones that have been received in natural animal sex cycle. using techniques of electroporation the dependence of specific electric conductivity of mouse oocytes, zygotes, -cell and -cell embryos on the external electric field intensity has been studied. it is shown that the whole pool of oocytes that were obtained in the result of ohs consists of two groups of oocytes that don't differ from each other morphologically, but differ by their electric parameters and resistance to electric breakdown. at the zygote stage, dividing of embryos into two groups is preserved, but is less expressed. at the stage of -cell and -cell dividing of embryos into two groups on their electric conductivity disappeared but certain scattering of the parameters due to individual embryo peculiarities is observed. the obtained data show that ohs may lead to latent changes of oocyte state that in their turn affect embryo quality. many microbes synthesize and accumulate granules of polyhydroxyalkanoates (pha, biodegradable storage materials alternative to traditional plastics), which help them survive under stresses. in particular, the plant-growth-promoting rhizobacterium azospirillum brasilense, that is under investigation worldwide owing to its agricultural and biotechnological significance, can produce poly- hydroxybutyrate (phb) [ ] . in our work, phb synthesis in a. brasilense cells was studied under various stresses using diffuse reflectance ftir spectroscopy. phb in cells was determined from the band intensity ratio of the polyester m(c=o) at * cm - to that of cell proteins (amide ii band at * cm - ), showing a. brasilense to be able to produce phb up to over % of cells' dry weight. stresses induced phb accumulation, enhancing ir absorption in phb specific regions. analysis of a few structure-sensitive phb vibration bands revealed changes in the degree of intracellular phb crystallinity (related to its enzymatic digestion rate) at different stages of bacterial growth, reflecting a novel trait of the bacterial adaptability to an enhancing stress, which is of great importance to agricultural biotechnology. the aim of this work is to furnish enzymes with polymerization ability by creating fusion constructs with the polymerizable protein, flagellin, the main component of bacterial flagellar filaments. the d domain of flagellin, exposed on the surface of flagellar filaments, is formed by the hypervariable central portion of the polypeptide chain. d is not essential for filament formation. the concept in this project is to replace the d domain with suitable monomeric enzymes without adversely affecting polymerization ability, and to assemble these chimeric flagellins into tubular nanostructures. to test the feasibility of this approach, xylanase a (xyna) from b. subtilis was chosen as a model enzyme for insertion. with the help of genetic engineering, a fusion construct was created in which the d domain was replaced by xyna. the flic(xyna) chimera exhibited catalytic activity as well as polymerization ability. these results demonstrate that polymerization ability can be introduced into various proteins, and building blocks for rationally designed assembly of filamentous nanostructures can be created ( table ) . the support of the hungarian national office for research and technology and the hungarian scientific research fund (otka) (grants ck , nk , nanoflag) is acknowledged. cluster phases of membrane proteins an alternative scenario for the formation of specialized protein nano-domains (cluster phases) in biomembranes dna fragmentation induced in human fibroblasts by accelerated fe ions of differing energies swift heavy ion irradiation of srtio under grazing incidence'' proc. natl. acad. sci. usa forespore engulfment mediated by a ratchet-like mechanism a channel connecting the mother cell and forespore during bacterial endospore formation a feeding tube model for activation of a cell-specific transcription factor during sporulation in bacillus subtilis the scanning ion-conductance microscope imaging proteins in membranes of living cells by high-resolution scanning ion conductance microscopy nanoscale live-cell imaging using hopping probe ion conductance microscopy beta -adrenergic receptor redistribution in heart failure changes camp compartmentation simultaneous noncontact topography and electrochemical imaging by secm/sicm featuring ion current feedback regulation timasheff in protein-solvent interactions the roles of water in foods on motor and electrical oscillations in urinary tract: computer evaluation daniele martin , , viktor foltin , , erich gornik , rumen stainov , , tanya zlateva nü rnberg. icsd e.v. postfach (pob) , d- mü nchen method: parameters: motor patterns (guinea-pig) -frequency/f, amplitudes/a (% init. length, isot. & intracell. rec.) of spontaneous phasic/spc & tonic/stc contractions, also electrical spikes/s, bursts/b, burst plateaus/bp (neu et al. biophys.j. / a/jan stretch ( - mn), k-/ca-influence induced specific changes in motor/electrical parameters. special computer programme reflects exactly biophysical parameters. conclusion: acc. to earlier/recent results mechano-sensitive ca ?? -activated k ? -channels participate in electrical oscillations of detrusor/ureteral myocytes. further experiments/evaluations incl effect of hydrophobic mismatch on the light-induced structural changes in bacterial reaction centers s. s. deshmukh, h. akhavein, and lá szló ká lmá n department of physics mechanism of proton transfer in nitric oxide reductase: computational study andrei pisliakov riken advanced science institute, wako-shi proteins , . acknowledgments this work was supported by project grant ptdc/qui/ / (cas) and doctoral grants sfrh th anniversary conference aicr this work was supported by the italian association for cancer research (airc), the istituto toscano tumori and the associazione noi per voi differential hydration, void volume: which factor provides the main contribution to dv u ? inserm umr fr; roumestand@cbs.cnrs.fr introduction: globalization needs new organizational models also for biophysics. reports on necessity of int. institutes for biophysics (iib) c/o int. universities (proposed by british nobel laureate b.russell) are given conception: proposals for ebsa-discussion: . enlargement of executive committee by a. honorary & presidents (permanent - : moral support & - fixed term), b. interdisciplinary commission: scientists from biology, medicine, physics, etc. (feps/iups, iuphar, iupab, etc). . implication of interdisciplinary topics to esba/iupab congressprogrammes, . also for biophysical journals. . organization of common interdisciplinary sessions not only to biophysical, but also to other congresses. . co-operation between esba/iupab with int. interdisciplinary organisations (waas, icsd/ias, eur. academies) for creation of iib by network of national ones: successive common personnel, possibility for whole life work, etc. conclusion: realization of proposals .- . could increase scientific/political authority of ebsa/iupab, leading to model for renewal of scientific organizations collective migration of neural crest cells: a balance between repulsion and attraction roberto mayor university college london goodilin , , olga v single-molecule cut-and-paste surface assembly (smcp) has been also used to build up a biotin scaffold that streptavidin utilizing specific molecular interactions, for example between dna-binding proteins and dna or antibodies and antigens, this technique is capable of providing a scaffold for the controlled self-assembly of functional complexes. furthermore, this allows for the introduction of smcp into protein science. we aim to employ dna-binding zinc-finger variants and gfp-binding nanobodies as shuttle-tags fused to the proteins of interest. thus a fully expressible system that can be used for the step-wise assembly of individual building blocks to form single-molecule cut-and-paste surface assembly optically monitoring the mechanical assembly of single molecules nanoparticle self-assembly on a dna-scaffold written by single-molecule cut-and-paste torsional motion analysis of group ii chaperonin using diffracted x-ray tracking nanomechanical manipulation of mason-pfizer monkey retroviral rna fragment with optical tweezers melinda simon , zsolt má rtonfalvi , pasquale bianco , beá ta vé rtessy , mikló s kellermayer micro-viscosimeter generated and manipulated by light andrá s buzá s , lá szló oroszi , ló rá nd kelemen , pá l ormos temesvá ri krt proc. natl. acad. sci. usa neural signal recordings with a novel multisite silicon probe gergely má rton , anita pongrá cz , lá szló grand , , É va vá zsonyi pé ter pá zmá ny catholic university, faculty of information technology, h- , /a prá ter st multiscale pattern fabrication for life-science applications francesco valle , beatrice chelli , michele bianchi , eva bystrenova , marianna barbalinardo , arian shehu , tobias cramer , mauro murgia , giulia foschi miroslava kuricova , jana tulinska , aurelia liskova , eva neubauerova , maria dusinska , , ladislava wsolova acceleration neuronal precursors differentiation induced by substrate nanotopography gianluca grenci , jelena ban , elisabetta ruaro , massimo tormen , marco lazzarino , and vincent torre light-induced structural changes are reported near the primary electron donor of bacterial reaction centers (brc) dispersed in detergent micelles and in liposomes from lipids with different fatty acid chain lengths. in this study we present evidence for the correlation between the light-induced increase of the local dielectric constant, determined by the analysis of the electrochromic absorption changes, and the lifetime of the charge-separated state at physiologically relevant temperatures. the increase of the local dielectric constant induced a significant decrease of the oxidation potential of the primary electron donor and a slow proton release, which appears to be the rate limiting step in the overall process. systematic selection of the head group charges of detergents and lipids, as well as the thickness of the fatty acid chains of the liposome forming lipids can increase the lifetime of the charge-separated state by up to orders of magnitude. such extensions of the lifetime of the charge-separated state were reported earlier only at cryogenic temperatures and can provide new opportunities to utilize the brc in energy storage. ontogenesis of photosynthetic bacteria tracked by absorption and fluorescence kinetics m. kis, e. asztalos, p. maró ti department of medical physics and informatics, university of szeged, hungarythe development of photosynthetic membrane of rhodobacter sphaeroides was studied by absorption spectroscopy and fast induction of bacteriochlorophyll fluorescence in different phases of the growth, under various growing conditions (oxygen content, light intensity etc.) and in synchronous cell population. the results are: ) the newly synthesized components of the membranes were imbedded immediately into the proteinous scaffold independently on the age of the cell (no ,,transient'' membranes were observed). ) under aerobic conditions, the pigments were bleached and under anaerobic conditions the pigment systems showed greening. the relative variable fluorescence (f v /f max ) had small age-dependent (but not cellcycle-related) changes. the fluorescence induction kinetics was sensitive marker of the aerobiosis: the f v /f max ratio dropped from . to . and the photochemical rate constant from Á s - to Á s - with an apparent halftime of about - hours after change from anaerobic to aerobic atmosphere. ) the electrogenic signal (absorption change at nm) reflected the energetization of the membrane which showed cell-cycle dependent changes. that included periodic production and arrangement of protein-lipid components of the membrane synchronized to the cell division. interfacial water in b-casein molecular surfaces: wide-line nmr, relaxation and dsc characterization t. verebé lyi , m. bokor , p. kamasa , p. tompa , k. tompa research institute for solid state physics and optics, hungarian academy of sciences, pob. , budapest,hungary, institute of enzymology, biological research center, hungarian academy of sciences, pob. , budapest, hungarywide-line proton nmr fid, echoes, spin-lattice and spin-spin relaxation times were measured at . mhz frequency in the - °c to ? °c temperature range, in lyophilized bcasein and aqueous and buffered solutions, and dsc method were also applied. the motivation for the selection of b-casein is the uncertainty of structural order/disorder. naturally, the nmr and thermal characteristics were also evaluated. the melting of hydration water could be detected well below °c and the quantity of mobile water molecules (hydration) was measured. the hydration vs. melting temperature curve has informed us on the bonding character between the protein surfaces and water molecules. the generally used local field fluctuation model and the bpp theory were applied in the interpretation, and the limits of the models were concluded. the torsional properties of dna play an important role in cellular processes such as transcription, replication, and repair. to access these properties, a number of single-molecule techniques such as magnetic tweezers have been developed to apply torque to dna and coil it. i will briefly refer to investigations of dna-protein interactions using these techniques, and describe what has been learnt. i will then focus on the development of novel magnetic techniques that go beyond standard magnetic tweezers, such as the magnetic torque tweezers and the freely-orbiting magnetic tweezers . these approaches allow one to quantify conjugate variables such as twist and torque. for example, the magnetic torque tweezers rely on high-resolution tracking of the position and rotation angle of magnetic particles in a low stiffness angular clamp. we demonstrate the experimental implementation of this technique and the resolution of the angular tracking. subsequently, we employ this technique to measure the torsional stiffness c of both dsdna molecules and reca heteroduplex filaments. lastly, i will describe novel applications of the optical torque wrench , . the optical torque wrench is a laser trapping technique developed at cornell capable of applying and directly measuring torque on microscopic birefringent particles via spin momentum transfer. we have focused on the angular dynamics of the trapped birefringent particle , demonstrating its excitability in the vicinity of a critical point. this links the optical torque wrench to non-linear dynamical systems such as neuronal and cardiovascular tissues, non-linear optics and chemical reactions, which all display an excitable binary ('all-or-none') response to input perturbations. based on this dynamical feature, we devise a conceptually novel sensing technique capable of detecting single perturbation events with high signal-to-noise ratio and continuously adjustable sensitivity.for the first time we report a comparative approach based on surface enhanced raman spectroscopy (sers) and raman spectroscopy to study different types of haemoglobin molecules in living erythrocytes. in erythrocytes there are two fractions of haemoglobin: cytosolic (hb c ) and membranebound (hb m ). the concentration of hb m is less then , % and therefore it is impossible to study hb m with traditional optical techniques. modifications of cellular membrane can affect conformation of hb m . therefore, it can be used as a sensitive marker of pathologies. firstly, we investigated enhancement of sers signal of hb m depending on ag nanoparticles' size. we found that the intensity of sers spectra of hb m and enhancement factor increase with the decrease in ag nanoparticles' size. secondly, we investigated the dependence of haemoporphyrin conformation in both hb c and hb m ion ph values. we observed different sensitivity of hb c and hb m to the ph and found that conformational movements of haemoporphyrin (vibrations of pyrrol rings and side radicals) in hb m are sterically hampered comparably with hb c . our observation is an evidence of a benefit of application of surface enhanced raman spectroscopy to investigate properties of the hb m in erythrocytes and provide new information about conformational changes and functional properties of hb m . rna nanotechnology is an emerging field with high potential for nanomedicine applications. however, the prediction of rna three-dimensional nanostructure assembly is still a challenging task that requires a thorough understanding the rules that govern molecular folding on a rough energy landscape. in this work, we present a comprehensive analysis of the free energy landscape of the human mitochondrial trna lys , which possesses two different folded states in addition to the unfolded one. we have quantitatively analyzed the degree of rna tertiary structure stabilization, firstly, for different types of cations, and, secondly, for several naturally-occurring nucleotide modifications in the structural core of the trna lys . , thus, notable variations in the rna binding specificity was observed for the divalent ions of mg ? , ca ? and mn ? , that can be attributed to their sizes and coordination properties to specific ligands. furthermore, we observed that the presence of m g modification together with the principal stabilizing m a modification facilitates the rna folding into the biologically functional cloverleaf shape to a larger extent than the sum of individual contributions of these modifications. in order to elucidate the mechanism of the recognition, we used diffracted x-ray tracking method (dxt) that monitors real-time movements of individual proteins in solution at the single-molecule level. we found that peptides move distinctly from i-a k , and the rotational motions of peptides correlate with the type b t cell activation. in the case of diabetogenic i-a g , immediately after peptide exchange, all the peptides moves magnificently but the motion ceased in a week, then new ordered motion appears; the rotational motion of peptides correlate to t cell activation, which is analogous to the peptide in i-a k . the rotational motion of peptides may create transient conformation of peptide/mhc that recognized by a population of t cells. dxt measurement of peptide/mhc complex well correlated to other biological phenomenon too. our finding is the first observation that fluctuations at the level of brownian motion affect to the functions of proteins.mason-pfizer monkey virus (mpmv) is an excellent model for the analysis of retrovirus assembly and maturation. however, neither the structure of the viral rna, nor its modulation by capsid-protein binding are exactly known. to explore the structure of the mpmv genome, here we manipulated individual molecules of its packaging signal sequence with optical tweezers. the -base-long segment of mpmv rna corresponding to the packaging signal, extended on each side with -base-long indifferent gene segments for use as molecular handles, was cloned into a pet b vector. rna was synthesized in an in vitro transcription system. rna/ dna handles were obtained by hybridization in a pcr with complementary dna initiated with primers labeled with either digoxigenin or biotin. the complex was manipulated in repetitive stretch and relaxation cycles across a force range of - pn. during stretch, transition occurred which increased the rna chain length and likely corresponds to unfolding. the length gain associated with the unfolding steps distributed across three main peaks at * , , nm, corresponding to * , , bases, respectively. often reverse transitions were observed during mechanical relaxation, indicating that refolding against force proceeds in a quasi-equilibrium process. structural investigation of gpcr transmembrane signaling by use of nanobodies jan steyaert , structural biology brussels, vrije universiteit brussel, pleinlaan , brussel, belgium, department of structural biology, vib, pleinlaan , brussel, belgiumin , scientists at the vrije universiteit brussel discovered the occurence of bona fide antibodies devoid of light chains in camelidae. the small and rigid recombinant antigen binding fragments ( kd) of these heavy chain only antibodies -known as vhhs or nanobodies -proved to be unique research tools in structural biology. by rigidifying flexible regions and obscuring aggregative surfaces, nanobody complexes warrant conformationally uniform samples that are key to protein structure determination by x-ray crystallography:• nanobodies bind cryptic epitopes and lock proteins in unique native conformations • nbs increase the stability of soluble proteins and solubilized membrane proteins • nbs reduce the conformational complexity of soluble proteins and solubilized membrane proteins • nbs increase the polar surface enabling the growth of diffracting crystals • nbs allow to affinity-trap active protein i will focus my talk on the use of nbs for the structural investigation of gpcr transmembrane signaling to illustrate the power of the nanobody platform for generating diffracting quality crystals of the most challenging targets including gpcrs and their complexes with downstream signaling partners. dynamics of the type i interferon receptor assembly in the plasma membrane stephan wilmes, sara lö chte, oliver beutel, changjiang you, christian paolo richter and jacob piehler university of osnabrü ck, division of biophysics, barbarastrasse , osnabrü ck, germanytype i interferons (ifn) are key cytokines in the innate immune response and play a critical role in host defense. all ifns bind to a shared cell surface receptor comprised of two subunits, ifnar and ifnar . detailed structure-function analysis of ifns has established that the ifn-receptor interaction dynamics plays a critical role for signalling specificities.here we have explored the dynamics of receptor diffusion and ifn assembly in living cells. by using highly specific orthogonal posttranslational labelling approaches combined with tirf-microscopy we probed the spatio-temporal dynamics of receptor diffusion and interaction in the plasma membrane of live cells on the single molecule level. for this purpose, we employed posttranslational labelling with photostable organic fluorophores. this allowed us to map diffusion and lateral distribution of ifnar and ifnar with very high spatial and temporal resolution by using single particle tracking (spt) and single molecule localization imaging. observed events of ''transient confinement'' and co-localization with the membrane-proximal actin-meshwork suggest partitioning of ifnar / in specialized microcompartments. this will be investigated in terms influence on receptor assembly and recruitment of cytoplasmic effector proteins. cytoplasmic dynein moves through uncoordinated action of the aaa ring domains ahmet yildiz department of physics, and department of molecular and cell biology, university of california, berkeley, ca usa cytoplasmic dynein is a homodimeric aaa? motor that moves processively toward the microtubule minus end. the mechanism by which the two catalytic head domains interact and move relative to each other remains unresolved. by tracking the positions of both heads at nanometer resolution, we found that the heads remain widely separated and move independently along the microtubule, a mechanism different from that of kinesin and myosin. the direction and size of steps vary as a function of interhead separation. dynein processivity is maintained with only one active head, which drags its inactive partner head forward. these results challenge established views of motor processivity and show that dynein is a unique motor that moves without strictly coordinating the mechanochemical cycles of its two heads.o- self-controlled monofunctionalization of quantum dots and their applicaitons in studying protein-protein interaction in live cells changjiang you, stephan wilmes, sara loechte, oliver beutel, domenik lisse, christian paolo richter, and jacob piehler universitä t osnabrü ck, fachbereich biologie, barbarastrasse , osnabrü ck, germanyindividual proteins labeled with semiconductor nanocrystals (quantum dots, qd) greatly facilitate studying protein-protein interactions with ultrahigh spatial and temporal resolution. multiplex single molecule tracking and imaging require monovalent quantum dots (mvqd) capable of orthogonally labeling proteins with high yield. for this purpose, we prepared monovalance qd-trisnta by a chemical conjugation method. our results indicated that monovalent qd-trisnta was obtained in high yield by restricting the coupling by means of electrostatic repulsion. monovalent functionalization of the qd-trisnta was confirmed by assays in vitro and in vivo. two-color qd tracking of interferon receptors ifnar and ifnar based on mvqd-trisnta were realized on live cell [ ] .to broaden the multiplex toolbox of mvqds, we extended the electrostatic-repulsion induced self-control concept for mono-functionalizing quantum dots with different affinity moieties. as a first instance, we used negatively-charged biotin peptide to produce qd with biotin mono-functionalization. we confirmed our approach was a general method to rend qd monovalent by single molecule assays based on stepwise photobleaching. these mvqds facilitate obtaining spatiotemporal information of ifnars' organization in live cells. by orthogonal labeling u a cells stably expressing ifnar at low level with biotin mvqd and mvqd-trisnta-ifn, we verified colocalization and colocomotion of individual ifn and ifnar at minute scale. combined with super-resolution imaging of ifnars' cytosolic effecter stat , we observed the dynamic coming-and-going contact between the microcompartments of ifnar and stat .a micron sized viscometer was fabricated using the couette type geometry that is capable of measuring the complex viscosity of fluids. the viscometer was produced by two photon polymerization of su photopolymer using a femtosecond laser system, a high na objective and a piezo translator stage. the viscometer was manipulated by holographic optical tweezers and operated in the . - hz frequency range. video analysis algorithm was used to evaluate our measurements. we tested the viscometer with water-glycerol solutions. one of the main reasons for lack of reliability in protein analysis for disease diagnostics or monitoring is a lack of test sensitivity. this is because, for many tests, to be reliable, they need to be performed on a homogeneous, and therefore very small, sample. current in-vitro techniques fail in accurately identifying small differences in protein content, function and interactions starting from samples constituted of few or even single cells. a nanotechnology approach may overcome the current limits in low abundance protein detection. we aim at designing a microwell device for the trapping (in native environment) and the parallel characterization of rare cells (e.g. adult stem cells). such versatile device, based on soft and nanolithography, will promote cell adhesion and viability on differently functionalized bio-compatible materials, allowing for the morphological characterization of the cells, at a single cell level. in parallel, by facing our microwell device with a protein nanoarray, produced via atomic force microscopy nanolithography, we can run proteomic studies at a single/few cells level. moreover, we could foresee the possibility to deliver different stimuli to each cell, correlating the changes in chemistry/ morphology with the protein profile at a single cell level. using an electrophysiological assay the activity of nhaa was tested in a wide ph range from ph . to . . forward and reverse transport directions were investigated at zero membrane potential using preparations with inside out and right side out oriented transporters with na ? or h ? gradients as the driving force. under symmetrical ph conditions with a na ? gradient for activation, both the wt and the ph-shifted g s variant exhibit highly symmetrical transport activity with bell shaped ph dependencies, but the optimal ph was shifted . ph units to the acidic range in the variant. in both strains the ph dependence was associated with a systematic increase of the k m for na ? at acidic ph. under symmetrical na ? concentration with a ph gradient for nhaa activation an unexpected novel characteristic of the antiporter was revealed; rather than being down regulated it remained active even at ph as low as . these data allowed to advance a transport mechanism based on competing na ? and h ? binding to a common transport site and to develop a kinetic model quantitatively explaining the experimental results. in support of these results both alkaline ph and na ? induce the conformational change of nhaa associated with nhaa cation translocation as demonstrated here by trypsin digestion. furthermore, na ? translocation was found to be associated with the displacement of a negative charge. in conclusion, the electrophysiological assay allowed to reveal the mechanism of nhaa antiport and sheds new light on the concept of nhaa ph regulation. swimming motility is widespread among bacteria. however, in confined or structured habitats bacteria often come in contact with solid surfaces which has an effect on the swimming characteristics. we used microfabrication technology to quantitatively study the interaction of swimming cells with solid boundaries. we tracked bacteria near surfaces with various engineered topologies, including flat and curved shapes. we were able to study several surface related phenomena such as hydrodynamic trapping and correlated motion. we think that our results may help to understand how physical effects play a role in surface related biological processes involving bacteria such as biofilm formation. cell labeling efficiency of oppositely charged magnetic iron oxide nanoparticles-a comparative study raimo hartmann , christoph schweiger , feng zhang , wolfgang. j. parak , thomas kissel ,# , pilar rivera_gil ,# biophotonics, institute of physics, philipps university of marburg, pharmaceutical technology, institute of pharmacy, philipps university of marburg e-mail: kissel@staff.uni-marburg.de; pilar.riveragil@physik.uni-marburg.dethe interaction of nanomaterials with cells is a key factor when considering their translocation into clinical applications. especially an effective accumulation of nanoparticles inside certain tissues is beneficial for a great number of applications. predominantly size, shape and surface charge of nanoparticles influence their cellular internalization and distribution. to investigate this, two series of maghemite (c-fe o ) nanoparticles were synthesized either via aqueous coprecipitation or via thermal decomposition of organometallic precursor molecules. size and the spherical shape of both nanoparticle types were kept constant whereas the charge was changed by modifying the surface of the nanoparticles with polymers of opposite charge, in detail poly(ethylene imine) (pei) and a polymaleic anhydride derivative (pma). the positively and negatively charged c-fe o nanoparticles were characterized with respect to size, zeta potential, colloidal stability and magnetic properties. furthermore, the uptake rate and localization of both formulations into a carcinoma cells after fluorescent labeling of the carriers as well as the resulting alteration in mr-relaxation times were evaluated. membrane proteins are the target of more than % of all drugs and are encoded by about % of the human genome. electrophysiological techniques, like patch-clamp, unravelled many functional aspects of membrane proteins but usually suffer from poor structural sensitivity. we have developed surface enhanced infrared difference absorption spectroscopy (seidas) , to probe potential-induced structural changes of a protein on the level of a monolayer. a novel concept is introduced to incorporate membrane proteins into solid supported lipid bilayers in an orientated manner via the affinity of the his-tag to the ni-nta terminated gold surface . full functionality of surface-tethered cytochrome c oxidase is demonstrated by cyclic voltammetry after binding of the natural electron donor cytochrome c. general applicability of the methodological approach is shown by tethering photosystem ii to the gold surface . in conjunction with hydrogenase, the basis is set towards a biomimetic system for h -production. recently, we succeeded to record ir difference spectra of a monolayer of sensory rhodopsin ii under voltage-clamp conditions . this approach opens an avenue towards mechanistic studies of voltage-gated ion channels with unprecedented structural and temporal sensitivity. initial vibrational studies on the novel light-gated channelrhodopsin- will be presented . probing biomass-chromatographic bead interactions by afm force spectroscopy gesa helms, marcelo ferná ndez-lahore, rami reddy vennapusa, and jü rgen fritz school of engineering and science, jacobs university bremen, bremen, germany e-mail: g.helms@jacobs-university.dein expanded bed adsorption (eba), bioproducts are purified from an unclarified fermentation broth by their adsorption on chromatographic beads in a fluidized bed. the unspecific deposition of biomass onto the adsorbent matrix can severely affect the process performance, leading to a poor system hydrodynamics which then decreases the success of this unit operation. to quantify the bead-biomass interactions different chromatographic beads are attached to afm cantilevers, and force spectroscopy experiments are performed with these colloidal probes on model surfaces and cells in solution. the experiments are conducted under varying conditions to study uncovering physiological processes at the cellular level is essential in order to study complex brain mechanisms. using multisite signal recording techniques in the extracellular space, functional connectivity between different brain areas can be revealed. a novel microfabrication process flow, based on the combination of wet chemical etching methods was developed, which yields highly reproducible and mechanically robust silicon-based multielectrode devices. the fabricated shaft of the probe is lm wide, lm thick, has rounded edges and ends in a yacht-bow like, sharp tip. its unique shape provides decreased invasivity. the sensor contains platinum recording sites at precisely defined locations. murine in vivo experiments showed that the probes could easily penetrate the meninges. high quality signals, providing local field potential, multi-and single unit activities, were recorded. the interfaces between the tissue and the platinum contacts were further improved by electrochemical etching and carbon nanotube coating of the metal sites. the integrated optical mach-zehnder interferometer is a highly sensitive device, considered a powerful lab-on-a-chip tool for specific detection of various chemical and biochemical reactions. despite its advantages, there is no commercially available biosensor based on this technique. the main reason is the inherent instability of the device due to slight changes of environmental parameters. in this paper we offer a solution to this problem that enables the optimal adjustment of the working point of the sensor prior to the measurement. the key feature is a control unit made of a thin film of the lightsensitive chromoprotein bacteriorhodopsin deposited on the reference arm of the interferometer. after showing the transfer characteristics of such a device, we demonstrate its applicability to sensing of specific protein-protein interactions. we expect our method to become a rapid and cost-efficientthe combination of unconventional fabrication technology and biomaterials allows both to realize state-of-the-art devices with highly controlled lateral features and performances and to study the main properties of the biomolecules themselves by operating at a scale level comparable with the one crucial for their activity. soft lithography and microfluidic devices offer a tool-box both to study biomolecules under highly confined environments [ ] and to fabricate in an easy way topographic features with locally controlled mechanical and chemical surface properties, thus leading to a finer control of the interplay of mechanics and chemistry. i will present an application of this technology to the control of cell fate that is becoming a key issue in regenerative medicine in the perspective of generating novel artificial tissues. patterns of extracellular matrix (ecm) proteins have been fabricated, by a modified lithographically controlled wetting (lcw), on the highly antifouling surface of teflon-af to guide the adhesion, growth and differentiation of neural cells (shsy y, n , ne- c) achieving an extremely accurate guidance [ ] . local surface topography is also known to influence the cell fate [ ] , thus, integrating this parameter in the substrate fabrication could increase the complexity of the signals supplied to the cells. in this perspective we have developed a novel fabrication technique, named lithographically controlled etching (lce), allowing, in one step, to engrave and to functionalize the substrate surface over different lengthscales and with different functionalities. i will conclude showing how we have been developing ultrathin film organic field effect transistors (ofets) as label-free biological transducers and sensors of biological systems. ofets are low dimensional devices where ordered conjugated molecules act as charge transport material. unconventional patterning techniques and microfluidics have been adapted to proteins and nucleic acids to dose the molecules on the ofet channel with a high control of the concentration. in another set of experiments, we have also been addressing the signalling from neural cells and networks grown on pentacene ultra-thin film transistosr [ , ] .advances in nanotechnology are beginning to exert a significant impact in medicine. increasing use of nanomaterials in treatment of diseases has raised concerns about their potential risks to human health. in our study, the effect of poly(lactic-co-glycolic acid) (plga) and titanium dioxide (tio ) nanoparticles (nps) on function of b-and t-lymphocytes was investigated in vitro. human blood cultures were treated with plga and tio nps in concentrations: . ; and lg/cm for h. lymphocyte transformation assay was used to assess the effect of nps on lymphocyte function. lymphocytes were stimulated with mitogens: concanavalin a, phytohaemmagglutinin (t-cell response) and pokeweed mitogen (b-cell response). our findings indicate immunomodulatory effect of plga nps. proliferative response of t-and b-lymphocytes exposed in vitro to the highest dose of plga for h was suppressed significantly (p. , p. ). on the other hand, we observed stimulative effect of exposure to middle dose of plga nps on b-lymphocyte proliferation (p. ). no alteration was found in lymphocyte proliferation treated in vitro with tio nps for h. in conclusion, proliferation of lymphocytes in vitro might be one of the relevant tests for evaluation of nps immunotoxicity.embryonic stem (es) cell differentiation in specific cell lineage is still a major challenge in regenerative medicine. differentiation is usually achieved by using biochemical factors (bf) which concentration and sides effects are not completely understood. therefore, we produced patterns in polydimethylsiloxane (pdms) consisting of groove and pillar arrays of sub-micrometric lateral resolution as substrates for cell cultures. we analyzed the effect of different nanostructures on differentiation of es-derived neuronal precursors into neuronal lineage without adding biochemical factors. neuronal precursors adhere on pdms more effectively than on glass coverslips but the elastomeric material itself doesn't enhance neuronal differentiation. nano-pillars increase both precursors differentiation and survival with respect to grooves. we demonstrated that neuronal yield was enhanced by increasing pillars height from to nm. on higher pillar neuronal differentiation reaches * % hours after plating and the largest differentiation enhancement of pillars over flat pdms was observed during the first hours of culture. we conclude that pdms nanopillars accelerate and increase neuronal differentiation. key: cord- -vm btiue authors: walwyn, david r. title: turning points for sustainability transitions: institutional destabilization, public finance and the techno-economic dynamics of decarbonization in south africa date: - - journal: energy res soc sci doi: . /j.erss. . sha: doc_id: cord_uid: vm btiue existing socio-technical systems tend to be intransigent to change. decarbonisation, on the other hand, is an imperative, leading to an obvious conflict between the need for, and highly effective resistance to, change. moreover, the abandonment of fossil fuel-based technologies in favour of more sustainable alternatives will require substantial reallocation of government’s operational expenditure, particularly in countries like south africa with high per capita greenhouse gas emissions and low per capita income. in this article, it is argued that reallocation will require more than niche experimentation and destabilisation of the present socio-technical regime. based on a study of south africa’s budget processes, it is concluded that change will only occur when four separate pre-conditions converge, namely a rapidly growing environmental problem capable of leading to civil unrest, a supportive and recently developed policy framework, decreasing techno-economic costs for its solution, and strong political support from an effective ministry or minister. turning points for transition, although infrequent, can be reached through strategic attention to these pre-conditions. a modified kingdon multiple streams approach, which introduces the additional dimension of techno-economic feasibility, is proposed as a useful framework for anticipating when and how to act in order to mobilise sufficient public resources for decarbonisation. the decarbonisation of energy and other systems is essential for the transition to a low-carbon future [ ] . many countries have committed to binding targets for greenhouse gas (ghg) emissions, including the attainment of net zero emissions by [ ] . the paris agreement is clear on what countries need to achieve in terms of such emissions, and over what time period these nationally determined contributions (ndcs) must be realised [ ] . however, the costs of decarbonisation have not been similarly specified in the agreement. individual countries are only now beginning to fully understand and quantify what investment will be required to reach the ndcs, and how these funds might be secured. some initial assessments have been reported in the literature [ ] [ ] [ ] [ ] . for the united kingdom, it has been estimated that the plan to reach net zero ghg emissions by will cost $ . trillion or $ , per metric tonne (mt) carbon dioxide equivalent [ ] . a similar value has been estimated for the united states of america, where the cost of replacing fossil fuels in the energy sector is estimated at $ . trillion or $ , per mt carbon dioxide equivalent [ ] . these values can be more easily comprehended by firstly calculating a total cost based on the present carbon emissions, then annualising this cost by assuming that the transition to low-carbon will take place over a -year period ( to ), and finally expressing the annual cost as a proportion of gross domestic product (gdp). the normalised costs of decarbonisation for the united kingdom and the united states of america are estimated at . % and . % of gdp respectively, whereas for south africa the value is about %. the issue of cost will be particularly acute for south africa [ ] . it is an upper middle-income country, heavily dependent on coal as a source of both electricity and liquid fuel [ , ] . decarbonisation of the energy sector will be a formidable undertaking, whose solution is made more difficult not only by the extent of the economic disruption and social dislocation that may result, but also by the constrained resources with which to address the issue [ , ] . a similar conclusion about the limited readiness of south africa for a low-carbon future has been reached by the world economic forum, which has placed the country in the th position out of countries based on the energy transition index [ , ] . already south africa has been criticised for its insufficient progress towards the attainment of the country's ndc targets [ ] . there is concern about its renewable energy programme, including ongoing delays [ ] , a poor outcome relative to the intended targets for economic development [ ] , and general deficiencies in the implementation of off-grid solar home systems [ ] [ ] [ ] . the government has also significantly alleviated the impact of a recently introduced carbon tax, and is failing to adequately resource the realisation of its ndcs [ ] . moreover, the covid- pandemic has caused a massive shock to the economy, reducing tax revenue collection by % and cutting at least % from the gdp [ ] . in short, the south african government appears unwilling and increasingly unable to resource its low-carbon transition. the issue of public sector resource allocation is critical for sustainability transitions. budget decisions within governments have direct and often irredeemable consequences. once such decisions are made, the resultant allocation of funds allows some programmes to be pursued and compels others to be halted. although there are several publications on green financing within south africa, such as its broader challenges and necessary design features [ ] and the role that public financial intermediaries have already played in the country's energy transition [ ] , there have been no specific studies on how to mobilise and reorient government expenditure for sustainability transitions, and particularly the decarbonisation of its energy sector. the unique contribution of this paper is its analysis of budget processes, leading to the proposition that four preconditions should be met before a significant reallocation of government's operational budgets, in support of decarbonisation, can be achieved. notably, in addition to the three factors of problem, policy and politics, which are central to kingdon' s multiple streams approach (msa) and are already welldescribed [ ] , the techno-economic value of the proposed solution must be addressed. the analysis in this paper seeks not only to support its claim for the four streams approach, but also to recommend ways of dislodging lock-in and re-directing government expenditure. the study was exploratory in its approach. it drew on interviews with ex-government officials, examples of previous re-allocations and government documents relating to the budget process. its objective was to identify the causal factors that could lead to profound changes in these budgets, and then to present these factors as preconditions that should be concurrently fulfilled. in its analysis, the study uses a theoretical framework which combines msa [ ] with historical instutionalism [ ] , as explained in the second section. the third section presents the relevant background on south africa's budget processes. the methodology, results, discussion and conclusions follow in sections , , and respectively. kingdon's msa postulates that change happens at single points in time or 'policy windows', when a number of causal chains or streams converge [ , ] . typically, the three streams comprise of the problem itself, a relevant policy framework and the political process through which change can be realised. msa further stresses the importance of policy entrepreneurs, who must attempt to couple the three streams through process of power brokerage and manipulation of problem contexts [ ] . msa specifically acknowledges the complex and chaotic nature of policymaking, and the difficulty of operating within an environment of ambiguity, irrationality and unpredictability [ ] . changes in policy are seen in msa to emerge spontaneously and stochastically, and can be missed by policy entrepreneurs due to the absence of well-developed policy solutions [ ] . msa's emphasis on windows of opportunity and convergence is also a central aspect of historical institutionalism, which refers to windows of opportunity as critical junctures and convergence as a process of conjuncture [ ] . historical institutionalism emerged in the s as a means of conceptualising and theorising how reform takes place at the meso-level, introducing such terms as path dependence and selfreinforcing processes, whose identification are critical to understanding and hence overcoming intransigence to transition and change. it adopts a longitudinal approach, perhaps over several decades, the analysis of which is used to identify the relationships of lock-in and dependence that comprise the socio-technical landscape [ ] . the hierarchies of micro, meso and macro, as developed within historical institutionalism, have become widely accepted and applied in the sustainability transitions literature [ , ] , as exemplified by its adoption of the multi-level perspective (mlp). the latter defines the three levels as the socio-technical landscape, consisting of government policy and inter/national systems, the socio-technical regime and the niche level, the latter including firms and networks of individual actors. although arguably a simplification of the broad diversity of individuals, collectives, organisations and systems, this layered hierarchy is fundamental to an understanding of transition, and how different processes take place within each level. in much of the mlp literature, change is considered to begin through niche innovations and policy experimentation, undertaken by an array of minor actors. the efforts of these actors may eventually become aligned and sufficiently powerful to destabilise an extant socio-technical regime, which has been wellestablished at the meso-level over a long period [ ] . a possible weakness of this model in the context of a developing country is the extent to which agency is possible within a highly resource-constrained political system. indeed, in the wider debate about structure vs agency, it can be argued that individuals and small networks of actors are disempowered by the broader structural context. it is precisely this consideration which makes the use of historical institutionalism, with its focus on the meso-level, as a highly relevant analytical framework for a country such as south africa. although operating as a democracy, it is clear that the societal environment in south africa still acts as a major constraint on the micro-level actors, given the low level of education and human capability [ ] . furthermore, the highly rigid approach of the pre- system of apartheid resulted in an extensive entanglement of the technological systems and the state, creating an almost irreversible degree of lock-in [ ] . in some senses, historical institutionalism is about theories of continuity, providing an explanation for why regimes remain stable, even though they are contested, rather than why they change. in order to address this gap, roberts and geels [ ] supplemented the insights of historical institutionalism with those of mlp as a means to further develop a theory of change. in their analysis of conditions for politically accelerated transition, informed by two case studies in the united kingdom, they argue that conditions for change must include both a weakening of the socio-technical regime, which they refer to as a push factor, and a strengthening of niche actors, which they label as a pull factor. following the insights from historical institutionalism, in which major policy change arises from struggles for power at the meso-level, they conclude that at least one mechanism of change is a macro-or landscape level shock. this severely disrupts the incumbent regimes and allows the emergence of niche actors as a new socio-technical system [ ] . in summary, historical institutionalism has two specific advantages when used to understand sustainability transitions, firstly its focus on meso-level institutions and policy regimes, and secondly the recognition that power struggles between political collectives or groups over scarce resources lie at the centre of politics and are critical to policy change [ , ] . institutional structures and arrangements, typically referred to as a political community or polity, engage in power struggles through the process of politics. in this sense, historical institutionalism is more appropriate in understanding south africa's budget processes and has been used in this study. it is argued that important decisions relating to resource allocation, which have the capacity to alter the course of sociotechnical systems, are the consequence of conjunctures taking place at critical moments. such turning points have a low probability of occurrence, given the dominant approach to policy stability and lock-in. the state can play an important role in transitions, either through exogenous changes at the level of the socio-technical landscape, or by strategic and planned initiatives to change the institutional environment [ ] . it is precisely the issue of the role of the state, and how it can be internally directed, that is the subject of this article. the study considers the separate roles of the executive, the administration and the legislature. the nature of the power balances between these three arms is explored by analysing their relative roles with respect to a core public sector process, namely the allocation of funds from the national fiscus to individual departments. an overview of this process, as it normally takes place, is presented in the next section. the adoption of south africa's new constitution in necessitated a comprehensive reform of the management of public finances, including its budget procedures. the latter were amended in several respects, such as strengthening the link between policy and budget allocations, the introduction of systems to address fragmentation and lack of clarity, and measures to improve transparency and re-establish fiscal stability [ ] . although the new processes were more top-down, the changes improved the alignment between post- priorities and actual public expenditure [ ] . a core feature of the revised budget process was the introduction of the medium-term expenditure framework (mtef), which was intended as the means by which government could ensure budget stability and predictability while allowing changes "at the margin" [ , p ] , thereby managing the "tension between competing policy priorities and budget realities" [ , p ] . the mtef was positioned as a first step in the "wider overhaul of the budgetary process, emphasising transparency, output-driven programme budgeting and political prioritization", that provided the link between the "technical preparation of budgets and the need to reflect political priorities in expenditure plans" [ p ]. its key features include rolling baselines, which are the core budgets for each department and are substantially unchanged within a single mtef, and a contingency reserve, which is intended to cover unforeseen expenditure. a detailed review of the timeline for the budget process is not possible in this article. in summary, the process is initiated by a cabinet lekgotla, which takes place in february to march of each year (see fig. ). at this meeting, the cabinet reviews the macroeconomic and fiscal policies, and the extent of the required budget cuts, or the additional funding that may be available for new priorities. these changes are then incorporated in a set of mtef budget guidelines [ ] , which are issued to the various national departments and provinces. in response to the guidelines, the departments prepare budget proposals, which are then submitted to national treasury, where they are consolidated into an overall budget estimate. based on the alignment between the estimates and the guidelines, national treasury provides feedback and, if necessary, requests revisions from the departments. the adjusted budgets are then assessed in the ministers' committee on the budget (mincombud) technical committee (mtec), which is composed of senior officials from national treasury (nt), the department of planning, monitoring and evaluation (dpme), the department of cooperative governance (dcog) and the department of public service and administration (dpsa). mtec then prepares recommendations for mincombud and cabinet regarding budget allocations in the mtef, taking into account government priorities, funding available, exchange rates, alternative funding sources and the division of revenue amongst the three spheres of government [ ] . once mincombud has approved the budgets, the minister of finance tables the medium-term budget policy statement in the whole process intentionally reinforces a central design principle of the post- reforms, namely the establishment and maintenance of a stable public finance environment, otherwise stated as a predictable expenditures and policies [ ] . one effect of this approach is that it severely limits the available funding for new policies or initiatives and reinforces a pattern of lock-in or pathway dependence within the budget allocations. however, there are two mechanisms through which small amounts of money can be sourced for new policies, if there is sufficient political support. before the estimated income is divided between the different departments and spheres of government, a contingency reserve is 'top-sliced' and retained by national treasury for emergency needs or novel policy instruments. this reserve provides a level of flexibility to the budget allocations, although, as shall be discussed later, it is a small amount relative to the demand for new funding from the various departments. the other mechanism, known as virement, involves the shifting of funds from one subdivision of a budget vote to another. there are a number of restrictions to this practice, including the requirement that it may not exceed % of the total allocation in the source subdivision (from which the funds are taken), that it may not involve the shifting of funds from capital to recurrent expenditure and that it cannot be used to increase remuneration without special approval from national treasury. clearly, expenditure predictability and budget stability are important objectives, especially in respect of financial markets and the cost of borrowing. however, both elements act against the needs of transition or change, particularly where the change has a significant cost or budget implication. the prospects of sufficient allocations for major new policy directions are limited by the mtef and the system weakens the role that government can play in enabling such changes. this imbalance has led to the growing realisation by south africans that reform can only take place in response to external pressure, widespread protest, and even violence. the / student protests regarding "free" higher education are an illustration of this perspective. provoked by a statement from the department of higher education and training (dhet) on transformation in higher education [ ] , the students embarked on nationwide protests, demanding that promises of free education be fulfilled. after a number of incremental changes, president zuma finally announced on december that higher education for the poor and working class students would be free [ ] . the president's decision was taken against the advice of national treasury, especially since it ran counter to the agreed budget processes and mechanisms [ ] . moreover the impact was profound; funding for the national student financial aid scheme (nsfas), the primary vehicle for the implementation of the new policy, a 'mere' r . billion in / , increased to r billion in - [ ] , as shown in fig. . certainly, in respect of this decision, it can be concluded that the executive directed, the administration was out-manoeuvred and the legislature side-lined. the intent that such decisions should be the outcome of evidence-based information and a collective consensus between the three arms, as stipulated by a range of policy documents, was over-ruled. thus, issues of policy conflict and priorities are often resolved through a political process which may relate to the power or influence of key positions within the executive arm of government (cabinet), or to the ability of the operational and legislative arms to determine the expenditure allocations. despite such processes being of critical importance to an understanding of how decisions on resource allocation are made, there is little published research in south africa on this topic. as illustrated by the earlier example of the nsfas, budget re-allocations are possible and do take place. accelerated sustainability transitions, as will be necessary for the timely implementation of the paris agreement, will require significant government investment, especially in new systems of transport and energy. to disregard or overlook these outlays under the pretext of lacking the necessary funds, appears disingenuous. it is a matter of priority and policy, and of avoiding the disrpution of existing, exclusive institutions [ ] . in summary, the analysis of budget processes leads to the following propositions about how change, and in particular realignment to operational budgets in favour of sustainability transitions, could take place. • for change to take place, the problem must be highly visible, a coherent and aligned policy framework must be in place, the solution should be affordable, it must require a political intervention, and failing to act must have severe consequences (such as social unrest). • the dynamics of each aspect are the problem must be accelerating in its severity; the solution must have falling cost implications; the window of change is generally very brief, opened by the sway of politics and rising popular dissent; and the political response must be rapid and effective. • the simultaneous convergence of these four aspects, described as a process of conjuncture resulting in a turning point or critical juncture, is essential for change to take place. in order to explore the validity of these propositions, a research project, involving a series of interviews over an -month period, was designed and initiated. the overall objective of the study, as already noted in the introduction, was to identify the factors that can lead to profound changes in government expenditure, and hence how greater priority can be mobilised for the support of sustainability transitions. further details of the research method are provided in the next section. the research followed a qualitative, inductive and exploratory approach with a purposive sampling strategy [ ] . the population consisted of ex-members of national treasury, and other departments, who had occupied senior positions within government in the recent past, including director-general, deputy director-general and chief director, and had more than years' experience of budget processes in the public sector. approval of the project by the faculty ethics committee was subject to two explicit conditions, namely that only exmembers could be interviewed and the responses had to be anonymised. the latter is a standard requirement and was fulfilled by following the normal procedures. however, the former condition added lengthy delays to the project due to the difficulty in identifying suitable respondents meeting the two criteria of having recently left a senior post in government. altogether, six interviews were completed. the respondents were interviewed using a semi-structured questionnaire in order to understand, primarily, how policy priorities are assessed and balanced within the public sector. the questionnaire was divided into four separate sections, with the first section covering the participant's view on the role of the state with respect to the environment and how this is presently being fulfilled, followed by three sections on the process of budget allocations and determination of funding priorities, the resolution of cases involving competing priorities, and general recommendations on accelerating support for sustainability transitions, respectively. in each case the interviews were recorded, transcribed and then analysed using atlas.ti. the coding structure was developed based on the research questions, with the coding groups covering the core questions of role, examples of policy conflict, the determination of priorities, details of the budget process, moments of change and lessons for sustainability transitions. it is acknowledged that the small sample size limits the external validity or generalisability of the results [ ] . the credibility and exclusivity of the data is, however, robust in that the respondents had all occupied senior positions and accumulated more than fifteen years of experience in budget processes within the public sector. although there were differences in perspectives, mostly there was agreement on the main determinants of the central question that this article seeks to address, namely how to influence such budget processes. as a result, it has been possible to extract a set of useful insights from the data. as mentioned earlier, south africa faces a huge decarbonisation challenge. its electricity sector is the most carbon intensive of all the g countries, it has a bankrupt power utility without resources to finance its normal operations, let alone decarbonisation, and it has a high level of unemployment, which places even more pressure on the imperative for a just transition [ ] . the global decarbonisation imperative will place huge financial pressure on state income. it has been estimated that the cumulative impact on south africa of a low-carbon transition, referred to as the 'transition risk' will be $ billion, which includes the loss of export revenue, and the loss of local markets for coal and liquid fuels [ ] . in addition, south africa will need to invest in new energy infrastructure, the extent of which can be illustrated by considering investment cost normalised for gdp. using data for carbon emissions and gdp data from the world development indicators database [ ] , and assuming that the average investment cost will be $ , per mt of carbon, it is calculated that south africa must source $ billion, or % of the gdp to finance the transition, as shown in fig. . it is clear from this comparison that the cost of decarbonisation will be more acutely experienced by countries with lower gdp and higher carbon emissions. for instance, iran, russia, india and saudi arabia have high relative ghg emissions and can expect a larger cost, normalised to gdp, than other countries. the combined impact of this investment cost and the transition risk, which represent a major challenge to the south african government, has a number of budgetary implications. as will be the case in many countries, the public sector will be required to implement mitigation strategies, such as the retraining of mining sector employees, introduction of new policy instruments to support the renewable energy sector, changes to legislation, interim approaches to reduce the impact of revenue changes on municipalities, measures to build resilience to climate change, programmes in research and development focussed on the necessary diversification of the economy and the remediation of environmental damage from fossil fuel usage. although several departments will be affected by transition risk, this study has looked specifically at the department of environment, forestry and fisheries (deff), which has overall responsibility for environmental management and is represented at executive level by the minister and deputy minister. the department has an annual budget of about $ billion, an amount which has not changed much over the last seven years, and under the medium-term expenditure framework allocations is projected to stay at a similar level in the immediate future (see fig. ). allocations to the department were considered by the interviewees to have grown from a low base, with the department being one of those to have benefitted from the growth in overall government revenue over the period to . opinions as to the adequacy of the present budget in meeting the department's responsibilities were mixed, although it was generally considered that the major constraint was not funding, but capacity to deliver. this sentiment is not echoed by the present minister, who indicated a need to leverage the resources of other sectors in order to ensure that the department could deliver on its present mandate [ ] . the respondents agreed that deff's role is to ensure the protection and preservation of the environment, or in broad terms 'environmental sustainability', and that this role was being fulfilled through the combined actions of regulation and enforcement. however, it was noted by the respondents that the department's efforts can be compromised by the very nature of public policy, which is to represent a broad range of interest groups, and the diversity of government's activities, some of which can impinge on the environment and have direct environmental costs. for instance, the granting of coal mining or fishing licenses was cited as an example of the tension between protection of the environment and economic development. in the case of fishing licenses, the desire to ensure the economic livelihoods of small fishing enterprises was considered to have led to over-harvesting of fish stock, with subsequent depletion, and in some cases collapse, of fish populations. similarly, the granting of additional coal mining licenses as a means of transforming the sector, or the use of pesticides to control malaria, had led to significant degradation of the environment in the affected areas. it can also be argued that environmental protection through restrictions on mining and other resource extraction activities can be justified economically, and that defining sustainability vs. development as a dichotomy is unproductive and misleading. longer term economic development is about environmental sustainability, a perspective which is expressed by the present minister, barbara creecy, who stated [ ] : "what i want to bring to this portfolio is the understanding that caring for the environment, caring about climate change and threats to biodiversity may well be an emotional concern for some people, but -million south africans are directly dependent on our natural resources." it is clear that conflict between the mandate of deff and other departments arise on a regular basis, and that in some cases, the deff is successful in preventing decisions leading to adverse environmental impacts. although no clear pattern emerged from the discussions, the interviewees were questioned about how such tension was managed, and, typically, which areas were prioritised. the results of these questions are presented in the next section. disagreements on government choices are generally resolved through the existing policy framework, which forms the blueprint for government action. however, in ambiguous situations, where proponents of each position can find relevant policies in support of their perspectives, decisions are taken based on the power of coalitions, politics, the charisma of individuals and the strength of lobby groups. such decisions often take place in the executive, which, in the view of one respondent, is a forum with weak environmental representation, given that there is one minister of environmental affairs, but five or six economic ministries, and eight or nine social ministries. in order to ensure a positive result for the environmental portfolio, it is therefore essential that the minister attempts to build an alliance with other ministries and civil society, and is then able to depend on this alliance in supporting a particular issue. as noted by one respondent: [ : ] "a flat-footed environment ministry that refuses to play the politics that is required ain't going to get anywhere." (respondent ) the choice of alliance partners is one of several important components in the construction of such a coalition. it was indicated that likely partners for environmental issues included the security cluster (defence, police, justice) and one or several of the cross-cutting departments (finance; foreign affairs; public service and administration; and planning, monitoring and evaluation). it was noted that the former was often supportive of longer-term perspectives as a means of averting social unrest and inter-nation conflict. apart from being a forum in which the environmental mandate is outnumbered, the executive is also a constitutional body which lacks a referee, particularly if the president does not assume at least some responsibility for non-partisanship. this one-sidedness is especially relevant in decisions relating to the construction of infrastructure or the expansion of one of its public enterprises, where the government acts as both the regulator and the player, and may too easily be able to bend the rules in its favour, or make a trade-off, as described by one respondent: [ : ] government always has to make trade-offs between various sectors and importantly with respect to the environment it is constantly making trade-offs between environmental and economic/social objectives, and really that is its function. so, it is not just a regulator of the environment. in regulating the environment, it balances multiple competing objectives …. i think that is just the hierarchy of decisionmaking that it needs to do (follow). (respondent ) it is not clear how such a hierarchy of decision-making is established, other than that it is political and highly contextual. a propensity for persistent and patient negotiation is an important prerequisite for the minister of environment, forestry and fisheries, especially in pursuing a pro-environment agenda which may be perceived as anti-employment or economic growth. in the next section, the respondents' comments on how such agendas have evolved, and particularly the reasons for any successes, are discussed. during the interviews, several incidents of decisions relating to significant policy changes were discussed, including the implementation of the sugar tax, the introduction of plastic bag regulations, the proposed carbon tax and the reforms on student fees. in the earlier discussion on historical institutionalism, it is argued that such moments of change require, inter alia, strong political support. one important strategy for obtaining this support, which emerged from the discussions, is the inclusion of revenue collection, which predictably secures agreement of arguably the most powerful government department, the department of national treasury. according to the respondents, the ability to extract additional fiscal revenue accounts for the relative ease with which changes such as the sugar tax were introduced, compared to other policy initiatives. the respondents noted, however, that even in the case of additional tax revenue, obtaining consensus from the executive always required a great deal of finesse. the nature of this 'footwork' is central to this study, but inevitably the most elusive aspect to define. it depends on the issue, the context, the personalities, the relevant institutions, the affected sectors, and a number of other factors. it is what distinguishes an effective from an ineffective minister, a progressive from an intransigent or reactionary administration. the respondents noted that in government change generally takes place with difficulty, if at all. the processes and systems are designed to maintain the status quo, perhaps for important reasons. policy stability, particularly macro-economic, is both a desire and a goal in governments, and change, especially when it is driven by narrow political interests outside of established governance processes, can be damaging to countries and economies. such momentum in government action is reflected acutely in budget allocations. the respondents commented that these allocations are mostly unchanged from one year to the next, giving little flexibility to the executive in being able to implement new initiatives. for instance, [ : ] "… every now and then there are some shifts, but those shifts might be fairly significant in the context of one particular department, but i'm not sure that they are significant in the context of an entire sector and usually those shifts are either … i don't think that they are ever more than maybe % of any government's allocation from one year to the next." (respondent ) in other words, there is no new money within treasury and budgets remain substantially unchanged from one year to the next. moreover, the re-allocation of funds from one budget line to another, within the rules of virement, is tightly controlled and requires national treasury approval. individual line managers are able to re-allocate unspent funds within a programme or line item, such as from one project to another, but these changes are relatively minor and cannot be applied to the funding of new initiatives. the challenge for the funding of sustainability transitions is, therefore, to persuade government departments to redirect their existing budgets over time, and mainstream the sustainability development goals in their daily practices. this challenge is recognised by deff as being central to its mandate and it engages regularly with other departments on the need to include such goals with some success. for example, the establishment of the biodiversity centre of excellence by the department of science and technology (now the department of science and innovation), the creation of the green fund and the implementation of the renewable energy independent power producers procurement programme were mentioned by the respondents as resulting from such inter-departmental discussions. there is one important exception to the overall situation of budget stasis, which is the power afforded to the president of south africa. although not explicitly stated in the constitution or the legal system, it appears that the president is able to circumvent the standard budget processes, as happened in the example of the student fees crisis of [ ] . the appropriateness of this use (or abuse) of power was questioned by a respondent: [ : ] "you know if the politicians don't accept the institutional rules of the game, then it is very easy for them to wreck the institutions. … there has to be a basic acceptance, a collective acceptance that this is the rules and this is how you do things. if people don't accept that, they can just wreck institutions very, very easily." (respondent ) in summary, the interviewees concurred on the budget situation as being highly constrained with the opportunity of finding new funding to support sustainability transitions (in pursuance of the sustainability development goals) as being not just remote, but impossible. the redirection of existing funds is the only option, although the budget process is ill-suited to such changes and in general only incremental adjustments over a long period are possible. the implication of these results for sustainability transitions are discussed in the next section. decarbonisation of south africa's energy sector is largely a problem for the state. more than % of the country's carbon emissions derive from the fleet of coal-based power stations owned and operated by the state-owned power utility, eskom [ ] . replacement of these facilities with renewable energy alternatives will require significant new finance. given its present financial circumstances, eskom itself is highly unlikely to be at the forefront of this reinvestment and the reform of the electricity sector. as already noted, the utility is deep in debt, and embroiled in a series of technical and political crises which severely constrain its ability to raise finance from capital markets or the state [ ] . this outlook suggests that much of the investment finance for the energy transition will be provided by the non-government sources. however, government will be required to provide financial support for a range of operational activities, including energy research and development, retraining workers affected by the energy transition, building of capacity to manage energy markets, and incentives to support local manufacture of renewable energy equipment. such funding will be mostly additional to the existing budgets of government departments, and will require either new funding or the re-allocation of budgets from other activities. the likelihood of new funding is minimal. government finances, already stretched to the limit before covid- , are now in a highlyborrowed predicament, with the level of government debt rising to % of gdp in the / financial year [ ] . the re-allocation of monies from other expenditure items within the operational budgets will be an imperative if government funding for decarbonisation and the energy transition is to be made available. the premise of this article has rested on the assumption that high-level decisions for such changes reflect not only the extent of the need to fulfil each department's public mandate, but also the strength or weakness of the interdepartmental power relationships. an understanding of these dynamics is therefore critical to the implementation of emergent and experimental policies within government, such as policies to support decarbonisation, sustainability transitions and the transformation of socio-technical systems. this study has intimated that the budget process affords limited opportunity for negotiations on departmental budgets. however, there are points in the process, referred to as windows of opportunity, when such changes are possible if a number of preconditions have been met. the concept of a window of opportunity is by no means unique. as already outlined, it is shared by both msa and the multi-level perspective; the latter focuses on the two streams of regime destabilisation and niche consolidation, whereas msa requires the conjuncture of problem, policy and politics. the south african context supports both approaches, but identifies the fourth precondition of an affordable solution. conditions in developing countries are frequently resourceconstrained, implying that regime changes lead to extreme changes for the affected parties. as a result, the conflict can be more apparent, the outcomes more divisive and the moments of change take place more unpredictable. in other words, the higher financial stakes determine the nature of the struggle and the resultant change, should it occur. the critical junctures or windows of opportunity are less frequent, more contested, require more significant alignment of interest groups and are characterised as strong conjunctural processes. the pre-conditions for these windows of opportunity are also different. it is argued here that one further stream is required, namely favourable techno-economic value or improvements in the benefit to cost ratio of the potential solution, as exemplified by the introduction of the sugar tax. this amendment to the msa is essential to understand how change can emerge, and hence to engineer or accelerate sustainability transitions. furthermore, each of the four streams needs to be characterised based on its dynamics or rates of change. the four streams are now discussed in more detail. policy changes rarely seem to take place in response to opportunities. politics is a domain that appears to be mainly reactive, and often takes the form of crisis management. given the multiple demands on the public sector, and the huge scale of the issues, this reactiveness is perhaps not surprising. one general precondition for policy change seems to be clear: there must be a shared understanding of a clearly defined problem for such changes to be considered. the mere existence of a problem is also not on its own sufficient; policy windows are more likely to open when the problem is both growing in scope and the rate of growth is accelerating, to the extent that it is mobilising extensive political support outside of government, most notably in civil society with its ability to mobilise civil unrest. the power of civil society to destabilise the landscape depends on the extent of mass mobilisation and public support for a particular issue, and the degree to which this support is able to gain momentum within nation states. government may then act to repress or outlaw civil society protest, as happened repeatedly under apartheid south africa [ ] . the inclusion of regime destabilisation as a necessary step in the processual framework of this model is also part of the msa and also the multi-level perspective. examples of such changes include the occurrence of pandemics, the environmental crisis, the advent of widespread road transport and the rise of wheat mono-culture [ ] . opinions on how to change a dominant regime have been offered by many authors. milton friedman, well known for the development of stabilisation policy, surprisingly had a perspective on how to change a socio-technical regime. he considered that only a crisis produces change, and that the most important pre-condition was to be wellprepared [ , p ix] . this requirement of being well-prepared is partly captured in the theories of policy experimentation and strategic niche management [ , ] . these argue that transition requires initial experimentation with new approaches, some of which may fail, but the successful interventions may eventually gather sufficient momentum to result in changes at the meso level. importantly, there are no clear predictors of success with early experiments, and policy frameworks may themselves not achieve the desired outcomes. it is therefore critical that such interventions be accompanied by a transparent and insightful monitoring and evaluation framework, which can inform whether the policy should be continued, strengthened or withdrawn. the stream of techno-economic value refers to the perceived cost benefit ratio of a public policy or intervention [ ] . such analyses are a legislative requirement within the south africa public service, and are typically framed with questions such as "what is the value for money?" or "what is the cost/benefit ratio?" [ ] . cost/benefit analysis is used as a means of informing budget decisions based on the economic viability or socio-economic benefit of a particular intervention. there are multiple approaches to the analysis, including the use of standard financial techniques such as discounted cash flows, net present values, internal rates of return and payback periods [ ] . mostly, the analysis requires the monetisation of the resultant social welfare and economic benefits of the intervention, the sum of which are then compared to the overall costs. measurement of the net social welfare is complicated by the large variety of possible benefits, the relative value of each intervention and the prediction of its impact. although costs are mostly explicit and relatively straightforward to calculate, the quantification of benefits is subject to assumptions on critical issues such as the contribution of energy to the quality of life, or the magnitude of the social discount factor, or the value of possible externalities (such as an improvement in the quality of the natural environment) which are associated with a particular intervention. some of these limitations can be avoided by applying cost/benefit analysis only as a comparative technique. in this way, the same assumptions apply to all the options being reviewed, and allow for the selection of the most cost-effective alternative amongst a portfolio of analogous projects. a useful example of how the techno-economic value can influence resource allocation decisions is the case of the declining cost of renewable energy technologies. although the cost of electricity from photovoltaic panels and onshore wind turbines was, prior to , generally more expensive than energy obtained from fossil fuels, this situation has now reversed and renewable energy technologies are now consistently cheaper [ ] , due mostly to improved manufacturing efficiencies and greater production capacity within the sector [ ] . the lower levelised cost of energy has been an important influence on the planning of national energy systems and the decisions to incorporate higher levels of renewable technologies within such systems [ ] . as for the problem stream, a positive impact on social welfare is not a sufficient condition; the benefit should have, at least, the prospect of growth. in other words, the potential savings should be increasing, or the cost of implementation should be decreasing, as indicated in the example of renewable energy technologies. accepting the validity of the normative process that policy determines strategy, strategy determines operational plans and operational plans drive budget allocations, it is reasonable to assume that budget allocations match the overall policy framework. however, government as a punctualised actor is in fact not a single entity; it is a loose agglomeration of multiple actors with overlapping but also conflicting policy objectives. for instance, industrial policy may contradict environmental policy, fiscal policy may oppose energy policy, health policy may conflict with trade policy, and defence policy may act against science and technology policy. in terms of decarbonisation, and the likelihood that departments of the environment will be able to secure funds from the national fiscus in support of decarbonisation, it is clear that attention to the politics is critical. for south africa, which faces a million metric tonne per annum decarbonisation challenge with limited ability to raise the necessary resources, this aspect of the environmental campaign will be vital. using the estimated value of $ , per mt carbon dioxide equivalent, the decarbonisation cost for south africa will amount to $ , billion, or % of gdp over a -year period ( to ). in comparison, the decarbonisation costs for the united kingdom will be . % of gdp, as previously indicated in the introduction. south africa, therefore, has limited options in addressing the decarbonisation challenge, other than to consider budget reallocation. the issue of securing resources to support sustainability transitions, and particularly public funding, is also highlighted in the literature on technological innovation systems, where resource mobilisation is identified as one of several critical functions for the establishment of systems to support such transitions [ ] . technological innovation systems as a conceptual model for transition is also a processual framework and in its discussion of resource mobilisation, it is argued that this function needs to be sufficiently covered from the early stages in the development of a new system. identifying resource allocation as an important part of the overall transition process is in itself insufficient in supporting the transition. it is equally important to understand the source of the funds and how they can be secured. public funding is critical in such transitions since the initial stages will be characterised by high levels of risk and limited participation from private funders. however, public budgets are subject to intense competition from other priorities including health, security, education and infrastructure, and the prospects of successfully lobbying for the re-direction of public funds from existing portfolios are severely restricted. this article has argued that such moments do exist, but in resource constrained setting such as south africa, they occur only within narrow windows of opportunity characterised by an escalating problem, the adoption of a recent policy framework, the prospects of an improving benefit to cost ratio, and a willingness to engage in realpolitik. previous studies have concluded with a number of policy recommendations for low-carbon transitions including the need for dynamic policy mixes dealing with demand-and supply-side instruments, a focus on politics in addition to policy, and active steps to phase-out existing technologies in addition to supporting niche-innovation [ ] . the results of this study suggest a number of more specific recommendations for resourceconstrained countries as follows. low-carbon futures are unlikely to be realised without public funding. as is argued in the case of public-funded research and development, such transitions will be subject to market failure and under-investment in the absence of public support. it is therefore important that government establish dedicated funding for sustainability transitions, in the same way that they have created budgets for industrial development or energy infrastructure. developing the structure and rationale for these budgets is an important step towards securing the actual funding. in resource-constrained settings, it will be hugely challenging to secure funds without re-allocation from existing portfolios, the prospects of which are unlikely on a daily basis. however, there will be windows of opportunity or critical junctures, which will arise when there is alignment between an escalating problem, a supporting policy framework and a rising benefit to cost ratio. an appreciation of such moments, and a mechanism for recognising their opening, is an important insight of this study. although leadership and agency are critical, engaging in politics is more that individual action. all actors, but especially politicians and senior public servants, need to enter into relationships with each other, and with civil society, labour and advocacy organisations. in the final decision on budgets, the most important factor may not be the strength of the evidence for the intervention, but the strength of the alliances which are willing to support the re-allocation proposal. conclusion and recommendations in addition to an overall cultural change, transitions to sustainability in south africa, and perhaps other countries, will require new programmes and projects within government. importantly, these programmes will require new funding. however, the prospects for this are severely limited by the present situation of decreasing government revenues and the overall path dependence of budget allocations. the lack of new funding will hinder such transformation and requires the development of new strategies. this research has sought to define the main components of the budget process in south africa and hence develop a strategy to overcome funding limitations for necessary and important operational programmes. it concludes that the conjunction of four streams is key to successful changes in budget allocations, namely a growing problem, preferably accelerating, a recently developed policy framework, a strong techno-economic justification whose prospects likely to become more attractive, and a strengthening political stream consisting of powerful alliances able to drive budget changes through the executive. these conclusions align with the insights of historical institutionalism, which include the supposition that change takes place as a result of contestation, and at the meso level, the destabilisation of the existing socio-technical regime combined with significant challenge from an alliance of niche players. this result implies that sustainability transitions in south africa will require highly strategic leadership of the environmental portfolio, which can direct the processes of conjuncture and act at critical moments in order to effect transformation. in particular, this leadership needs to sharpen the arguments for public funding to support sustainability transitions, develop the necessary budget structure, learn to recognise critical junctures or windows of opportunity, and network with power actors willing to support budget reallocations. waves of disruption in clean energy transitions: sociotechnical dimensions of system disruption in germany and the united kingdom these countries have committed to a net-zero emissions goal -could it solve the climate crisis? /l. /rev. : adoption of the paris agreement the analyses on the economic costs for 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commerce students regarding "free" higher education in south africa zuma announces free higher education for poor and working class students zuma ignored treasury, nec on higher education s promise of free higher education honoured sustainability transitions in the developing world: challenges of socio-technical transformations unfolding in contexts of poverty promoting novelty, rigor, and style in energy social science: towards codes of practice for appropriate methods and research design brown to green: the g transition to a net-zero emissions economy world development indicators bringing a climate of change tensions in the transition: the politics of electricity distribution in south africa south africa sees government debt exceeding % of gdp state-civil society relations in post-apartheid south africa capitalism and freedom policy experimentation and innovation as a response to complexity in china's management of health reforms strategic niche management and sustainable innovation journeys: theory, findings, research agenda, and policy applied welfare economics: cost-benefit analysis of projects and policies socio-economic impact assessment system guidelines, department of planning monitoring and evaluation renewable energy gathers steam in south africa functions of innovation systems: a new approach for analysing technological change the socio-technical dynamics of low-carbon transitions the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. key: cord- -qw pf r authors: greaves, peter title: vii digestive system date: - - journal: histopathology of preclinical toxicity studies doi: . /b - - / - sha: doc_id: cord_uid: qw pf r publisher summary this chapter deals with the digestive system. the major and minor salivary glands and their secretions also represent and integral part of the protective mechanism of the oral cavity, and derangement of saliva production may lead to loss of integrity of the oral mucosa. drug-induced abnormalities of taste sensation are also well-described phenomena occurring in man although human studies are necessary for the detection of these effects. inflammation of the oral cavity may involve the buccal mucosa, the gingiva (gingivitis), the tongue (glossitis), and the peridontal tissues (peridontitis). therapeutic agents can induce inflammatory lesions in the tongue. moreover, a protective layer of mucus, a visco-elastic material containing high molecular weight glycoproteins produced by the major and minor salivary glands, covers the stratified squamous mucosa of the oral cavity. salivary secretions also possess digestive enzyme activity although in herbivores and carnivores, it is usually low in contrast to high digestive enzyme activity in omnivorous species. the oral mucosa can be damaged by excessive local trauma from foreign materials, hard fragments in food and damaged teeth may produce ulceration of the mucosa with subsequent infection. however, the oral mucosa may show manifestations of local or systemic disease or derangement produced by therapeutic agents. excessive contact by therapeutic agents such as aspirin, potassium supplements and corticosteroids have been reported to produce local ulceration in the mouth (zentler-monro and northfield, ) . the increased use of mouthwashes over the last years has resulted in a number of reported adverse effects to the buccal mucosa in people (gargari and kabani, ) . systemic disorders produced by anticoagulants or chemotherapeutic drugs may also be evident by bleeding or ulceration in the oral cavity (goepp, ) . buccal ulceration is also described as part of a generalised hypersensitivity reaction to drugs (zentler-monro and northfield, ) . the major and minor salivary glands and their secretions also represent and integral part of the protective mechanism of the oral cavity and derangement of saliva production may lead to loss of integrity of the oral mucosa. drugs that effect motor co-ordination can give rise to drooling and disruption of cricopharyngeal co-ordination (wyllie et al., ) . drug-induced abnormalities of taste sensation are also well-described phenomena occurring in man although human studies are necessary for the detection of these effects. indeed, many alterations in the oral mucosa are those that are more readily detected by careful clinical and macroscopic observation rather than exhaustive histopathological examination of the buccal mucosa in laboratory animals -provided the basic toxicity profile of a novel agent is adequately assessed in the usual preclinical studies. oral irritation studies are used in the testing of products for use in the oral cavity, mainly for surgical, dental and hygiene purposes but also therapeutic agents administered by the sublingual route. this route may be selected for substances that are broken down in the stomach or show a rapid first pass effect. as it is technically not feasible to perform full preclinical toxicity studies by the sublingual route, conventional oral or parenteral routes are preferred for systemic toxicity studies on such compounds. the choice of the best route will to a large extent be dictated by pharmacokinetic considerations. however, it is necessary to assess local irritancy potential to oral mucosa using a laboratory animal model. test species for oral irritation studies are usually rats, hamsters (cheek pouch), guinea pigs, dogs or primates using gross and histopathological assessment. a similar scheme to that employed in the histological assessment of skin irritancy is appropriate. inflammation of the oral cavity (stomatitis) may involve the buccal mucosa, the gingiva (gingivitis), the tongue (glossitis) and the peridontal tissues (peridontitis). although inflammatory lesions are found sporadically in untreated laboratory rodents, dogs and primates, stomatitis can be induced by systemic administration of high doses of therapeutic agents. anticancer and antimitotic agents are particularly liable to induce stomatitis. a notable example is bleomycin that is capable of producing stomatitis as part of its general effect on squamous cells (thompson et al., ) . in humans, the adverse effects on therapeutic ionising radiation on the salivary glands may also give rise to inflammatory changes in the oral cavity (fox, ) diuretics and other agents, which are capable of producing severe electrolyte disturbances and uraemia at excessive doses, can also produce stomatitis when then are administered in high doses to laboratory animals (garthoff et al., ) . these lesions may be analogous to the well-described association of ulcerative stomatitis and uraemia in man and laboratory animals (boyd, ; barker and van dreumel, ) . the dog appears very sensitive to the ulcerogenic effects of uraemia in the oral cavity, although as there is a poor correlation between actual levels of blood urea and stomatitis, other biochemical factors are undoubtedly involved. compounds, which effect pigmentation of the skin, can produce similar changes in pigmented oral mucosa. a number of drugs including chlorpromazine, quinacrine, chloroquine, amodiaquine and pyrimethamine cause pigmentation of the oral mucosa in man notably over the hard palate. chloroquine and pyrimethamine have also been shown to significantly increase numbers of active melano-cytes within the palatal mucosa of pigmented da rats when treated orally for weeks (savage et al., ) . melanocytes in treated rats were shown to be enlarged and packed with melanin pigment and to possess extensive arborisation of cell processes between squamous cells. an experimental inhibitor of platelet aggregation, which produced pigment loss in the dark hair of long-evans rats and the skin of beagle dogs, also induced pallor of the normally pigmented oral mucous membranes in dogs (gracon et al., ; walsh and gough, ) . apart from loss of pigment, the histology of the mucous membranes and skin was normal. the tongue is conveniently sectioned for histological study, although reliance is often placed on careful visual inspection, because the usefulness of systematic histological examination of the tongue in routine preclinical safety studies has not been clearly established. a few lesions occur which are fairly specific to the tongue. amyloid may become deposited in the muscular and connective tissue of the tongue in amyloid-prone species, particularly mice (dunn, ) . mice, especially dba and dba/ ncrj strains, are liable to develop calcification in the lingual muscle spontaneously, even at a young age (imaoka et al., ) . calcified lesions are seen in the longitudinal muscle under the dorsolateral epithelium and the central part of the tongue, which, when severe, are associated with inflammation, granulation tissue, polypoid change, hyperplasia of the overlying squamous epithelium and ulceration. the histogenesis of this lesion is uncertain. in the dba/ ncrj mice, mineralisation of the tongue is associated with myocardial and aortic mineralisation (doi et al., ) . therapeutic agents can induce inflammatory lesions in the tongue. an example is provided by the investigational anticancer immunotoxin, zd , a mouse monoclonal antibody (c ) against colorectal carcinoma antigen conjugated to recombinant ricin a-chain. when administered to wistar-derived rats, this agent produced myocyte necrosis and inflammation specifically located below the ventral subepithelial surface of the tongue (westwood et al., ) . as the changes were different to the low grade myositis seen elsewhere in treated animals, these authors speculated that the changes in the tongue may have been related to the particular receptor profile of this area targeted by the monoclonal antibody. in common with other changes induced in the digestive tract of rats and cynomolgus monkeys by the administration of recombinant human epidermal growth factor, the tongue showed squamous epithelial hyperplasia characterised by a uniform increase in the thickness of the squamous epithelium in both species (breider et al., ; reindel et al., ) . at high doses, the squamous epithelium of the tongue of the primates was twice the thickness of control mucosa associated with elongation of rete pegs. teeth are usually only inspected by naked eye in conventional toxicity studies and this is appropriate for the assessment of a mature dentition. however, there has been an increasing awareness of dental lesions in toxicity studies, particularly as the teeth are visualised when the maxilla is examined histologically in inhalation studies. study of the rodent dentition in inhalation studies has shown that spontaneous lesions of the dentition are quite common. in one laboratory, malformations (dental dysplasia) of the maxillary incisors were observed in % of female and % of male cd- mice and . % female and . % sprague-dawley rats in and month inhalation studies respectively (losco, ) . the rat incisor and its pathology has been the subject of an excellent review (kuijpers et al., ) . unlike in humans, the rodent incisor continues to grow and differentiate throughout life and is renewed every - days. located at the centre of the tooth is the vascular pulp. this is surrounded by proliferating ondotoblasts which form predentin which when calcified becomes dentin. surrounding ameloblasts when induced by the presence of dentin produce the overlying enamel layer. it is these active cellular layers, which can be modified or damaged by xenobiotics, vitamin deficiencies, calcium, phosphate or magnesium deficiency, parathyroidectomy, hypophysectomy, hyperparathyroidism, adrenal insufficiency and fluorosis (kuijpers et al., ) . although in humans the mature dentition is no longer growing, in children the dentition is in a growth phase that starts in utero and lasts into the second decade. as increasing numbers of children survive malignant disease, damage to the mature dentition can occur as a result of cytotoxic therapy during childhood. clinical study of the teeth of children treated for malignancy have shown increased incidence of enamel hypoplasia and missing teeth (welbury et al., ) . histological examination of teeth from children treated with vincristine or combination chemotherapy for malignant disease has demonstrated prominent incremental lines in dentine correlating with the number of times the intravenous cytotoxic agents were administered (macleod et al., ) . it has also been shown that vincristine, a drug which interferes with the assembly of microtubules and reduces secretory activity in a number of cells including osteoblasts and chondroblasts, also effects dentine formation in the rat incisor (stene and koppang, ) . two weeks following a single intravenous dose of vincristine to young adult rats, a faint incremental line in the dentine was observed, probably a reflection of a direct effect of the drug on the dentinogenic tissue at the time of injection. at higher doses, focal niche-like or punched out defects in dentine were observed, expression of more severe injury to highly sensitive dentinogenic populations at the time of injection (stene and koppang, ). the precise mechanism of damage is not fully understood although decreased secretion of dentine matrix by odontoblasts has been demonstrated. calcification appears unaltered (stene and koppang, ) . administration of the alkylating anticancer agent cyclophosphamide or a sin-gle exposure to ionising radiation, produces localised niche-like or punched out defects in the rat incisor, rather than the more diffuse changes induced by vincristine. this presumably reflects more localised injury to a sensitive subpopulation of dentinogenic cells (koppang, ; vahlsing et al., ) . anticonvulsant drugs also produce changes in the dentition of man and experimental animals. in humans, reported alterations include tooth root resorption, small teeth, delayed shedding of deciduous teeth and retarded eruption of permanent teeth, features similar to those found in hypoparathyroid or pseudohypoparathyroid conditions (robinson et al., ) . tooth root alterations were also reported in a study in which young male wistar rats were treated with diphenylhyantoin for month. treated rats showed evidence of molar root resorption lacunae that penetrated the cementum and involved the dentine. the lacunae contained a dense infiltrate of cells contiguous with similar cells in the surrounding periodontal ligament. robinson and harvey ( ) showed that the changes were similar to those occurring in parathyroidectomized rats but not those in rats made hypocalcaemic with a calcium deficient diet. they suggested that the changes induced by diphenylhydantoin in rats were similar to those in pseudohypoparathyroidism in which resistance of tooth roots to resorption is reduced. discoloration of teeth and bone is a well-described side effect of tetracycline administration and it has also been reported in patients treated with the semisynthetic derivative, minocycline (cale et al., ) . interestingly the ameloblastic epithelium of the enamel forming tissues of growing incisors in wistar rats treated with high doses of human recombinant epidermal growth factor showed hyperplasia characterised by pseudostratification, increased nuclear-cytoplasmic ratio and increased cytoplasmic eosinophilia (breider et al., ) . this finding is consistent with the presence of epidermal growth factor receptors in the cells of the enamel organ (martineau et al., ) . periodontitis is a common and important disease in man and animals although overt cases are not usually seen in toxicity studies. however, periodontitis of a degree sufficient to disrupt chronic rat toxicity and carcinogenicity studies has been reported. robinson ( ) described periodontitis in alpk/ap rats in which there were erosive granulomatous cavities adjacent to molar teeth with fistulas opening into the nasal cavity. these changes were associated with penetrating food fibres in the gingival sulcus and it was suggested that the presence of long pointed food fibres in the powdered diet was the main reason for occurrence of periodontitis. peridontitis in rodents also results from the effects of dental pathology such as fractures, malformation or malposition of incisors (losco, ) . drug-induced overgrowth of the gingival tissues is a well-described phenomenon in both humans and laboratory animals including dogs, cats, and rats. in man, these changes have been associated with diphenylhydantoin (phenytoin) (beghi et al., ) nifedipine, calcium channel blockers (ledermann et al., ) , cyclosporin a (barthold, ) and valproic acid (syrjamen and syrjamen, ) . cyclosporin a, diphenylhydantoin and calcium channel blockers have been associated with similar changes in laboratory animals (do'nascimento et al., ; latimer et al., ; waner et al., ) . in most instances there is swelling of the gingiva by firm nodular overgrowths around the teeth. histologically, these overgrowths are characterised by marked acanthosis of the squamous epithelium overlying connective tissue that is infiltrated by large numbers of chronic inflammatory cells. fibrovascular proliferation may be marked. in patients treated with cyclosporin, myxomatous degeneration is described in association with dense infiltration of plasma cells and lymphocytes (barthold, ) . secondary acute inflammation in association with food debris and hair shafts is described in dogs treated with oxodipine (waner et al., ) . the forces behind these changes are unclear. studies of changed induced by nifedipine and hydantoin have shown increases in extracellular ground substance and increased numbers of fibroblasts containing sulphated acid mucopolysaccharides (kantor and hassel, ; lucas et al., ) . these drugs may alter fibroblastic proliferative and synthetic activity, possibly by selection of a subpopulation of fibroblasts (hassel et al., ) . it has also been suggested that an underlying mechanism in phenytoin-induced gingival hyperplasia involves the decrease in salivary iga that develops in some patients (beghi et al., ) . study of cyclosporin a-induced changes have suggested that impairment of t lymphocyte function may permit overgrowth of oral bacteria and bacterial products which may influence fibroblast function (barthold, ) . a spontaneous form of gingival hyperplasia has been described in non-human primates (macaca mulata). this is characterised by an enlargement of the marginal and alveolar gingiva by connective tissue consisting of relatively poorly cellular bundles of collagen fibres. the lesions show little inflammatory alterations and the overlying squamous epithelium shows mild hyperkeratosis only (schiødt et al., ) . this pathology is similar to hereditary gingival fibromatosis in humans. sessile or pedunculated squamous papillomas and infiltrating squamous carcinomas are occasionally found in the oral cavity of most laboratory animals including rodents (odashima, ; emminger and mohr, ; leiniger and jokinen, ; takahashi and okamiya, ; mohr, ) , rabbits (sundberg et al., ; sundberg and everitt, ) , and beagle dogs (watrach et al., ) . the microscopic structure of these neoplasms in rodents resembles those occurring in squamous epithelium in other sites. although a number of agents induce squamous neoplasms in the oral cavity, spontaneous squamous carcinomas are generally uncommon spontaneous lesions in laboratory animals. however, some strains of rodent may develop squamous neoplasms more commonly. for instance, in life time studies ad libitum fed brown-norway rats, % of males and % females developed oral squamous cell carcinomas although only % and % in food-restricted animals respectively (thurman et al., ) . it was suggested that certain pedigrees possessed a genetic predisposition to these neoplasms. papillomas occurring in rabbits and dogs are of note because they can occur in quite young animals, apparently as a result of infection with viruses of the papilloma group. viral inclusions may be seen in histological sections. the implications of papilloma viruses in laboratory species are that the progression of virally induced papillomas to malignant squamous carcinomas can be potentiated by nonviral factors including application of xenobiotics (howley et al., ) . in rabbits, the prevalence of oral papillomas varies considerably but they are quite common in some laboratory strains. they are overlooked because of their small size and a distribution limited to the ventral surface of the tongue (sundberg et al., ) . microscopically, they are typical squamous papillomas composed of irregular acanthotic squamous epithelium and a fibrovascular stalk of variable size. squamous cells at the margins of papillomas at the junction with normal mucosa, often show large, oval nuclei, marginated chromatin and central, basophilic, intranuclear inclusions, which electron microscopic examination shows to contain viral particles. oral papillomas in dogs develop as multiple growths, regressing spontaneously after a few months. they are also caused by a virus of the papilloma group, which possesses a high degree of specificity for the mucosa of the oral cavity and adjacent skin (watrach et al., ) . histologically, they are composed of proliferative masses of epithelial cells, keratinised on the surface and resting on an irregular connective tissue stroma or pedicule. large vesicular cells with basophilic intranuclear inclusions are also found in the granular cell layer, identifiable as virus arrays by electron microscopy (cheville and olson, ) . malignant change has been described in these canine lesions and this can occur in young beagle dogs (watrach et al., ) . although many types of papilloma viruses have been identified in both man and animals (pfister, ) , common antigenic determinants exist between viruses in different species. this immunological cross-reactivity can be exploited in the immunocytochemical localisation of papilloma viruses in epithelial lesions of many animal species. papilloma virus antigen has been demonstrated in oral papilloma of dogs and rabbits using antisera to bovine papilloma virus type i (sundberg et al., ) . cells positive for virus and viral inclusions are located in the upper layers of the epithelium, especially within cells of the granular layer. spontaneously developing odontogenic tumours are rare in rodents but they have been induced in laboratory animals given carcinogens such as nitrosoureas or exposed to ionising radiation (gössner and luz, ) . a range of tumours originating from dental tissues with epithelial, mesenchymal or mixed appearances has been reported in rodents (kuijpers et al., ) . the classification of odontogenic tumours is complex and confusing. they range from benign anomalies and cystic structures through to malignant neoplasms. the ameloblastoma comprises cords, nests, anastomosing strands or islands of ondontogenic epithelial cells within a fibrous stroma. the tumour cells resemble ameloblasts with the cords of spindle shaped cells similar to the stellate epithelium bounded by a peripheral layer of cuboidal or columnar cells resembling the inner enamel epithelium. other tumours of the odontogenic epithelium show induction of the mesenchymal elements or develop a complete sequence of odontogenic epithelium, odontogenic mesenchyme and dental hard tissues including dentine, enamel and cementum. in the rat these have been classified as odontoma characterised by the presence of all dental hard tissues and odontogenic fibroma composed of undifferentiated or primitive mesenchymal cells of developing dental tissue (mohr, ) . odontogenic tumours developing in fischer rats treated with aflatoxin were located in the upper jaw associated with the incisor teeth and were composed of proliferating fibroblast-like cells within which ovoid calcified bodies resembling cementum were seen (cullen et al., ) . occasional inclusions of solid epithelial nests were also seen. no metastatic deposits were found although the neoplasms were locally aggressive. in addition, squamous tumours and neoplasms of mesenchymal origin typical of other organs, bones and soft tissues are found in this region. although salivary glands may not represent vital organs in the same sense as the kidneys or heart, severe derangement of their secretions can alter both the quality and quantity of saliva. depending on the particular glands and cells affected, dry mouth, mucositis, and dental caries may develop . the severe oral complications of irreversible salivary damage and dysfunction, which can occur patients with head and neck cancer as a consequence of local irradiation, may have a significant impact on the efficacy of therapy, quality of life and survival (fox, ) . a protective layer of mucus, a visco-elastic material containing high molecular weight glycoproteins produced by the major and minor salivary glands, covers the stratified squamous mucosa of the oral cavity. these mucins usually contain more than % carbohydrate in the form of neutral and acidic oligosaccharide chains, o-glycosidically linked to threonine or serine. mucins possess several roles including mechanical flushing of the oral cavity, protection and lubrication of soft and hard tissues, modulation of oral microbial flora, buffering activity, regulation of calcium/phosphate equilibrium, digestion and extracellular post translation processing of molecules present in saliva . the heterogeneity of salivary glycoproteins suggests that they act as a defence against pathogenic microorganisms by competing with microbial binding sites of similar structure on the surface of cells lining the digestive tract (schulte, ) . minor salivary glands may also play an important part in the local immunosurveillance of the oral cavity for their ducts are anatomically closely associated with lymphoid tissue (nair and schroeder, ; nair et al., ) . salivary secretions also possess digestive enzyme activity although in herbivores and carnivores, it is usually low in contrast to high digestive enzyme activity in omnivorous species (junqueira et al., ) . the phylogenetic association of the salivary glands with the thyroid gland is evident functionally because salivary glands are capable of concentrating iodide in their secretions, although this is not under control of thyroid stimulating hormone (ingbar, ) . it has been shown that thyroxine accelerates the differentiation of the granular convoluted tubule cells and the appearance of epidermal growth factor in the submandibular gland of the neonatal mouse (chabot et al., ) . the structure of the salivary glands differs among laboratory species, between different glands in the same species and between sexes. it is usually considered that there are three major salivary glands, the parotid, the sublingual and the submandibular (submaxillary) glands. minor salivary glands are scattered in other locations throughout the mouth and oropharynx. in dogs and other carnivores, the zygomatic (infra-orbital) gland, located just below the zygomatic arch and the buccal (molar) gland are also often referred to as major salivary glands. microscopically, salivary glands are composed of secretory glands or 'endpieces' attached to a connecting system of intralobular and extralobular (secretory) ducts. secretory endpieces may be acinar or tubulo-acinar in nature. the secretory cells have been subdivided into serous, mucous, seromucous and special serous types. controversy remains about the precise nature of the secretory cells found in the various salivary glands of different species and this makes critical interspecies comparisons difficult (see detailed discussion of this problem by pinkstaff, ). the duct system is less complex. this comprises an intercalated duct which leads from the secretory endpiece into a striated (secretory or intralobular) duct, so termed because their lining cells are striated by delicate eosinophilic cytoplasmic rods. the striated ducts converge into interlobular ducts and a main excretory duct system. in rats, mice and hamsters, an overall similarity in gross and microscopic anatomy of the various salivary glands exists although there are histochemical differences (munhoz, ; glucksmann and cherry, ; dawe, ; pinkstaff, ; emmiger and mohr, ) . moreover, it has been demonstrated that salivary glands in rodents as well as a number of other species show morphological and histochemical sexual dimorphism (pinkstaff, ) . the sublingual gland in rats, mice and hamsters is composed principally of mucous acini, with indistinct serous demilunes. acini open into fairly long intercalated ducts lined by flat or cuboidal cells devoid of granules. the parotid gland is composed of serous-type secretory cells containing zymogen granules and prominent hyperchromatic basal cytoplasmic poles. the submandibular gland is anatomically the most complex salivary gland in rodents. secretory endpieces are composed of small or moderately sized cells with foamy cytoplasm and basophilic basal poles. the most striking feature is the presence of an additional duct segment interposed between the intercalated and striated ducts. this segment is lined by cylindrical epithelium with basal nuclei and eosinophilic cytoplasm containing secretory granules. this duct segment is termed the granular duct or granular convoluted tubule. these granular cells are of special interest because they contain a large number of heterologous biologically active peptides including nerve growth factor, epidermal growth factor, renin, and kallikrinins (barka, ; mori et al., ). the precise physiological role of many of these peptides in salivary gland remains uncertain. epidermal growth factor was originally isolated from the mouse salivary gland. it initiates premature eyelid opening and incisor eruption when injected into the neonatal mouse (cohen, ) . study of the mouse submandibular gland has shown that both epidermal growth factor and nerve growth factor are released into saliva following the administration of phenylephrine, sympathomimetic amine acting mainly on αreceptors and isoprenaline (isoproterenol), a β-adrenergic agent (murphy et al., ) . immunohistochemical study also demonstrates that epidermal growth factor becomes depleted in mouse salivary tissue following administration of phenylephrine and similar agents (tsukitani and mori, ) . phenylephrine has been shown to cause marked secretory activity accompanied by loss of granules from granular cells, as well as loss of immune reactive carbonic anhydrase, an enzyme which participates both in membrane transport of bicarbonate ions into saliva and glandular secretion (noda, ) . morphological studies have shown that both acinar and granular tubular cells participate in this response to adrenergic agents (murphy et al., ) . this is in contrast to the effects of pilocarpine, a cholinergic agent, which elicits the secretion of saliva deficient in serous proteins with little or none of the growth factors, as its effects are more limited to acinar cells. glycoprotein secretion of rodent salivary glands has stimulated histochemical study using both conventional mucin histochemical techniques and labelled lectins which possess affinity for specific sugars or sugar sequences (tables and , pages and ). studies of rat, mouse and hamster salivary glands using batteries of labelled lectins have shown a greater heterogeneity of oligosaccharides in salivary glands than seen by classical histochemical techniques. there are considerable species differences and variations between murine strains and sexes of the same strain as well as heterogeneity among morphologically similar cells within one gland (schulte and spicer, , ; schulte, ) . the results of histochemical studies are in excellent agreement with studies using biochemical methods but suggest a significant influence of genetic and hormonal factors on the synthesis of salivary glycoproteins. less attention has been paid to the structure and cytochemistry of the dog salivary glands. there appears to be little variation between the structure of salivary tissues between beagles and other strains although variation with age has been reported (reifel and travill, ; nagoyo and tandler, ) . munhoz ( ) has described the histochemical features of the dog parotid gland. the dog parotid is of seromucinous type secreting both acidic and neutral mucosubstances, in contrast to the more neutral mucosubstances secreted by rodent glands. the salivary glands of non-human primates are similar to those in man. they possess parotid glands of serous or seromucous type, submandibular glands with both serous and mucous acini and sublingual glands of mainly mucous type. the salivary glands of the non-human primate react to adverse stimuli such as ionising radiation in a similar manner to human salivary tissue . focal chronic inflammation of the salivary glands occurs sporadically in untreated rats, mice, hamsters, dogs or primates employed in toxicology although severity and prevalence is variable. sialoadenitis as a result of a corona virus, the sialodacryoadenitis virus, is a well-known and fairly ubiquitous condition in rats, first described by innes and stanton ( ) . the condition is characterised histologically by oedema and congestion of submandibular and parotid salivary glands as well as extra-orbital lachrymal and harderian glands. it is accompanied by inflammation of variable severity and chronicity in both glandular and connective tissue as well as degeneration and necrosis of duct epithelium (fig. ) . the regenerative hyperplasia of the duct epithelium may be quite intense about a week after infection but all changes regress after about weeks and glands are essentially normal after or weeks (carthew and slinger, ; percy and wojcinski, ) . there may be a delay in the appearance of inflammatory cells and the onset of repair in rats immunosuppressed with cyclophosphamide (hanna et al., ) . depletion of salivary gland epidermal growth factor also occurs during the infection (percy et al., ) . suppurative infections in the neck region of the rat such as those produced by klebsiella aerogenes also cause acute and chronic inflammation of salivary glands with fibrosis and glandular proliferation of salivary tissue (arseculeratne et al., ) . sialadenitis occurs spontaneously in autoimmune-prone strains of mice such as the nzb/nzw and sl/ni strains and it has been reported in ageing female, but not male bdf mice (hayashi et al., ) . the non-obese diabetic mouse known for its spontaneous insulin-dependent diabetes mellitus also develops immune mediated damage to submandibular glands (fujino-kurihara et al., ; törnwall et al., ) . in ageing bdf females the submandibular gland was shown to be involved by a destructive inflammatory process characterised by an intense infiltration by small and medium sized lymphocytes, associated with mild inflammation in other organs such as the parotid and sublingual glands, pancreas and kidney. immunocytochemistry showed that most of the lymphocytes were t cells (thy- . and lyt- positive) of the helper/inducer subset (l t or cd positive) and less than % were of suppresser/cytotoxic (lyt- or cd fig. . section from salivary tissue from a sprague-dawley rat during an infection with the sialodacryoadenitis virus showing intense ductular inflammation. (he, × .) positive) type (see haemopoietic and lymphatic systems, chapter iii). circulating anti-salivary duct antibody of igg was also detected in afflicted mice. it was suggested that helper/inducer t cells played a key role in the production of this change, unlike induced autoimmune sialoadenitis in which cytoxic t-cell subsets may directly destroy glandular tissue. it has been suggested that this process in ageing females is related to the decline in the number of splenic lyt- cells in mice with advancing age (hayashi et al., ) . these cells are believe to be the most susceptible to ageing (see haemopoietic and lymphatic systems, chapter iii) in the non-obese diabetic strain of mouse derived from jcl-icr mice, a periductal chronic inflammatory infiltrate is found in the submandibular gland at about the same time that immune-mediated insulitis is most marked. this suggests that there is an extension of the autoimmune process to salivary tissue (fujino-kurihara et al., ) . it is probable that helper/inducer cd t cells are essential components of this infiltrate and a number of cytokines and their receptors such as ip- (interferon-γ inducible protein ) and rantes (regulated upon activation normal t cell expressed and secreted) may have an important role (törnwall et al., ) . an autoimmune type of sialoadenitis can also be experimentally induced certain strains of mice. crj:cd mice, thymectomized at days, a time point at which lyt- positive cells (cd suppresser t lymphocytes) can be maximally reduced, followed by immunisation at and days after birth with homogenates of salivary gland and complete freund's adjuvent, develop a distinctive sialoadenitis in the submandibular and to some extent the parotid glands (hayashi et al., ) . this sialoadenitis is characterised by degenerative changes in salivary glandular tissue associated with an extensive and intense infiltrate of small and medium sized lymphocytes. these cells appear shortly after immunisation but increase in number with time. immunocytochemical study has shown that many of these cells are reactive to antisera to thy- . and lyt- (cd ) features of suppresser/cytotoxic t lymphocytes. later appearing cells demonstrate features of plasmacytoid lymphocytes and contain immunoglobulin of mainly igg class (hayashi et al., ) . these authors therefore suggested the sialoadenitis appeared as both a result of cytotoxic/suppresser t-cell activity and an antibody-dependent cell-mediated cytotoxicity. in the hamster salivary glands, interstitial infiltrates of lymphocytes and plasma cell are quite common and may become more marked with advancing age (mcmartin, ) . whereas necrosis of the parotid gland of uncertain aetiology sometimes occurs in the dog, mild focal chronic inflammation is quite a common incidental finding in canine salivary glands and has been reported in about % of normal beagle dogs (kelly et al., ) . although the inflammation in salivary tissue which results from ionising radiation is only indirectly relevant to drug safety evaluation, it is of interest in view of the notable species differences in sensitivity to this form of insult. serous acinar cells in man and rhesus monkey appear least resistant to the effects of ionising radiation, where damage is characterised by widespread degranulation and degeneration of acini, infiltration by polymorphonuclear cells followed by lymphocytes, plasma cells and subsequent atrophy and fibrosis . these changes contrast with the lesser effects of ionisation radiation on the rodent salivary glands in which there is little or no acute inflammatory response. lymphoid bodies are sharply circumscribed collections of lymphoid cells generally located between the parotid and sublingual glands close to a cervical lymph node in mice. they are apparently normal aggregates of lymphoid tissue. like many other glandular organs, the size of the secretory tissue of the salivary gland is responsive to functional demand and is subject to age-related changes. in man, the gland parenchyma frequently becomes atrophic and replaced by connective tissue or fat with advancing age, possibly partly related to vascular changes (waterhouse et al., ; scott, ) . in ageing rats, the extent and height of granular ducts and their content of mature secretory granules has also been shown to decrease with age (sashima, ) . dietary factors influence salivary gland size. decreased food consumption or protein starvation can reduce the weight of salivary glands in rats. there is shrinking of mucous and serous glands and loss of zymogen granules associated with decreased rna but unchanged dna content, attributable to the reduced requirements for protein synthesis , mcbride et al., . as salivary gland function is responsive to adrenergic stimulation, it is not surprising that atrophy occurs following adrenergic blockade. the weights of the submandibular gland in mice were shown to decrease following administration of the β-adrenergic blocking agent, propranolol (smith and butler, ) . this was associated with a reduction in stainable neutral mucins and a decrease in the thickness of the acinar cells making the gland lumens appear larger than normal. the cytotoxic agent, alloxan, known primarily for its specific effect on pancreatic b cells, has also been shown to produce weight loss of the rat submandibular gland, associated with lipid inclusions in the acinar cells, capillary basement membrane thickening and reduced salivary flow (reuterving et al., ) . it is probable that alloxan exerts a cytotoxic effect on the acinar cells of the rat submandibular gland (sagström et al., ) . methotrexate, a folic acid antagonist, has also been reported to cause vacuolization of acinar and ductular cells with reduction of secretory granules in rat salivary glands (mcbride et al., ) . ligation of the main excretory ducts has frequently been used as an ex-perimental model for study of salivary gland atrophy as well as the regeneration that follows removal of the ligature. there is marked atrophy of all cell types but most markedly the acinar cells through apoptosis. although overt necrosis has been reported following ligation of the excretory duct, it appears that this may have been the result of constriction of the vasculature for acinar cells are relatively intolerant to a decrease in oxygen and nutrient (denny et al., ) . a number of therapeutic agents increase salivary gland size in man, although the scarcity of biopsy data precludes a critical assessment of the precise mechanism in many cases. drugs reported to produce salivary gland enlargement in man include iodide-containing radiological contrast media, isoprenaline and anti-inflammatory agents phenylbutazone and oxyphenbutazone. enlargement may also occur after endotracheal anaesthesia and upper gastrointestinal tract endoscopy in man (riddell, ) . some of these agents and procedures may produce spasm of large salivary ducts and retention of secretions. several pharmacological agents, particularly sympathomimetic amines, have been shown to produce increases in salivary gland size in rodents following repeated dosing (brenner and stanton, ) . there is an intimate relationship of sympathomimetic amines with the control of the secretory process in salivary tissue. whereas a single injection of isoprenaline (isoprotorenol) in the range of - mg/kg induces discharge of preformed secretory granules followed by gradual re-synthesis and reconstitution, repeated injections produces an increase in the size of salivary glands (simson et al., ) . histologically, the enlarged glands are composed of secretory cells of increased size that contain increased amounts of secretory substances in the cytoplasm (simson et al., ) . although these histological features are principally those of diffuse cellular hypertrophy, the increase in dna content and radioactive thymidine uptake described in the salivary tissue following repeated administration of isoprenaline suggests that a degree of hyperplasia also occurs (barka et al., ) . these effects do not depend on the integrity of the autonomic nerves because they occur after ablation of the autonomic ganglia (barka et al., ) . they appear to be mediated by an effect on adrenergic β-receptors. the effects can be blocked by propranalol, a β-receptor antagonist but not by phenoxybenzamine, an α-receptor antagonist (brenner and stanton, ) . as theophylline and caffeine also elicit salivary gland enlargement in rats, a role for cyclic ', -adenosine monophosphate (camp) in salivary gland enlargement has been postulated (brenner and stanton, ) . detailed study of hypertrophy, protein synthesis, and intracellular camp activity in the salivary glands of rats treated for days with isoprenaline (isoproterenol), a series of β-adrenergic receptor agonists and the phosphodiesterase inhibitors, theophylline and caffeine, showed that similar effects occurred with all agents although differences in the degree of hypertrophy, the nature of pro-tein and glycoprotein synthesis and golgi membrane enzyme activity were recorded (wells and humphreys-beher, ) . the parotoid gland showed the most pronounced hypertrophy followed by the submandibular gland but the sublingual gland appeared to be unaffected by treatment. the degree and nature of the changes induced by the various β /β receptor agonists suggested that most of these effects were mediated through β receptors which are present in greatest numbers on the parotid and salivary cells. it was suggested that the effects of β-adrenergic agonists on salivary gland are produced by a receptor-mediated stimulation of adenylate cyclase activity causing an increase in levels of intracellular camp. however, other factors may be important for wells and humphreys-beher ( ) also showed that although isoproterenol and caffeine increased salivary cell camp to comparable levels, the hypertrophy was greater with isoproterenol. cardioactive phosphodiesterase inhibitors were shown to produce submaxillary hypertrophy in rat subacute toxicity studies (rogers et al., ; jayasekara et al., ; smith et al., ) . parotid and submaxillary glands were those most affected by the inotropic phosphodiesterase inhibitor ici , (westwood et al., ) . as the agents produced their positive inotropic action via selective inhibition of the cardiac phosphodiesterase subfraction iii specifically requiring camp as its substrate, it was suggested that the salivary gland hypertrophy was a result of phosphodiesterase inhibition (smith et al., ) . other classes of drugs can also produce salivary gland enlargement in rats in repeated dose studies. doxylamine, a representative of the widely used ethanolamine group of antihistamines, has been reported to produce marked cytomegaly in the fischer rat parotid gland. enlarged cells were characterised by a basophilic and coarsely granular or vacuolated cytoplasm (jackson and blackwell, ) . the b c f mouse did not develop these changes after a similar treatment schedule. in view of the presence of considerable amount of epidermal growth factor in salivary glands, it is of interest to note the effects of its administration to laboratory animals. salivary gland weights were increased in rats and cynomolgus monkeys infused with high doses of recombinant human epidermal growth factor (breider et al., ; reindel et al., ) . however histological features seen are primarily those of ductular epithelial hyperplasia (see below under hyperplasia). epithelial cells characterised by abundant granular eosinophilic cytoplasm as a result of the accumulation of mitochondria are often referred to as oncocytes, a term used by hamperl ( ) to describe similar cells in hürthle tumours of the thyroid gland. they may be found in various focal nodular and neoplastic states of the salivary glands in both man and laboratory animals. the precise significance of these cells is uncertain. the mitochondria usually appear unremarkable except for lack of dense granules and it has been suggested that the mito-chondrial changes represent an adaptive phenomenon or compensatory hyperplasia (ghadially, ) . in human salivary tissue their prevalence seems to increase with advancing age and they can be associated with hyperplastic lesions or neoplasms such as oxyphil adenomas and adenolymphomas. eosinophilic cells also occur in the salivary glands of certain strains of aged rats (bogart, ) and in mice with experimentally induced autoallergic sialoadenitis (takeda et al., ) . in the study of takeda et al. ( ) the eosinophilic cells appeared to arise predominantly in the secretory (glandular) ducts of the submandibular glands, although eosinophilic cells can apparently develop from either duct or acinar cells. well-defined, unencapsulated foci of enlarged acinar cells occur spontaneously in the salivary glands, particularly the parotid of rats, mice (chiu and chen, ) , and hamsters although their reported incidence varies between laboratory. the enlarged cells possess greatly expanded cytoplasmic volume that retains a vesicular, vacuolated or foamy appearance or possesses a pale eosinophilic granular texture. the basal parts of the cells usually stain intensely blue in haematoxylin and eosin stained sections and contain large, dense, irregular hyperchromatic or pyknotic nuclei showing little evidence of mitotic activity. although there has been little ultrastructural study of these foci, the cytoplasmic alterations appear to be distinct from those of so-called oncocytes that characterised by granular eosinophilic cytoplasm packed with mitochondria. the biological nature of these foci is uncertain. the lack of any prominent mitotic activity, cell proliferation or expansive growth suggests that they are most aptly regarded as hypertrophic lesions (chiu and chen, ). although they increase in prevalence with increasing age in certain strains of rat, there is no evidence to suggest that they represent pre-neoplastic lesions or possess any relationship with development of neoplasia in salivary tissue (dawe, ) . hyperplasia and squamous metaplasia of the salivary ducts are common features of many inflammatory and reactive conditions in the salivary glands of rodents, dogs, non-human primates and man and can be associated with the presence of stones and calculi with the duct system. squamous metaplasia and regenerative change in the ducts occurs in rats afflicted with sialodacryoadenitis (carthew and slinger, ) . it is also described specifically located in the ducts of the sublingual glands in the wistar rat in the absence of obvious sialodacryoadenitis or evidence of any specific disease. similar regenerative hyperplastic duct changes are also seen in necrotic and inflammatory conditions in the dog salivary gland . detailed morphological examination with immunocytochemical study of epi-dermal cytokeratins of the rat salivary gland after arterial ligation has shown that the acinar units can also undergo squamous metaplasia (dardick et al., ) . it appears that the acinar-intercalated duct complexes can rapidly reprogram to produce epidermal cytokeratin filaments in ischaemic or inflammatory states. hyperplasia of the ductular epithelium appears to be the principle result of the administration of epidermal growth factor to rats and cynomolgus monkeys. in rats histological features were primarily of ductular epithelial hyperplasia without evidence of significant acinar hyperplasia (breider et al., ; reindel et al., ) . in primates the changes were most striking in the interlobular and large intralobular ducts where the epithelium showed multilayered and papilliform projections. however, mitotic activity was evident throughout the duct epithelia and acinar cells showed hypertrophy with depletion secretory granules and the presence of large vesicular nuclei . focal duct and acinar hyperplasia, showing minimal compression of the surrounding parenchyma and distinct from focal hypertrophy is also described in the classification of rat salivary lesions (mohr, ) . primary neoplasms of salivary glands are uncommon in the usual strain of rats and mice employed in carcinogenicity bioassays (haseman et al., ) . acinar and tubular adenomas and adenocarcinomas as well as squamous carcinomas are reported in rats (mohr, ) , mice (frith and heath, ) and hamsters (takahashi and okamiya, ) . some carcinomas showing squamous or glandular differentiation may be observed infiltrating the salivary gland that originate in other local structures of the head and neck region. occasionally, salivary gland neoplasms show adenomyomatous differentiation. mixed glandular and lymphoid tissue patterns resembling wartin's tumour in man are also sometimes seen. neoplasms of soft tissues also develop in and around the major salivary gland in rodents (see integumentary system chapter i). in humans the oesophagus is not considered a common site for drug-induced injury although some studies have suggested that medication-induced changes are more prevalent than previously supposed (bonavina et al., ) . severe damage can occur following prolonged contact between mucosa and ingested tablets or capsules which results in local high concentrations of potentially irritant substances (bott and mccallum, ; brors, ; kikendall, ; levine, ) . damage as a result of local contact may be more common in elderly subjects as the amplitude of oesophageal contractions decrease with age and capsules more liable to lodge in the lumen of the oesophagus (bonavina et al., ) . however patients of all ages may be affected. women have been injured more frequently than men probably because of the greater likelihood of their being treated with potentially injurious drugs (kikendall, ) . the shape and surface coating of tablets may influence their tendency to adhere to the mucosa and lodge in the oesophagus (marvola et al., ) . a wide variety of drugs have been implicated. in the united states the majority of cases appear to be caused by ingestion of tetracycline or doxacycline (levine, ) . some of the causative agents such as potassium chloride, aspirin and other non-steroidal anti-inflammatory drugs are also implicated in ulceration lower in the gastrointestinal tract. over recent years the bisphosphonate, alendronate has been one of the most commonly reported causes of adverse effects in the oesophagus with severe injury being reported. although injury is linked to ingestion without water or failing to remain upright after swallowing the medication, alendronate is particularly caustic (kikendall, ) . oesophagitis due to candida albicans is a well-described complication of antibiotic therapy. administration of immunosuppressive drugs may predispose to viral infections in the oesophagus. a number of agents affecting neuromuscular co-ordination may also predispose to gastro-oesophageal regurgitation and reflux oesophagitis (bott and mccallum, ) . in laboratory rodents spontaneous lesions of the oesophagus are occasionally seen. oesophageal impaction has been described in untreated srl:bhe rats. this is characterised by massive dilatation of the oesophagus with food or bedding (ruben et al., ) . histologically, the muscle fibres in the wall of the oesophagus show varying degrees of degeneration including swelling or shrinking of fibres, myofibrillar fragmentation, cytoplasmic vacuolation and mineralisation. so-called megaoesophagus, characterised by enlargement of the oesophagus, degeneration of muscle fibres and ganglion cells in the myenteric plexus has also been described in certain strains of rats and mice (harkness and ferguson, ; randelia and lalitha, ) . its cause is unknown. a commonly occurring lesion reported in fischer rats is oesophageal hyperkeratosis, which occurs at all ages . in the study by maeda et al. ( ) , it occurred more commonly in rats fed a protein-restricted, calorie unrestricted diet than in rats fed ad libitum with normal diet. it was suggested that the particular high prevalence of oesophageal hyperkeratosis observed in all groups in this particular study was related to acidification of drinking water . another pathological findings in rodents is perforation of the oesophagus as a result of a gavage accident. under these circumstances there is a variable inflammatory and purulent exudate localised around the perforation or spread within the pleural or occasionally the pericardial cavities. the oesophagus and surrounding tissues need careful examination by the pathologist for it is not always clear from clinical findings that oesophageal damage has occurred. spontaneous oesophageal lesions are uncommon in laboratory beagles, even though emesis and vomiting are frequent responses of this species following dosing in toxicity studies. local oesophageal irritancy potential of drugs has been assessed in a number of animal models, notably the cat and pig (carlborg and densert, ; olovson et al., ) . in these models, the test drugs are placed in the upper oesophagus using endoscopic techniques for periods of several hours to allow dissolution of the preparation. subsequently, the animals are followed for - days and histopathological assessment performed on the oesophagus. the degree of inflammation, erosion of mucosa or deep ulceration is recorded in a semiquantitative manner. the degree of ulcerogenic activity of drugs in these models seems to correlate with reported ulcerogenic activity in the human oesophagus (carlborg et al., ) . systemic administration of drugs with radiomimetic or antimitotic activity can cause hypoplastic changes in the oesophageal mucosa as well as the remaining gastrointestinal tract mucosa (tucker et al., ) . conversely, hyperplasia with increased keratinization has been reported in the oesophagus of the rat following chronic high dose administration of alcohol (mascrès et al., ) . acanthosis with hyperkeratosis and parakeratosis has been reported in the oesophagus but not stomach of rats treated for up to months with mesuprine hydrochloride, a β-adrenergic receptor stimulator (nelson et al., ) . as part of its effects on the gastrointestinal tract, the oesophagus in rats and primates has been reported to develop uniform hyperplasia of the squamous epithelium following infusion of recombinant epidermal growth factor (breider et al., ; reindel et al., ; vinter-jensen, ) . in the rat, mouse and hamster the forestomach occupies about two-thirds of the proximal stomach area and is lined by cornified stratified squamous epithelium. the limiting ridge is a distinct elevated mucosal fold at the junction between the forestomach and the mucosa of the glandular part of the stomach. as humans lack a forestomach, the relevance of changes produced by drugs and chemicals in the rodent forestomach is disputed. studies in rats in which the forestomach has been removed have suggested that the forestomach acts as a storage organ releasing relatively undigested food into glandular stomach in response to energy demand (gärtner and pfaff, ) . hence, the forestomach mucosa may be exposed to xenobiotics mixed in undigested food for far longer periods than elsewhere in the gastrointestinal tract. the interpretation of forestomach changes should take into account physiological factors, residence time and exposure differences to drugs between the rodent forestomach and human oesophagus. however the squamous mucosa lining the oesophagus in species without a forestomach may react to xenobiotics in a similar way to the forestomach mucosa of rodents if equivalent exposure levels are attained. inflammation and ulceration of the forestomach mucosa are some of the commonest spontaneous gastrointestinal lesions in laboratory rats, mice and hamsters. the prevalence of these gastric lesions varies between species, strains of laboratory rodents as well as between different laboratories. the precise causes of forestomach ulceration remain unclear although a variety of factors have been associated with its development including advanced age, infection, parasitism, diet, feeding regimens and stress. in rats, conflict-induced ulceration occurs in the forestomach and there is an age-related susceptibility, older rats developing more ulcers than younger rats (sawrey and sawrey, ) . in rats and mice dying of spontaneous disease, ulceration of the forestomach is also quite frequently observed. protein restriction or starvation has also been shown to produce forestomach ulceration in rats . ulceration of the forestomach in rodent toxicology studies may be incidental, particularly if the lesions are few and show no clear relationship to dose. if limited to high dose groups, ulceration may be a result of non-specific toxicity and stress-related. however, administered chemicals may have direct local effects of sufficient severity to cause focal damage to the forestomach mucosa. histological features of ulcers and inflammatory lesions of the forestomach are similar in rats, mice and hamsters. in mild cases, a scattering of acute inflammatory cells is seen in the intact squamous mucosa. ulcers can be single or multiple and are characterised by loss of squamous epithelium with a variable accumulation of neutrophils, mononuclear cells, cellular debris, fibrin and hair fragments in the ulcer crater. the inflammatory process may extend deeply into the stomach wall and be associated with intramural inflammation, oedema, endarteritis and fibrosis. haemosiderin pigment is also found in the ulcer margins. profuse haemorrhage may follow erosion of large blood vessels and complete perforation of the stomach wall with peritoneal involvement also occurs (greaves and faccini, ) . in long-standing cases of ulceration, hyperplasia of the adjoining squamous epithelium occurs, characterised by irregular acanthosis and down-growths of squamous epithelium into the submucosa . xenobiotics may produce inflammatory changes in the forestomach mucosa following initial dosing but subsequently, repair occurs even though treatment continues. an example of this phenomenon is illustrated by butylated hydroxyanisole. after week of administration of this agent in a % mixture in diet to rats, a vesicular inflammatory reaction characterised histologically by the presence of subepithelial vesicles containing inflammatory cells and exudate was seen (altmann et al., ) . after further treatment, only hyperplasia of the squamous epithelium was evident, presumably as an adaptive response to the effects of the continued insult. hyperkeratosis associated with hyperplasia of the squamous epithelium is seen sporadically in untreated aged rodents. these changes may be localised to the margins of chronic forestomach ulcers or they can be associated with diffuse inflammation of the mucosa. occasionally, the forestomach mucosa of untreated, aged rodents exhibits hyperkeratosis with hyperplasia without inflammation (fig. ). such changes may be diffuse or focal, but they are often localised to the zone adjoining the glandular stomach mucosa. there may be evidence of basal cell proliferation and downgrowth of the epithelium into the underlying stroma. dietary factors also influence the thickness of the forestomach mucosa. vitamin a deficiency, known to produce squamous metaplasia in glandular tissues may produce forestomach hyperplasia and hyperkeratosis in rats. when spf fischer rats were maintained in a vitamin a deficient state for over months, hyperplasia with hyperkeratosis, not unlike that produced by known carcinogens was reported (klein-szanto et al., ) . administration of a wide range of both industrial chemicals, therapeutic agents including both genotoxic and non-genotoxic carcinogens produces hyperkeratosis and hyperplasia of the forestomach epithelium which may be followed by preneoplastic lesions and squamous carcinoma (see below). histologically, the changes are characterised by hyperkeratosis, parakeratosis with varying degrees of acanthosis and papillomatosis (greaves and faccini, ) . the changes can be florid and it may be difficult to make a clear distinction between severe hyperplasia and neoplasia. nevertheless, it has been shown that the florid hyperplasia of the forestomach epithelium without evidence of cellular atypia can be completely reversible following the withdrawal of an inciting stimulus, ethyl acrylate (ghanayem et al., ) . hence, a critical feature may be the presence of cellular atypia in view of its association with agents with potent (genotoxic) carcinogenic activity. neoplasms arising in the forestomach of rodents are usually squamous carcinomas although basaloid features are also seen (fukushima and leiniger and jokinen, ; mohr, ; tatematsu, ) . squamous carcinomas, as at other sites, show variable differentiation being composed of proliferating squamous epithelium with moderate to marked cellular atypia, pleomorphism and mitotic activity with clear evidence of invasion into the muscularis. although they are relatively uncommon spontaneous lesions in aged rodents, they can be induced in rodents by administration of nitroso compounds (tatematsu ) as well as a range of non-genotoxic agents (see below). some authors report basal cell carcinoma when basaloid features are pronounced (tatematsu, ) . a wide range of agents is capable of producing squamous hyperplasia of the rodent forestomach and a number of these also induce squamous carcinomas. in kroes and wester reviewed over genotoxic and non-genotoxic compounds that were reported to produce hyperplasia and carcinoma in the forestomach of rats, mice or hamsters. a well-studied example is butylated hydroxyanisole (bha) an important food antioxidant (reviewed by whysner and williams, ) . structurally related phenols and acids produce similar changes (rodrigues et al., ) . ethyl acrylate, used in the production of materials for dental and medical devices is also capable of inducing marked squamous hyperplasia, papillomas and carcinomas after long-term treatment of f rats and b c f mice (ntp, ; ghanayem et al., ) . sk&f , an experimental histamine h receptor antagonist produced atypical forestomach hyperplasia in rats following administration by gavage for year by a mechanism which appeared unrelated to the inhibition of the h receptor (betton and salmon, ) . other therapeutic agents associated with squamous hyperplasia and neoplasia include the -hydroxy- -methylglutaryl coenzyme a (hmg-coa) reductase inhibitors (kloss et al., ; bueld et al., ; akiba et al., ; physicians' desk reference, ) and aristolochic acid (göggelmann et al., ; schmeiser et al., ) . some cytoprotective prostaglandins appear capable of inducing hyperkeratosis and hyperplasia without neoplasia presumably through a mechanism related to their pharmacological activity (levin, ) . this occurs in rats treated with misprostol, a synthetic prostaglandin e methyl ester analogue with gastric anti-secretory and anti-ulcer activity (kotsonis et al., ) , cl , , a synthetic analogue of prostaglandin e type (kramer et al., ) and , -dimethyl prostaglandin e (reinhart et al., ) . even the extensively used antibiotic, ampicillin has been associated inflammation, ulceration with acanthosis and hyperkeratosis in mice but not rats treated for years (national toxicology program technical report, ) . sodium saccharin is also reported to produce hyperplasia without neoplasia of the forestomach in f rats (hibino et al., ) . among its wide range of pharmacological effects on the gastrointestinal tract, the forestomach has also responds to recombinant epidermal growth factor when infused into rats (breider et al., ; vinter-jensen, ) . histological examination showed hyperkeratosis and hyperplasia of the squamous epithelium. the large body of studies performed with butylated hydroxyanisole illustrates the various factors that can influence the development of treatment-induced hyperplasia and neoplasia of the rodent forestomach and subsequent assessment of human risk. butylated hydroxyanisole possesses little or no mutagenic activity in vitro but when administered to rats for years as a % mixture in the diet, it produced squamous hyperplasia, squamous papillomas and squamous carcinomas of the forestomach. at . % in the diet butylated hydroxyanisole induced only hyperplasia . it also produces proliferative lesions in the forestomach of both mouse and hamster (ito et al., ) . studies in which butylated hydroxyanisole was fed in the diet to rats for shorter periods have shown that squamous epithelial hyperplasia occurs after only week of treatment preferentially over the lesser curvature, the site at which carcinomas developed in the -year studies (altmann et al., ) . after weeks' treatment, mucosal hyperplasia characterised by pronounced hyperkeratosis, parakeratosis and acanthosis most pronounced over the lesser curvature, was present in rats given % butylated hydroxyanisole in diet but not in rats given . , . and . % mixtures . abundant mitoses were found in the basal cells layers and tritiated-thymidine labelling confirmed that the changes were accompanied by a high rate of cell proliferation. following cessation of administration of butylated hydroxyanisole after weeks, the tritiatedthymidine labelling index rapidly reverted to control levels within about week although hyperplasia took longer to regress. nearly complete regression of the hyperplasia occurred after about weeks of normal diet . the distribution of squamous hyperplasia induced in the rodent stomach by butylated hydroxyanisole is influenced by the mode of administration. whereas following feeding of rats with butylated hydroxyanisole mixed in the diet lesions tended to be located near the limiting ridge, altmann et al. ( ) showed that gavage of butylated hydroxyanisole in corn oil produced similar changes at the apex of the forestomach. it was suggested that this difference was due to incomplete mixing of butylated hydroxyanisole in the stomach lumen when given by gavage and prolonged contact of the gavage mixture with the upper segment of the forestomach (altmann et al., ) . more recently, it was shown that fischer , shr, lewis and sprague-dawley rats differ in their response to the hyperplastic and carcinogenic effects of % butylated hydroxyanisole in pelleted diet. the most sensitive was the shr strain followed by the f rats and the differences correlated with the cytotoxic effects of butylated hydroxyanisole in the different strains (tamano et al., ) . it was suggested that the presence of vascular damage in the stomachs of the shr rats might have contributed to the response to cytotoxicity and subsequent carcinogenicity. residence time of administered compounds in the forestomach may influence the development of lesions. although it has been demonstrated that butylated hydroxyanisole does not produce hyperplasia in the oesophagus of animals without a forestomach, have shown that high-doses given to primates are capable of producing an increase in mitotic activity in the lower end of the oesophagus similar to that occurring at equivalent exposure levels in the rat. the implication is that these interspecies differences may simply be a question of differences in exposure of the squamous mucosa to compound. this underlines the fact that mechanisms of action and exposure levels of xenobiotics attained in the gastrointestinal tract of rodent and non-rodent species as well as of man need to be carefully assessed when hyperplastic changes are induced in the forestomach mucosa of rodents. such information can be helpful in facilitating regulatory decisions in this area (moch, ) . on balance, the evidence suggests that the tumour development by butylated hydroxyanisole in rodents represents an epigenetic phenomenon related to largely reversible cytotoxicity and increased cell proliferation (whysner and williams, ) . in view of the low levels of exposure to butylated hydroxyanisole that occurs with the usual use of this agent, carcinogenic hazard for the human stomach is therefore probably very small. similar phenomena have also been reported in studies of phenols and acids that are structurally related to butylated hydroxyanisole (rodrigues et al., ) . these agents include n-butyl and n-propyl- -hydroxybenzoic acid esters, propionic acid and -methoxyphenol. however, these studies suggested that certain areas of the forestomach epithelium react differently to structurally related chemicals, possibly due to the variable levels of activating enzymes within different zones of the forestomach epithelium. co-administration of acetylsalicylic acid was shown to abrogate some of these effects, suggesting that prostaglandin synthetase may be involved in the hyperplastic response (rodrigues et al., ) . a number of hmg-coa reductase inhibitors with different chemical structures including marketed products such as lovastatin, simvastatin and fluvastatin are also associated with the development of squamous hyperplasia of the rodent forestomach. the hyperplasia is time and dose dependent and may be associated with oedema and some inflammation of the submucosa. some, but not all of these agents are also capable of producing squamous neoplasia of the forestomach mucosa of rats, or mice or both after long-term treatment (kloss et al., ; bueld et al., ; akiba et al., ; physicians' desk reference, ) . the mechanism of action remains unclear although the degree of hyperplasia seems related to pharmacological potency. their carcinogenic potential in rodent bioassays does not seem to relate to the degree of hyperplasia in shortterm studies. moreover, the development of hyperplasia depends on local high concentrations of drug because when administered by non-oral routes, hyperplasia does not occur (kloss et al., ) . as most of these drugs are non-mutagenic, these findings are presumably also epigenetic in origin and possess relatively little risk for humans when given in the usual therapeutic doses. a contrasting example is provided by aristolochic acid, a nitrophenanthrene derivative of the ancient medicinal plant aristolochia clematis which was used as an anti-inflammatory component in a number of medicinal preparations in germany until (göggelmann et al., ; schmeiser et al., ) . aristolochic acid is a direct acting mutagen in salmonella typhimurium. when fed to rats at doses of . and mg/kg/day, aristolochic acid produced severe papillomatosis of the entire forestomach within a period of months. this was characterised histologically by the presence of branched squamous papillomas up to mm high with focal dysplastic features. invasive squamous carcinomas with metastases were found subsequently, or months later without further treatment (mengs et al., ) . even at a low dose of . mg/kg/day papillomas and squamous carcinomas developed months after a -month period of treatment. quite clearly the complexity of the hyperplastic response of the rodent stomach to xenobiotics, the association of hyperplasia induced by non-mutagenic compounds with the development of forestomach carcinomas, and the similarity of response in the forestomach to that of the oesophagus, dictates the need for a careful analysis of hyperplasia induced by novel drugs in the forestomach. the prelude to this assessment is careful histopathological characterisation of the changes. unlike the mouth and oesophagus through which tablets, capsules, gavage fluids and drug/diet mixtures pass relatively rapidly, the human stomach mucosa remains in contact with high local concentrations of administered compounds for much longer periods of time. administration of compounds in liquid or solid form, particle size, fasting and feeding all affect the gastric motility pattern. in the fasted state there is a cyclical pattern of motility consisting of three main phases. the first is a quiescent phase, followed by a phase of irregular contractions that increase in amplitude and frequency to reach a maximum in a third phase. feeding results in the replacement of this cyclic pattern by regular tonic contractions that move food towards the antrum and mix it with gastric secretions. these patterns have been well studied in both dog and man and appear to be qualitatively similar in the two species (sarna, ) . these motility patterns may have an impact on the length of time drugs remain in contact with stomach mucosa. for instance, the residence time of large non-disintegrating capsules or tablets administered in the fasting state is more dependent on the frequency of powerful phase iii contraction than if drugs are given as fluids or mixed with diet. for dosage forms released in the stomach, gastric residence time will influence drug supply to the main absorptive surfaces in the small intestine, which in turn may affect drug absorption (dressman, ) . gastric acid is also important in making ingested salts soluble. although the presence of food in the stomach is a stimulus of acid production, the ph in the forestomach of rats is highest in full stomachs and lowest when empty, presumably as a consequence of the buffering action of food (ward and coates, ) . the glandular stomach is conveniently divided into the fundus characterised by mucosal folds or rugae and the smoother antrum, which opens into the pylorus and duodenum. in species devoid of a forestomach, the proximal stomach mucosa or cardia is also lined by glandular mucosa. the glandular mucosa is covered by surface epithelium of regular columnar cells that extends downwards to form small gastric pits or foveolae. the gastric glands are simple tubular structures usually considered to comprise three segments. the base is the deepest part, the neck the mid-region, and the most superficial is the isthmus, continuous with the gastric pit. the upper part of the gastric gland contains mucous neck cells. small cuboidal chief or zymogenic cells, which secrete pepsinogen and stain blue or purple in haematoxylin and eosin sections, are located in deeper parts of the gland. the eosinophilic-staining parietal (oxyntic) cells, which produce hydrochloric acid, are distributed more randomly throughout the gastric glands. parietal cells can also be visualised by immunocytochemical staining with antibodies directed at h + k + -atpase (canfield et al., ) . the gastric glands situated near the limiting ridge in rodents, show a modified structure. in species not endowed with a forestomach, the mucosa near the cardia is composed of simplified branched glands lined by columnar epithelium. the antral mucosa is covered by a surface epithelium with gastric pits similar to that of the fundus but mucous secreting columnar glands line the glands. the stomach mucosa is richly endowed with endocrine cells, not all of which have been well characterised. enterochromaffin cells are quite numerous in the basal parts of the gastric glands of the fundus, particularly in the rat . they are generally argyrophilic, staining with silver staining techniques such as that of grimelius (grimelius, ; grimelius and willander, ) that utilise exogenous reducing agents. these cells contain histamine and histamine-related enzymes such as histidine decarboxylase in the rat and other species . endocrine cells which are argentaffin in type stain with silver stains such as that of masson ( ) because of the presence of endogenous reducing substances including -hydroxytryptamine and catechol-amines are also reported in the mucosa of the fundus of some species including man but apparently not in the rat . enterochromaffin cells are characterised ultrastructurally by the presence of numerous rounded or oval, vesicular, electron-lucent granules frequently containing a small eccentric electron dense core. gastric enterochromaffin cells can also be stained by immunocytochemical techniques using antisera to histamine and histidine decarboxylase as well as to non-specific enolase and chromogranin a betton et al., ) . immunocytochemical study of the rat fundus using a battery of antisera to a variety of gastrointestinal peptides has shown somatostatin containing (d) cells and glucagon staining cells but no cells with gastrin (g cells) or serotonin reactivity (bishop et al., ) . gastrin or g cells possess apical processes reaching the stomach lumen believed to be important in stimulation of gastrin release as a result in increases in antral lumen ph or the presence of amino acids or peptides . glucagon and serotonin containing endocrine cells have also been located in the rat antral mucosa (bishop et al., ) . increased gastric acid secretion is initiated by activation of central vagal efferent pathways but acid secretion is maintained by both neural and endocrine reflexes activated by the presence of food in the stomach. gastrin secreted from the g cells of antrum is the main stimulant of acid secretion. somatostatin is secreted from antral d cells when the luminal ph falls to below . to act by a paracrine mechanism to suppress g cell function thus forming a negative feedback loop (dockray, ) . the two main endocrine cell types from the body mucosa integrate neurohumoral stimuli rather than respond to luminal chemicals. although gastrin is capable of stimulating parietal cells directly, it has an even greater effect through stimulation of enterochromaffin cells to release histamine, a potent paracrine stimulator of parietal cells (hinkle and samuelson, ) . gastrin stimulates release of histamine from enterochromaffin cells of the body mucosa, which increases acid secretion through activation of parietal cell histamine-h receptors. both parietal and enterochromaffin cells are inhibited by somatostatin released from the d cells of the body mucosa in response to a variety of neurohumoral stimuli such as noradrenaline, vasoactive intestinal peptide, calcitonin gene-related peptide, and cholecystokinin. it should also be noted that gastrin has a role in kinetics and differentiation of both parietal and enterochromaffin cells (dockray, ) . gastrin acts at the gastrin/cholecystokin-b receptor that is expressed by gastric epithelial cells and by neurones in the central nervous system (kopin et al., ; wank, ) . the gastrin receptor is simply the cholecystokinin-b receptor located in the stomach. the other cholecystokinin receptor, cholecystokin-a has high affinity for cholecystokinin. stimulation of this receptor in the stomach mediates secretion of pepsin from gastric chief cells and release of somatostatin from d cells resulting in inhibition of acid secretion. in the central nervous system cholecystokinin and its receptors contribute to the regulation of satiety, anxiety, analgesia and dopamine-related behaviour (wank, ) . finally, it is worth recording that both progesterone and oestrogen receptors have been identified in both normal and pathological gastric tissues of humans (wu et al., ) . generative cells in the gastric mucosa as shown by uptake of tritiated thymidine for dna synthesis are distributed principally in the isthmus (inokuchi et al., ) . tracing of cells using thymidine labelling have shown that most of the cells in the generative zone migrate in a successive manner to the mucosal surface to form columnar epithelium. the life span of surface epithelium in the stomach of rats, mice and hamsters has been calculated to be about - days. studies of cell cycle and dna synthesis time in the proliferative zones in the stomach of rat, hamster and man have suggested that the generative cells in the isthmus undergo mitoses at about -hour intervals in rodents and -hour intervals in man (inokuchi et al., ) . although this process of migration from the proliferating cell zone of the isthmus renews surface epithelial cells rapidly, cell migration to the lower parts of the gastric glands is much slower and more complex. detailed studies have show that undifferentiated cells in the region of the isthmus represent a common source for surface mucous cells and mucous neck cells (karam and leblond, ) . electron microscopic and ultrastructural cytochemistry has suggested note: saccharide binding specifications are much more complex than the inhibition by simpler sugars outlined above suggests. see review by nicholson ( ) . *source: nicholson, ; goldstein and hayes, ; schulte and spicer, ; giannasca et al., . that chief cells in the adult rat stomach develop from undifferentiated stem cells in the isthmus (suzuki et al., ) . graft experiments in mice have also suggested that immature cells of the isthmus differentiate into chief cells as well as parietal cells (matsuyama and suzuki, ) . studies in transgenic mice have shown that mature parietal cells influence the fate of other gastric epithelial cells because targeted degeneration of parietal cells is associated with loss of chief cells suggesting interactions between these cell populations in determining their differentiation (li et al., ; canfield et al., ) . gastrin is also an important regulator of parietal cell and enterochromaffin differentiation and number (dockray, ; montgomery et al., ) . labelling experiments in the hamster stomach have shown that both chief and parietal cells possess a similar but quite long life span of about days (hattori, ; hattori and fujita, ) . it has been suggested that the relative distribution of chief and parietal cells in the gastric gland represents an expression of their different migration patterns downwards from the proliferative zones in the isthmus. this type of migration pattern in which cells are able to overtake each other has been termed a 'stochastic flow system' (inokuchi et al., ) . the origin and kinetics of endocrine cells of the stomach has also been the subject of debate but the available morphological, cytochemical and kinetic evidence suggests that the majority of these cells develop from the same stem cells as the other non-endocrine cells of the gastric mucosa, although self replication also occurs (matsuyama and suzuki, ; inokuchi et al., ; solcia et al., ) . much of our knowledge about mucins produced by the epithelial cells lining the gastrointestinal tract has been obtained using histochemical techniques and these approaches continue to be helpful in the understanding of spontaneous and drug-induced gastrointestinal disease (sheahan and jarvis, ; filipe, ; tsiftsis et al., ; jass and roberton, ) . for these reasons, mucin histochemical techniques represent useful tools for the characterisation and elucidation of experimentally or drug-induced changes in the glandular mucosa of gastrointestinal tract. techniques commonly employed are presented in table . the physiochemical properties of gastrointestinal mucins are dependent on their glycoprotein constituents. these glycoproteins are high molecular weight compounds with large numbers of sugar chains attached to a polypeptide backbone by o-glycosidic linkages between n-acetylgalactosamine and serine or threonine (berger et al., ) . the principle monosaccharides present are fucose, galactose, n-acetylgalactosamine, n-acetylglucosamine and sialic acid. traces of mannose may be present and ester sulphate residues are common (filipe, ) . due to this extensive glycosylation, mucins have a filamentous conformation, which is often negatively charged. this is believed to be important in forming a protective barrier to the cell. however, this property is a two-edged sword because when opposing cells have specific receptors for mucins, adhesion may become the predominant factor (van klinken et al., ) . although mucins are important in the gastrointestinal tract, it should be remembered that other products secreted by goblet cells might be important in mucosal defence. it has been recently recognised that trefoil proteins, a family of small proteins secreted by goblet cells and present on the mucosal cell surface, can also protect against a variety of deleterious agents, including bacteria, toxins and drugs (podolsky, ) . there are considerable regional variations in glycoprotein constituents in the gastrointestinal tract and these differences are probably related to physiological and functional factors. furthermore, synthesis and secretion of glycoproteins alter with changes in cell differentiation. alterations also occur in mucins in various inflammatory and neoplastic disease states as well as following administration of certain drugs and chemicals (ishihara et al., ) . terminal sugars or sugar sequences can be demonstrated histochemically by the use of labelled lectins, mostly plant proteins which combine non-enzymatically with particular sugar molecules, see table (goldstein and hayes, ; debray et al., ; rudiger, ) . magenta: all mucosubstances containing hexoses schiff d-pas (pearse, ) and deoxyhexoses with vicinal glycol groups. some non-sulphated acid mucosubstances. neutral mucosubstances. periodate-borohydride/ magenta: pas activity following periodate borosaponification/pas, hydride/potassium hydroxide indicates pb/koh/pas presence of o-acylated sialic acids. (reid et al., ; periodate borohydride reduces periodate culling et al., ; generated aldehydes. potassium hydroxide removes o-acylesters from potential vicinoldiols and sialic residues linked glycosidically to a potential vicinoldiol. alcian blue ph . basophilia: weakly sulphated mucins. carboxyl (pearse, ) groups of sialomucins alcian blue ph . basophilia: sulphated mucins (lev & spicer, ) alcian blue ph . -magenta: neutral mucins periodic acid schiff, ab/pas basophilia: acid mucins (mowry & morard, ) purple-blue: neutral and periodate reactive acid mucins high iron-diamine, brown-black: sulphated mucins hid (spicer, ) unstained: sialomucins high iron-diamine-alcian brown-black: sulphated mucins blue ph . , hid/ab basophilia: non-sulphated acid mucins (sialomucins) (spicer, ) source: adapted from filipe, . when gastrointestinal mucins were studied in several species using histochemical techniques under uniform conditions, species differences were most obvious in the stomach and duodenum (sheahan and jarvis, ) . neutral mucins generally predominate in the stomach, contrasting with acid mucins in the small intestine, and sulphated mucins in the colon. in the stomach neutral mucins staining purple with the pas/alcian blue stain, predominate in the surface and foveolar mucosa, whereas mucous neck cells and antral glands contain acidic mucins that stain blue with pas/alcian blue procedure. sulphated mucins, as shown by the high iron diamine technique (hid) are also found in the deep glandular mucosa of the antrum in rat, mouse and man (filipe, ; jass, ; greaves and boiziau, ) . extremely heterologous staining patterns are seen in the gastric mucosa with labelled lectins, each lectin staining quite different cell populations. there are considerable interspecies differences in staining patterns with the same lectins (kuhlmann et al., ; suganuma et al., ) . the so-called paradoxical concanavalin a stain, in which conjugated concanavalin a is used to label mucins before and after periodate oxidation, has also been used to classify the alterations in mucins in proliferative and neoplastic conditions of the rat stomach mucosa (kobayasi et al., ; tatematsu ). although gastric erosions and ulcers in the glandular mucosa occur quite commonly in laboratory animals in toxicity studies, it is often difficult to determine whether such lesions in treated animals indicate a real ulcerogenic risk for the test compound. there is little that is histologically specific to drug-induced ulceration of the gastric glandular mucosa. mucosal haemorrhage, depletion of mucin, erosions and ulcers with or without inflammation may all be found. erosions represent mucosal breaks superficial to the muscularis mucosa. ulcers are lesions that extend through the muscularis mucosa. whilst the histopathological features of gastric erosions and ulcers are themselves relatively non-specific, it is important to look for any associated pathology in the stomach such as mucus depletion, epithelial hyperplasia or dysplasia, intestinal metaplasia and vascular lesions (see below). in humans, biopsy data suggests that drug-induced ulceration is characteristically devoid of an inflammatory component, but the most usual histological appearances are those of underlying gastric pathology (riddell, ) . formation of gastric and duodenal ulcers is dependent on the presence of both acid and peptic activity in gastric juice because acid without pepsin appears to have little digestive power. important predisposing factors in human patients with peptic ulceration include helicobacter pylori (campylobacter pylori) infection of the antrum, cigarette smoking and ingestion of non-steroidal anti-inflammatory drugs (soll, ) . helicobacter pylori is believed to infect over half the human population and its presence in the gastric mucosa is associated with chronic atrophic gastritis and peptic ulceration (cover and blaser, ) . it is a microaerophilic, gram-negative organism that possesses potent urease activity crucial for its survival at acidic ph. genome sequence analysis has shown that helicobacter pylori has well developed sequences for motility, scavenging iron and dna restriction and modification systems used by bacteria to degrade foreign dna. the link between helicobacter pylori infection and peptic ulceration is related to increases in gastrin release, perhaps through bacterial products or cytokines released from activated lymphocytes (richter-dahlfors et al., ) . although helicobacter pylori can infect other species, apart from non-human primates, the usual laboratory animal models do not appear to develop the inflammatory disease seen in humans (nedrud, ) . erosions and ulcers also develop following stress, reflux of intestinal contents and bile, changes in acid secretion and hypoxia, all of which may develop under the conditions occurring in high-dose toxicity studies. the requirement to give the test compound in high doses may also dictate the need to administer exceedingly high concentrations of test agent. this may produce damaging high local concentrations on the mucosa not relevant to therapeutic doses used in clinical practice. it has been demonstrated that hyperosmolar solutions of quite innocuous substances such as glucose can cause haemorrhage, erosions and ulcers of the rat gastric mucosa (puurunen et al., ) . the well-known association of gastric erosions and haemorrhage with uraemia may also be manifest following administration of high doses of drugs such as diuretics which severely derange fluid and electrolyte balance (garthoff et al., ) . stress ulceration may be linked to temporary ischaemia of the mucosa (dubois, ) . synergism between the ulcerogenic action of drugs and stress is a well-described phenomenon (rainsford, ; beattie, ) . protein depletion and starvation is also capable of inducing gastric ulceration in rats . although gastric pathology represents the largest cause of morbidity and mortality in man following therapy with non-steroidal inflammatory agents (fowler, ) , the reasons for this are probably multifactorial. the acidic properties of some of these drugs may cause direct local damage of gastric epithelial cells, demonstrable by the fact that appropriate formulation can reduce gastric toxicity of these agents in man (brors, ) . anti-inflammatory agents are capable of decreasing synthesis of glycoproteins and this may adversely influence protective mucus production of gastric mucosa (azuumi et al., ; ishihara et al., ) . it has also been suggested that non-steroidal anti-inflammatory agents cause cellular damage to the gastric mucosa by back-diffusion of gastric acid into mucosal tissues (davenport, ) or by causing damage to the gastric capillary bed with subsequent mucosal infarction (robins, ) . the theory that has gained widespread acceptance is that the ulcerogenic potential of non-steroidal anti-inflammatory drugs is related to their pharma-cological activity. vane ( ) proposed that the ulcerogenic potential of these agents was largely a result of their ability to inhibit prostaglandin synthetase, thereby reducing the protective effects of prostaglandins. pharmacokinetic factors may also be important. lipid solubility in the low ph environment of the stomach may influence local penetration into the mucosa (mccormack and brune, ) . moreover, it has been proposed that certain anti-inflammatory agents may possess lesser ulcerogenic potential in man because their inhibition of prostaglandin production is more limited to sites of inflammation, sparing gastric mucosa (whittle et al., ; whittle and vane, ) . it has also been demonstrated that factors altering the enterohepatic circulation of drugs can influence the expression of gastric damage (overvold et al., ) . comparative studies of the ulcerogenic activity of indomethacin in beagle dogs and domestic pigs has suggested that the dog may be an excessively sensitive species as a result of extensive enterohepatic circulation of indomethacin in this species (hanhijarvi et al., ) . prediction of ulcerogenic potential for man based on data from animal models is clouded by the lack of good comparative data on the relative ulcerogenic potential of non-steroidal anti-inflammatory agents in man because of extensive differences in side effect reporting (fowler, ) . moreover proper comparisons in man require not only equivalent therapeutic doses but also comparable dosage forms (brors, ) . in laboratory animals, a variety of different patterns of drug-induced gastric damage have been described. the study by shriver et al. ( ) in which a wide variety of different anti-inflammatory drugs were administered to fasted sprague-dawley rats under identical conditions, suggested the drugs could be divided into three groups based on their profiles of gastrointestinal toxicity. immunological agents such as azathiaprine, cyclophosphamide, methotrexate and d-penicillamine produced gastric mucosal haemorrhage whereas aspirin and related agents produced gastric mucosal haemorrhage and ulcers. the powerful nonsteroidal anti-inflammatory drugs indomethacin and phenybutazone produced gastric mucosal erosion and ulcers as well as small intestinal damage. comparative single oral dose studies of several different non-steroidal antiinflammatory agents at three different dose levels by suwa et al. ( ) in the rat using histology and measurement of faecal blood loss with cr-labelled blood cells have also shown that different patterns of ulceration can be produced by different agents when administered under identical conditions. single oral doses of some non-steroidal anti-inflammatory drugs including aspirin produced widespread superficial damage and desquamation of gastric epithelium with little or no inflammation at hours following dosing which completely healed weeks later. this damage was associated with transient faecal blood loss. by contrast, indomethacin and ibuprofen produced both gastric damage and circumscribed, penetrating ulcers along the mesenteric border of the jejunum and ileum. furthermore, ulcers were still present after weeks and were associated with prolonged or biphasic blood loss (see small intestine). in addition feeding conditions can influence the distribution of erosions and ulcers in laboratory animals. in fasted rats, erosions due to indomethacin treatment are found in the body of the stomach whilst in conventionally fed rats they are most prominent in the small intestine. detailed studies by satoh et al. ( ) showed that rats fed for hour after a hour-fast and given a single dose of indomethacin within hours of re-feeding developed erosions and ulcers in the antrum primarily along the lesser curvature. indomethacin given to fasted rats produced erosions in the body mucosa. a further factor that needs to be kept in mind is that chronic administration of ulcerogenic compounds may produce quite different pathological appearances to those found following single dose administration. administration of aspirin to rats for weeks has been shown to stimulate epithelial proliferation of the gastric body but not antral mucosa, possibly by an effect on cyclic adenosine ', ' monophosphate (cyclic amp) or though increasing the rate of epithelial exfoliation (eastwood and quimby, ) . such a response may be the basis for increased resistance of the gastric mucosa to the chronic affects of these agents. it also may explain the tendency for ulcers to occur in the antrum following chronic administration of aspirin-like drugs as the proliferative response and presumably the adaptive potential appears less in this part of the gastric mucosa. both interspecies variations and strain differences have been reported in the response to ulcerogenic compounds. rainsford et al. ( ) showed that extravasation of red blood cells and greater vascular damage was observed in rats treated with aspirin or benoxprofen than in pigs given similar doses. sprague-dawley rats appear less susceptible to the ulcerogenic effects of cold-restraint stress than wistar rats (goldenberg, ) . diuretics and some angiotensin converting enzyme (ace) inhibitors and angiotensin ii antagonists have been associated with the development of gastric erosions and ulceration when administered in high doses to laboratory animals ( fig. ) (imai et al., ; garthoff et al., ) . however, these effects appear related to the severe electrolyte disturbances produced by excessive doses of these drugs. this is perhaps analogous to the well-known association of gastrointestinal tract erosion and haemorrhage with uraemia. dogs appear to have a particular predisposition to this effect where it may be associated with deposition of basophilic ground substance and mineral in connective tissues and blood vessels in the mucosa (barker and van dreumel, ) . although inflammatory conditions due to microorganisms are generally uncommon in the stomach, gastritis is reported in laboratory rhesus monkeys in association with the presence of helicobacter organisms (reed and berridge, ) . as in the analogous condition in man, the stomach of affected animals shows an infiltration of the central mucosa by small lymphocytes and plasma cells, associated with reactive or atrophic changes in the mucosa and the presence of small curved bacteria in glands, visualised best with the warthin-starry stain. infiltration of the stomach by lymphocytes in rats treated with human recombinant interleukin- without ulceration was reported as part of a multisystem involvement induced by this agent (anderson and hayes, ). decrease in gastric mucus secretion may accompany both spontaneous inflammatory conditions and drug-induced lesions in the stomach of man and experimental animals. mucus depletion is characterised histologically by the presence of an intact epithelial layer in which cells show loss of the normal clear cytoplasm replete with mucous substances by more basophilic cells that contain little or no mucin. qualitative changes in mucus composition can also accompany mucus depletion. gastric epithelium in man may show decreases in sulphated mucosubstances following stress, high alcohol consumption or after aspirin administration (filipe, ) . similar changes occur in laboratory animals subjected to ulcerogenic regimens. stress ulceration in the rat is accompanied by decreased sulphation of gastric glycoproteins, presumably an expression of the changes in gastric cellular activity accompanying stress (lambert et al., ) . administration of aspirin and other anti-inflammatory agents including adrenocortical steroids to laboratory animals also reduces the content of sulphomucins in the gastric mucosa, probably by reducing their synthesis (denko, ; gerard, ; ishihara et al., ) . rather surprisingly, administration of histamine h -receptor antagonists and fig. . section from the glandular stomach from a rat treated with ah high dose of an angiotensin ii antagonist that shows superficial degeneration and ulceration (erosion) of the mucosa. (he, × .) proton pump inhibitors associated with reduction of gastric acid output and increases in gastrin secretion have also been associated with alterations in gastric mucus. administration of omeprazole or famotidine to rats for weeks was shown to inhibit prostaglandins pge as well as the synthesis of both total and sulphated glycoprotein synthesis along with histochemical evidence of reduction in pas staining of the surface mucus (yoshimura et al., ) . although the mechanism for this change is unclear, the reduction in mucus, particularly sulphated mucus that is believed to be particularly resistant to peptic digestion, may have implications for mucosal defence. intestinal metaplasia of the stomach is characterised by the presence of differentiated epithelium, which resembles small intestine on the basis of light microscopic and ultrastructural morphology, mucin patterns and enzyme histochemistry (morson, ; planteydt and willighagen, ; lev, ; goldman and ming, ; . it develops in man in gastric mucosa altered by chronic atrophic gastritis and its significance is due to the fact that a link exists between intestinal metaplasia and gastric cancer. although intestinal metaplasia is found much less commonly in laboratory animals, it has also been reported to occur in association with gastric cancer induced by polychlorinated biphenyls (ward, ) . in view of this association with gastric cancer, it has been suggested that intestinal metaplasia represents a pre-neoplastic lesion. however, over recent years prospective clinical studies and experimental data have suggested that it is an epiphenomenon, coexisting with, but unrelated to the development of cancer. in man, several forms of intestinal metaplasia have been described. these variants fall into two main groups, an incomplete type and a complete form (teglbjaerg and nielson, ; jass and filipe, ; jass, ) . complete intestinal metaplasia is characterised by the presence of goblet cells, paneth cells and absorptive cells with brush borders and variably developed intestinal villi. incomplete forms are more heterogeneous characterised by goblet and mucous columnar cells but no absorptive cells and variable patterns of mucin. the routine alcian blue: ph . , periodic acid-schiff stain (ab/pas) ( table ) distinguishes between the intestinal acid mucins (blue) from the neutral mucins of gastric type. however, variable sialomucin and sulphomucin staining patterns are seen in intestinal metaplasia in man with the high iron-diamine/alcian blue stain (hid/ab) (jass, ) . the incomplete form of intestinal metaplasia, showing marked sulphomucin secretion, has been found more commonly in association with gastric cancer in man (jass and filipe, ; jass, ; wells et al., ) . however, prospective studies have tended to indicate that intestinal metaplasia with sulphomucin secretion may be an age-related form of chronic atrophic gastritis and not a premalignant lesion (ectors and dixon, ) . it has been suggested that intestinal metaplasia represents an adaptive response to long-standing chronic inflammation and reduced acid secretion. it may also represent an adaptive defensive response to long-standing helicobacter pylori infection because intestinal mucosa is more resistant to these organisms (steer, ; ectors and dixon, ) . intestinal metaplasia has been found in association with gastric cancer in laboratory animals. fischer rats treated with the polychlorinated biphenyl, aroclor , mixed in the diet for years developed foci of intestinal metaplasia in the stomach epithelium in association with gastric adenocarcinomas (ward, ) . these lesions were characterised by abundant mucin-containing cells and alkaline phosphatase activity typical of the small intestine (morgan et al., ; ward, ) . similar, but more diffuse intestinal metaplasia was reported in the stomach of primates treated with polychlorinated biphenyls, although unassociated with gastric neoplasia (allen, ; mcconnell et al., ) . intestinal metaplasia is also found in the stomach of laboratory animals treated with powerful genotoxic gastric carcinogens. although tsiftsis et al. ( ) showed hyperplasia and foci of atypical changes (dysplasia) but little or no intestinal metaplasia in rats following administration of n-methyl-n´-nitro-n-nitroguanidine, tatematsu et al. ( ) were able to show intestinal metaplasia in rats treated with the same agent. however, intestinal metaplasia can be induced in rodents by a variety of different procedures that are not associated with the development of gastric cancer. intestinal metaplasia can be induced in the glandular stomach of rodents by fractionated, localised, ionising radiation (watanabe, ; watanabe et al., ) , injection of xenogenic stomach antigens as well as propantheline bromide and the non-carcinogen, iodoacetamine shirai et al., ) . the characteristics of intestinal metaplasia in laboratory rodents are similar to those seen in man with early increases in intestinal enzyme activity (alkaline phosphatase, lactase, trehalase, sucrose and maltase), development of goblet cells containing neutral, sialo-, or sulphomucins, and intestinal crypts with or without paneth cells. both the fundus and antrum can show changes although as in man, males appear more prone to develop intestinal metaplasia than females . based on these experimental findings, have also proposed that intestinal metaplasia is not a precancerous condition but an adaptive response to a chronic elevation in ph in gastric secretion due to the early loss of parietal cell mass brought about by these various procedures. on balance therefore, the evidence to date suggests that although intestinal metaplasia is associated with cancer and may consequently be considered a helpful morphological feature in the evaluation of human gastric biopsies, the finding of isolated intestinal metaplasia in safety studies does not indicate a preneoplastic state. finally, a form of metaplasia in which hepatocytes have been found has been reported as rare incidental findings in the glandular stomach of mice sacrificed at the end of -year carcinogenicity bioassays (leiniger et al., ) . histologically, focal accumulation of well-differentiated hepatocytes were found in the submucosa and lamina propria adjacent to the limiting ridge with dilated adjacent gastric glands showing epithelial hyperplasia and mineralisation with herniation into the submusosa. whilst these foci were not believed to be related to treatment with xenobiotics, it is not clear whether represented metaplasia or congenital ectopia. the gastric glandular epithelium is predisposed to the deposition of calcium possibly as it is a site at which marked ion exchange normally takes place. focal aggregates or concretions of densely blue-staining mineral are fairly commonly observed in haematoxylin-stained sections from the stomachs of aged rats where they are associated with cystic dilatation of the gastric glands (greaves and faccini, ) . mice and hamsters occasionally show similar changes. small concretions are also observed in gastric glands in the beagle dog. these appear to represent aggregates of calcium around mucoid material. gastric mineralisation may become marked in rodents and dogs when there is disturbance of mineral metabolism, particularly in association with renal pathology. this has been well described in rats with severe renal disease (glomerulosclerosis) and parathyroid hyperplasia (snell, ) . a similar phenomenon has been described in the stomach of dogs in uraemic states (cheville, ) . identical changes result from the administration of drugs that induce prolonged azotemia or electrolyte disturbances. these changes are characterised by diffuse deposition of mineral in the intestinal tissue of the mucosa of the gastric body but not cardia, antrum or pylorus. mineral deposits develop around basement membranes surrounding epithelium and blood vessels. the lamina propria becomes expanded by oedema and fibroplasia of the interstitium also develops. the gastric glands themselves become distorted with swelling and degeneration of parietal cells and atrophy of chief cells. erosion of the glandular epithelium with haemorrhage occurs presumably as a result of the ischaemia caused by diffuse vascular injury and altered parietal cell function. focal atrophy of the gastric glandular mucosa is a sporadic occurrence in laboratory rodents, usually as a result of previous focal gastric inflammation, ulceration, mineralisation or vascular occlusion. these changes, characterised histologically by focal fibrosis of the mucosa, gastric glandular dilatation and atrophy variably accompanied by polymorphonuclear cells and mast cells are common in certain strains of rats when years or more in age (anver et al., ) . whereas diffuse mucosal atrophy occurs following severe inflammatory insult, diffuse atrophy of the stomach glandular mucosa without inflammation can be a result of surgically or drug-induced reduction in trophic factors necessary for the maintenance of normal gastric morphology and function. this is observed in man and experimental animals following antrectomy because this removes the peptide-producing cells of the antrum (gjurldsen et al., ; neilsen et al., ) . in the rat, antrectomy is accompanied by hypogastrinaemia, reduced weight and height of the oxyntic mucosa and a reduced number of argyrophil cells (håkanson et al., . this is in contrast to procedures such as antral exclusion that lead to hypergastrinemia and increased thickness of the oxyntic mucosa. mice with genetic deletion of the gastrin gene also show reduction in the thickness of the gastric mucosa. whilst all cell types are present, there is a most pronounced decrease in the numbers of parietal cells as well as enterochromaffin cells associated with an increase in surface mucous cells. these changes are linked to a profound decrease in acid secretion, which becomes unresponsive to histaminergic, cholinergic and gastrinergic stimulation (hinkle and samuelson, ) . analogous atrophic changes have been reported following pharmacological removal of trophic stimuli. for instance, administration of the cholecystokinin-b/gastrin receptor antagonist, ci- to cynomolgus monkeys for periods of up to weeks was associated with an initial phase of multifocal degeneration of gastric glands primarily in the fundus followed by diffuse reduction in the thickness of the glandular mucosa with little or no qualitative changes to the cell populations (dethloff et al., ) . although bilateral vagotomy produces profound functional changes in the stomach, notably reduction of gastric acid secretion, morphological changes in the fundal mucosa are not marked either in experimental animals or in man (crean et al., ; aase and roland, ) . studies in the rat have shown that diffuse atrophy of the gastric glands characterised by a decrease in the number and size of parietal, chief and mucous cells occurs transiently following truncal vagotomy but histological features return to normal by about month after surgery (nakamura, ) . by contrast, unilateral vagotomy in the rat leads to marked and persistent atrophy of the oxyntic zone on the denervated side. this is characterised histologically by reduced height of the mucosa and reduced numbers and staining intensity of argyrophil cells (hakanson et al., ) . håkanson and his colleagues argued that this unilateral atrophy was due to the removal of the trophic action of the vagus. the lack of lasting atrophy after bilateral but not unilateral vagotomy was explained by the subsequent rise in gastrin that occurs after bilateral vagotomy as a result of lack of acid feedback inhibition of gastrin release (håkanson et al., ) . removal or reduction in extra-gastric trophic factors or hormones may also reduce the thickness of the gastric mucosa. this is has been demonstrated in the rat by hypophysectomy which causes a reduction in thickness of oxyntic and antral mucosa, compared with pair-fed controls. although there was little or no change in peptic:parietal cell ratios, a significant decrease in cell volume and secretory activity of gastric glandular cells were demonstrated which suggested a widespread disturbance of synthesis and secretory mechanisms (bastie et al., ) . atrophic changes in the chief cells were observed in rats treated for months with high doses of omeprazole, an inhibitor of acid secretion. the findings were considered to represent disuse atrophy secondary to the inhibition of acid secretion (hansson et al., ) . another inhibitor of gastric acid secretion, the tricyclic agent pirenzepin, also produced atrophy of the fundic mucosa of rats following months but not month of treatment (lehy et al., ) . the atrophy was characterised by reduction in parietal cell numbers associated with lower numbers of gastrin-containing cells in the antrum, features unlike those following prolonged treatment with histamine h -receptor antagonists. an increase in the thickness of the gastric mucosa can be the result of hypertrophy or hyperplasia of the mucosal cells and this occurs both spontaneously or following administration of drugs and chemicals. in view of the different cell populations in the gastric mucosa and the variety of morphological alterations that occur, it is difficult to make a clear distinction between hypertrophy and hyperplasia without morphometric techniques. morphometric techniques have shown that hypertrophy of some mucosal cells can coexist with hyperplasia of other gastric cell populations. a distinction also needs to be made between diffuse or uniform hyperplasia involving one or more of the cell populations from the hyperplasia associated with proliferative or adenomatous overgrowth. adenomatous hyperplasia also needs to be evaluated for atypical cytological features (dysplasia), which are linked to development of gastric carcinoma (see below). cells of gastric glandular mucosa undergo increases in size or number in response to the effects of gastrointestinal trophic hormones or their synthetic analogues. similar changes also follow administration of compounds that inhibit gastric acid secretion or modify other trophic hormones or growth factors. when gastrin or its synthetic analogue, pentagastrin is administered subcutaneously to rats and mice for several weeks, there is both an increase in the number and size of parietal cells without concomitant increase in zymogenic chief cells (willems and lehy, ; crean et al., ; balas et al., ) . in addition, diffuse hyperplasia of enterochromaffin cells also occurs. by contrast, cholecystokinin, a trophic peptide found in the duodenum and sharing the same c-terminal tetrapeptide sequence as gastrin, increases in the number of chief cells but not parietal cells when administered to mice under similar conditions . drugs which inhibit or neutralise gastric acid secretion such as histamine h antagonists, proton pump inhibitors and antacids also induce hypertrophy or hyperplasia of the parietal cell population (witzel et al., ; crean et al., ; mazzacca et al., ; kaduk and hauser, ; betton et al., ; white et al., ) . these agents are associated with a rise in serum gastrin levels, probably as a result of loss of feedback inhibition of low antral ph on gastrin-producing g cells (witzel et al., ) . not all histamine h antagonists produce identical effects. other cytological changes have been reported with famotidine, another h -receptor antagonist. this agent produced a dose-related increase in the prevalence and degree of eosinophilic granularity in chief cells of the stomach in toxicity studies in rats but not dogs (burek et al., ) . electron microscopy showed an increase in electron density of zymogen granules and it was argued that these effects were the result of secondary inhibition of pepsin secretion or turnover due to inhibition of acid secretion. cytoprotective agents of prostaglandin type produce different forms of diffuse gastric hyperplasia. rats treated with , -dimethyl prostaglandin e hourly for weeks, not only developed forestomach alterations (see above) but also thickening of both the body and antral mucosa. in the body mucosa, these changes were the result of a proportional increase in the total mass of surface and foveolar mucous cells, mucous neck cells, chief cells, parietal and endocrine cells as well as connective tissue. this was largely as a result of increase in cell number, although parietal cells also increased in size (reinhart et al., ) . unlike treatment with gastrin and gastrin analogues there was an increase in number of surface and foveolar mucous cells associated with increase in mucus content. misprostal, a synthetic prostaglandin e methyl ester analogue also produced diffuse glandular hyperplasia, characterised by lengthening of gastric pits and increased mucous secretion in the preclinical safety studies in dogs and rats (kotsonis et al., ) . this glandular hyperplasia not only affected the body but also the antral mucosal. studies with tritiated thymidine showed that the labelling index was reduced in rats treated with misprostal, suggesting hyperplasia following administration of prostanoids is a result of an increase in cell survival and decrease in cell shedding rather than an increase in cell proliferation (fich et al., ) . levin ( ) has reviewed the effects of prostaglandins of the e series on the gastrointestinal tract of dogs and rodents. a dose-related diffuse hyperplasia of the gastric glandular mucosa has been reported in both rats and cynomolgus monkeys given human recombinant epidermal growth factor. the gastric mucosa was thickened and there was a increase in the number of undifferentiated cells particularly in the neck region and upper part of the gastric glands (breider et al., ; reindel et al., ) . mitotic figures were also numerous in the upper reaches of the mucosa. the lower parts of the gastric glands were generally less affected. the large increase in the number of undifferentiated cells may have a functional effect on gastric acidity and function (vinter-jensen, ) . administration of recombinant growth hormone has also been reported to induce thickening of the gastric glandular mucosa in dog toxicity studies along with typical growth hormone-induced changes in other organs, body weight increases and insulin-like growth factor (prahalada et al., ) . the pyloric and fundic mucosa showed histological evidence of hyperplasia of the mucous neck cells. thickening of the gastric glandular mucosa as a result of an irregular proliferation and cystic dilatation of gastric glands associated with inflammation characterises a number of non-neoplastic conditions in the stomach of man and laboratory animals. cystic change with chronic inflammation and foveolar hyperplasia is observed in biopsies taken from the edge of chronic gastric ulcers in man (franzin and novelli, ) . ménétrier's disease ('polydadenomes en nappes'), a rare disease found primarily in middle-aged men is also characterised by enlarged gastric folds, foveolar hyperplasia and gastric glandular cystic dilation (berenson et al., ; wilkerson et al., ) . although its pathogenesis remains elusive, increased expression of transforming growth factor α (tgfα) and the epidermal growth factor receptor has been described (demsey et al., ) . as tgfα is an epithelial cell mitogen that inhibits gastric acid secretion and increases gastric mucin, and transgenic mice that overexpress tgfα in gastric mucosa develop a similar condition (see below), it was suggested by demsey et al. ( ) that tgfα might have an important role in this condition. similar changes have been observed in animals in association with infestation of the gastrointestinal tract by parasites (jubb and kennedy, ; cook et al., ) . laboratory rodents may develop a similar pattern of changes spontaneously with advancing age, although the cause of this change remains uncertain. the distinction between adenomatous hyperplasia and adenoma is not clear cut. nevertheless, adenomas are usually defined as localised or focal proliferative lesions with well-ordered glandular patterns with a clear boundary with the surrounding normal mucosa. they are usually exophytic or polypoid in nature but adenomas with localised downward growth are described (mohr, ) . proliferation of the gastric glandular mucosa has been well characterised in the laboratory mouse because certain strains have a particular tendency to develop this condition spontaneously with advancing age (stewart and andervont, ; rowlatt et al., ) . hyperplasia also occurs spontaneously in conventional laboratory strains employed in carcinogenicity bioassays. its prevalence can be influenced by environmental factors such as housing (chvédoff et al., ) , food restriction (rehm et al., ) and the administration of xenobiotics (poynter et al., ; betton et al., ) . similar gastric changes have also been reported to occur in mice thymectomised shortly after birth (suzuki et al., ) . histologically, these changes in mice are characterised by hyperplasia of the foveolar and neck regions of the body mucosa (fig. ) . in advanced cases this is accompanied by elongated, tortuous, or dilated glands lined by simple columnar or cuboidal epithelium, devoid of parietal or chief cells. the abnormal cells show only mild cellular pleomorphism and mitotic activity. the abnormal glands dis-place normal glandular tissue and may penetrate through the muscularis mucosa to reach the muscularis externa and serosa. step sections demonstrate continuity between these glandular elements and a total absence of metastatic spread in the adjacent tissues and lymph nodes. the lamina propria also shows increased amounts of smooth muscle and collagen accompanied by variable numbers of lymphocytes and other chronic inflammatory cells. oedema may be observed and blood vessels are often dilated. the antral mucosa remains relatively unaffected. histochemistry has shown variable mucin secretion of the altered glands. some glands are devoid of mucin, others show an increase in sulphomucin as revealed by the high-iron diamine technique (greaves and boiziau, ) . it has been suggested that these features are similar to ménétrier's disease in man and might have a similar pathogenesis (dempsey et al., ; takagi et al., ) . the aetiology of the spontaneous condition in the mouse is uncertain. the occurrence of similar lesions in thymectomized mice has given rise to the suggestion that autoimmune damage to the gastric mucosa may be responsible (kojima et al., ) . the presence of circulating anti-parietal antibodies and the decrease in the number of parietal cells in thymectomized mice suggested that autoimmune damage can occur to parietal cells with compensatory chronic stimulation and proliferation of the generative zones (suzuki et al., ) . however, based on findings in female han nmri mice, rehm et al. ( ) showed that this proliferative condition can develop in mice in the absence of antiparietal antibodies. they demonstrated that this change is associated with an increase in the number of antral gastrin cells, raising the possibility of a hormone or paracrine mechanism. a similar proliferative form of gastropathy has been reported in mice which overexpression transforming growth factor α (tgfα), a potent mitogen and member of the epidermal growth factor family of peptides. tgfα acts by binding to and activating the tyrosine kinase of the epidermal growth factor receptor. transgenic mice overexpressing tgfα develop severe adenomatous and cystic hyperplasia of the gastric glandular mucosa starting from about months of age along with loss of mature parietal cell numbers and a diminution in gastric acid production (takagi et al., ) . the degree of change was to some extent dependent on the genetic background on which the transgene operated. an increased prevalence of similar changes have been reported in cd- mice treated with the novel histamine h -receptor antagonist sk&f for months (betton et al., ; . although treated mice developed hyperplasia of gastric neuroendocrine cells similar to that observed in rodents treated with other antisecretory agents, they also showed an increase in the severity of glandular hyperplasia. like the spontaneous condition, these changes were characterised by thickening of the mucosa by hyperplasia of the foveolar and neck regions, and downward proliferation of glandular elements into gastric glands (betton et al., ) . poynter et al. ( ) have also reported similar glandular hyperplasia in the mouse stomach associated with histamine h -blockade with the agent ioxtidine. these findings were similarly associated with hyperplasia of neuroendocrine cells. adenomatous polyps have also been reported in the pyloric antrum of c bl/ j mice treated for weeks with the synthetic progestin, cyproterone acetate (tucker et al., ) . these were single, pedunculated and well-differentiated lesions showing little evidence of dysplasia. the mechanism for the induction of these polyps is unclear although they may have been hormonally mediated as progesterone receptors have been identified in gastric tissue (wu et al., ) . although usually less prevalent and less exuberant than in mice, the aged rat also develops proliferative gastric glandular changes spontaneously. these changes are characterised by hyperplasia of the foveolar and mucin-secreting cells of the body mucosa, development of cystic glands lined by simple mucous or flattened cells, accompanied by chronic inflammatory cells, prominent blood vessels and smooth muscle in the lamina propria (greaves and faccini, ) . the antrum remains relatively unaffected. proliferative alterations can be induced by administration variety of xenobiotics as well as following surgical procedures that induce chronic reflux of normal intestinal contents. for instance, hyperplasia of the gastric mucosa, notably over the lesser curvature has been described in rats following the so-called bilroth ii gastrectomy that allows reflux of intestinal and biliary secretions into the stomach (kobayashi et al., ) . a proliferative condition of the gastric mucosa has been shown to develop following long-term treatment of rats with an ulcerogenic regimen of aspirin. female sprague-dawley rats given mg/kg of aspirin in % methylcellulose once daily orally by gavage for months followed for periods of up to months without treatment, developed focal proliferative changes at the sites of healed ulcers, mainly in the mucosa of antrum or antral-body junction (st john et al., ) . these lesions were characterised by the presence of proliferating gastric glands lined by columnar, cuboidal or flattened epithelial cells in the mucosa, which also extended through the muscularis mucosa. mucus content of these glands was variable but when present was principally acidic in type, as shown by staining with alcian blue at ph . . the lesions were accompanied by increased collagen in the lamina propria, endarteritis and an infiltration of lymphocytes, plasma cells and mast cells. the lesions were not associated with the development of carcinoma following months' observation and it is probable that they were the result of the chronic damage and repair induced by aspirin treatment. hyperplasia of the gastric glandular mucosa also occurs in rats following the administration of powerful genotoxic carcinogens, although characteristically in association with of atypical histological changes and ultimately carcinoma. these changes have been best characterised in sequential studies with the rat using the carcinogen n-methyl-n´-nitro-n-nitrosoguanidine at doses low enough to avoid overt gastric ulceration and regenerative hyperplasia. it was shown that hyperplasia developing under these conditions occurs diffusely both in the body and antral mucosa. furthermore, the changes occurred earlier in the antrum than in the body and were focal or polypoid in character (tsiftsis et al., ) . involvement of the antrum in this way is quite unlike the spontaneous hyperplasia of the rat gastric mucosa. histologically, this form of hyperplasia is characterised by lengthening of the foveolae and neck regions both in the antrum and body. hyperplastic pits or foveolae show increased secretion of sialomucins and sulphomucins with a concomitant loss of neutral mucins. polychlorinated biphenyls such as arochlor , which produce intestinal metaplasia and adenocarcinoma in the stomach of rats, also induce proliferative alterations characterised by proliferative cystic lesions in the mucosa associated with inflammation and fibrosis (morgan et al., ) . in common with lesions induced by genotoxic agents, these changes are found primarily in the antrum and pyloric regions, zones of predilection for the development of gastric carcinoma in man and experimental animals. it is important to distinguish between the various hyperplastic and adenomatous conditions found in the gastric glandular mucosa in laboratory animals that are not associated with neoplasia from those which precede the development of carcinoma. this distinction is complicated by the fact that proliferative changes associated with the development of cancer both in man and laboratory animals possess features in common with lesions not associated with neoplasia. however, a key distinctive feature is the degree of epithelial dysplasia. dysplasia is considered to be the lesion common to gastric conditions in man such as atrophic gastritis and gastric polyps that have been linked with a significantly increased risk of gastric cancer. although the term dysplasia may be less widely employed in experimental pathology, similar dysplastic changes to those occurring in man have been characterised in laboratory animals in which precancerous gastric lesions have been studied (tsiftsis et al., ) . it therefore represents a unifying concept in the assessment of proliferative changes in the gastric glandular mucosa of laboratory animals. as defined by an international group concerned with the diagnosis of preneoplastic conditions in the stomach of man, the principle features of dysplasia are (i) cellular atypia; (ii) abnormal differentiation; and (iii) disorganised mucosal architecture (morson et al., ; nagayo, ) . cellular atypia is characterised by nuclear pleomorphism, hyperchromasia and stratification of nuclei, increased nuclear-cytoplasmic ratio and loss of cellular and nuclear polarity. abnormal differentiation is shown by reduction or alteration in the normal secretory products of the mucosa. disorganised mucosal architecture is shown by irregularity of crypt structure, back-to-back glands, budding and branching of crypts and intraluminal and surface papillary growths. it is important to assess gastric mucosa very carefully for the features of dysplasia when hyperplastic gastric changes are found in treated animals. in the rat gastric cancer model employing the carcinogen n-methyl-n´-nitro-n-nitrosoguanidine, dysplastic changes were shown to start in the proliferating neck region of hyperplastic zones (tsiftsis et al., ) . these changes were characterised histologically by irregular growth patterns of glandular cells showing reduced mucin secretion, numerous mitoses and enlarged pleomorphic nuclei. these atypical glands were observed to extend downwards, eventually replacing normal gastric glands and ultimately penetrating the muscularis mucosa forming infiltrating adenocarcinomas of variable differentiation. the antrum developed these changes earlier than the body mucosa (tsiftsis et al., ) . these considerations were important in the safety evaluation of the histamine h receptor antagonist, tiotidine (ici , ) a guanidino-thiazole derivative that also produced proliferative gastric lesions in the stomach of rats in a month carcinogenicity study (streett et al., ; . these changes were found mainly in the pyloric region and were characterised histologically by superficial erosions and irregular pyloric glands lined by cells with basophilic cytoplasm and enlarged hyperchromatic nuclei. some atypical glands penetrated the muscularis mucosae. dysplastic lesions situated primarily in the pyloric region were also associated with the development of invasive carcinoma in some rats. extensive histological sectioning of the stomach in rats treated with tiotidine for only months also revealed evidence of early proliferative changes (streett et al., ) . therefore, these lesions produced by tiotidine possessed more in common with those induced by powerful carcinogens such as n-methyl-n´-nitro-n-nitrosoguanidine than the benign, species-specific proliferative change of little or no relevance for human safety. interestingly, mice treated for months with tiotidine were devoid of dysplastic changes in the gastric mucosa (streett et al., ) . one of the most remarkable examples of drug-induced gastric alterations in rodent bioassays is the hyperplasia of enterochromaffin cells and development of carcinoid-like neoplasms in the stomach of rats treated with omeprazole (havu, ) . omeprazole is a substituted benzimidazole which inhibits gastric acid secretion by blocking the enzyme h + , k + -atpase, the proton pump of the parietal cells, in a specific and dose-dependent manner (fellenius et al., ) . although in rats there is a increase in number of gastric argyophilic cells with increasing age, rats treated with omeprazole for weeks showed a marked, dose-related and diffuse increase of argyophilic, non-argentaffin cells in the basal half of the oxyntic fundal mucosa (havu, ) . these changes were more marked in female than in male rats but were not observed in the bioassay in which cd- mice were treated with similar doses of omeprazole for weeks. these diffuse changes in the rat stomach were associated with focal hyperplasia of argyrophilic cells. these focal lesions were also associated with a doserelated increase in larger focal nodular lesions of argyrophilic cells, some of which were undoubtedly locally infiltrating carcinoid tumours. these nodular argyrophil lesions posed the usual problems of differential diagnosis of endocrine hyperplasia and neoplasia (see endocrine system, chapter xiii) and distinction of hyperplasia from neoplasia was uncertain. histologically, nodular lesions were composed of multifocal anastomosing solid or pseudoacinar cords of proliferating, regular cells with uniform nuclei and moderately abundant fine granular pale cytoplasm. these nodules showed little or no cellular pleomorphism or mitotic activity but clear evidence of submucosal infiltration without involvement of the muscularis externa was observed in some cases. the overall light microscopic features were similar to those of gastrointestinal carcinoid tumours reported in man. the incidence of gastric carcinoids was reported to be as high as % in females in the high dose group but only a few cases observed in similarly treated males (ekman et al., ; havu, ) . electron microscopy of the altered argyophil cells confirmed the presence of electron-lucent, vesicular granules, frequently with small irregular dense cores characteristic of enterochromaffin cells of the stomach. immunocytochemical study showed that these cells contained histidine decarboxylase which is found normally in gastric enterochromaffin cells which produce and store histamine . other findings reported in rats treated with omeprazole have been a proportional increase in the number and size of non-endocrine cells of the fundus (blom, ) , an increase in the number and immunostaining properties of the antral gastrin-containing g cells and hypergastrinaemia (bishop et al., ; creutzfeldt et al., ) . all functional and morphological changes following treatment for days were fully reversible after days' drug withdrawal (creutzfeldt et al., ) . as a result of these treatment-related increases of these normally rare gastric carcinoids in the rat bioassay with omeprazole, clinical trials with this agent were suspended until it was agreed that the endocrine alterations were a result of prolonged drug-induced achlorhydria. it was postulated that omeprazole causes a prolonged inhibition of acid secretion in the rat, which causes activation, and subsequently hyperplasia of antral gastrin cells and marked hypergastrinaemia. hypergastrinaemia in turn stimulates enterochromaffin cells of the fundus, which in time results in enterochromaffin hyperplasia . this argument is supported by the fact that similar morphological findings are reported in chronic atrophic gastritis and other achlorhydric sites in man (solchia et al., ; müller et al., ) and that antrectomy in the rat prevents the appearance of enterochromaffin hyperplasia following treatment with omeprazole . although mild dose-related gastric argyrophil cell hyperplasia was noted in dogs treated with omeprazole for year, neoplasms of the stomach were not observed during this time period. why mice neither developed neither argyrophil hyperplasia nor gastric carcinoids with a similar treatment regimen is not clear, as the mechanism of action of omeprazole is similar in rat, dog and mouse. however, as the duration of action of omeprazole is shorter in the mouse, it was postulated that sustained inhibition of gastric acid secretion over hours is necessary to activate increased gastrin secretion from antral cells (havu, ) . it has also been suggested that the mouse possesses fewer gastric enterochromaffin cells than the rat and shows a much lower serum gastrin response to omeprazole treatment (ekman et al., ) . duration of action or potency may also be the explanation for the lack of reports of carcinoid neoplasms in rats following inhibition of gastric acid secretion by the histamine h -receptor blockers cimetidine and ranitidine. neither of these drugs completely inhibits gastric acid secretion in the rat for hours (leslie and walker, ; larsson et al., ) , although mild gastric neuroendocrine hyperplasia has been recently described in cimetidine-treated rats (hirth et al., ) . however, the long-acting h -receptor antagonist sk&f produced gastric carcinoid neoplasms when administered at a high dose ( mg/kg) to rats for years (figs. and ) (betton et al., ; . although this dose level of sk&f did not entirely suppress gastric acid secretion and control gastric ph over hours, plasma gastrin levels remained elevated at - times control values over this period. in a -month oral carcinogenicity study in cd- mice at the same dose level ( mg/kg), a diffuse neuroendocrine cell hyperplasia and multifocal glandular hyperplasia or neoplasia was also observed (betton et al., ) . similarly, loxitidine, a potent, non-competitive, insurmountable histamine h -antagonist produced hyperplasia of neuroendocrine cells and carcinoid tumours in the gastric fundus of both rats and mice after years' treatment in diet and drinking water, respectively (poynter et al., (poynter et al., , . other histamine antagonists bl- and ici have been reported to produce neuroendocrine neoplasms in the stomach of rats and rats and mice, respectively (hirth et al., ; streett et al., ) . wormsley ( ) reviewed the considerations in the risk-benefit analysis of agents intended for long-term administration for peptic disease. drugs of other classes also cause hyperplasia of gastrin-containing cells. immunocytochemical study using antigastrin antibody revealed increased gastrin cell numbers in the antral mucosa of dogs given high doses of adrenocorticosteroids for weeks and these changes were accompanied by enhanced serum and tissue gastrin levels (delaney et al., ) . these results suggest that adrenocorticosteroids have a trophic effect on gastrin-containing cells. in human patients hypergastrinaemia is also produced by pharmacologically induced hypochlorhydria although this is usually only slight and hyperplasia of enterochromaffin cells has not been observed (soll, ) . a complicating factor in drug safety evaluation is the association of gastric cancer in both man and laboratory animals with n-nitroso compounds. some of the most effective stomach carcinogens in laboratory animals have proved to be nnitroso compounds particularly since sugimura and fujimura ( ) induced gastric adenocarcinomas in rats with n-methyl-n´-nitro-n-nitrosoguanidine dissolved in drinking water. furthermore, epidemiological evidence associating nnitroso compounds with human cancer is also fairly strong for the stomach (corea et al., ; pocock, ) . the formation of n-nitroso compounds is theoretically possible with a variety of compounds that contain amino groups. it has been suggested that the formation of nitrosamines occurs in vivo under the acidic conditions in the stomach following dietary ingestion of nitrite, nitrates and secondary amines (mirvish, (mirvish, , . low levels of preformed nitrosamines are also present in some commercial pelleted diets for laboratory animals, principally derived from fishmeal (edwards et al., ) . calculations based on dietary intake and nitrosatability of precursors and carcinogenicity of derivatives have suggested that the risk that arises from endogeneous nitrosation is highly variable but highest from ureas and aromatic amines (shephard et al., ) . a number of drugs in widespread clinical use have been shown to produce nnitroso products in acidic aqueous media, although the extent to which this occurs in actual therapeutic use is unclear (gillatt et al., ) . some clinical evidence suggests that nitrosation of therapeutic agents can occur in clinical practice. for instance piperazine, a cyclic secondary amine, widely used as an antihelmintic drug, has been shown to form small quantities of n-mononitrosopiperazine in the human stomach as measured by gas chromatography-thermal energy analysis (bellander et al., ) . however, n-mononitrosopiperazine has not been shown to be carcinogenic in rodents (love et al., ) . n,n´-dinitrosopiperazine, carcinogenic to the upper gastrointestinal tract in rodents (lijinsky and taylor, ) , was not detected in man after administration of piperazine under the same circumstances (bellander et al., ) . the possibility of nitrosation is not usually taken into account in the testing of carcinogenic potential of novel drugs as bioassays are usually only performed with parent compound. however, concerns about nitrosation have arisen in subsequent clinical practice. an example of this was the proposal that a few gastric cancers found in patients whilst being treated with the histamine h receptor antagonist cimetidine, were the result of treatment (elder et al., ; reed et al., ; hawker et al., ) . it now seems likely that all those observed cancers associated with cimetidine were incidental (penston and wormsley, ) . however, at that time concerns were increased by the theoretical possibility that cimetidine has the potential be nitrosated in vivo (elder et al., ) . a further factor was the concept that the treatment-induced gastric secretory inhibition with subsequent bacterial colonisation of the stomach rendered the conditions conducive to the generation of n-nitroso compounds from normal dietary constituents (reed et al., ; penston and wormsley, ) . these concerns appear to be unfounded. long-term surveillance studies with cimetidine have shown no causal link between its clinical usage and gastric malignancy (colin-jones et al., ; langman, ) . in addition, carcinogenicity bioassays performed with cimetidine, cimetidine plus nitrite and nitrosocimetidine have not shown any tumorigenic effect in the gastric mucosa (anderson et al., ) . a -year study in dogs in which multiple gastric biopsies were taken at intervals of approximately months have also shown no indication of gastric hyperplasia, dysplasia, intestinal metaplasia or neoplastic change . although complacency is certainly not warranted with respect to the nitrosation of therapeutic agents in vivo, the risks of development of gastric malignancy from such drugs when administered on a short-term basis are probably very small (world health organisation, ) . even for gastric antisecretory agents which may be administered for longer periods of time, the balanced view would also permit development of novel agents provided they are not obviously mutagenic or carcinogenic in the usual preclinical studies and are not particularly liable to undergo rapid nitrosation. most carcinomas of the glandular mucosa are adenocarcinomas, whether induced by the potent genotoxic carcinogens or therapeutic agents. these tend to develop in the antral region in rodents (streett et al., ; szentirmay and sugar, ) . they range from those with well differentiated tubular or papillary features to poorly differentiated forms with trabecular, mucoid or signet ring features (tatematsu, ) . squamous metaplasia within adenocarcinoma can also be observed. stroma may be abundant with pronounced chronic inflammatory infiltration and hyalinisation. metaplastic cartilage and bone has also been described (szentirmay and sugar, ; mohr, ) . gastric adenocarcinomas induced in dogs by n-methyl-n´-nitro-n-nitrosoguamide show similar histological features although their reported distribution in the stomach appears more variable (fujita et al., ) . histological criteria for the diagnosis of invasive adenocarcinoma in experimental animals may vary between individual pathologists. some retain the old criteria of stewart and co-workers ( ) who defined invasive cancer as a neoplastic growth reaching the serosa. it is now considered more appropriate to apply criteria of use in human diagnostic pathology (see mohr, ) . unequivocal invasion of the submucosa is sufficient evidence of an invasive and therefore malignant process (greaves and faccini, ) . the small intestine is of major importance in drug safety evaluation for it represents the primary site of drug absorption. in view of its length and the presence of villi, it possesses an enormous surface area of specialised absorptive epithelium. furthermore, ingested substances have an extended residence time in this part of the gastrointestinal tract. the canine model has been one of the most popular for the study of drug absorption because the dimensions of the canine gastrointestinal tract permit administration of dosage forms intended for clinical use in humans. for this reason, factors, which influence drug absorption, have been better studied in dog and man than many other species. however, data from dog and man not only suggest that diverse factors influence drug absorption from the small intestine but that there are considerable species differences. residence time is of particular importance for drugs, which are incompletely absorbed because differences in mucosal contact time can be expected to result in differences in the fraction absorbed. dressman ( ) has shown using the heidelberg capsule technique that small intestine transit time in dogs is varies from between and over minutes whereas in man equivalent times are between and minutes. these results suggest that absorption of poorly absorbable drugs is likely to be quantitatively less although more variable in dogs than in man. however, these differences do not explain why some poorly lipophilic drugs such as chlorothiazine, acyclovir and phosphalinic acid are more extensively absorbed in dogs than in man. intestinal ph is consistently higher in dogs than in man so that drugs with half maximal absorption ph in the range ph - may also be expected to be absorbed at different rates in man and dog (dressman, ) . physiological and anatomical differences in the small intestine of other test species particularly rodents and humans are also likely to have an impact on drug absorption although many of these factors are still poorly understood. in addition to the small intestine acting as an absorptive surface, it is becoming increasingly obvious that it plays an important part in the metabolism of drugs (breckenridge, ) . although monoxygenase activity is relatively low in the gut compared with the liver, conjugation mechanisms are efficient and activity of udp-glucuronosyltransferase and glutathione-s-transferase are as high or even higher than in the liver (hänninen et al., ) . in addition, the gastrointestinal microflora not only possesses metabolic capacity itself but also can influence the turnover rate of mucosal cells and subsequent exfoliation and release of enzymes into the lumen (hänninen et al., ) . gastrointestinal metabolising activity is important because that the mucosa is exposed to high concentrations of xenobiotics in toxicity studies, and this can influence their overall bioavailability (chhabra and eastin, ) . studies in untreated rats have shown that the concentration of total cytochrome p in small intestinal microsomes is only about % of that found in liver microsomes (bonkovsky et al., ) . however, it exists in at least two forms and as in the liver, its activity can be induced by xenobiotics. it has been shown that in the rat, the concentration of cytochrome p and drug metabolising enzyme activity increases in intestinal epithelial cells as they move from crypt to villous tips and they are found in greater concentration in the proximal two-thirds of the small intestine than in the distal third (hoensch et al., ; bonkovsky et al., ) . bonkovsky et al. ( ) also showed that the phenobarbital-inducible form of cytochrome p represents less than % of total p in the small bowel, but as in the liver, phenobarbital treatment can increase this form to about % of total cytochrome p in small intestine cells. furthermore, it has been shown that drug metabolising activity in the tips of the villi in the duodenum is greater in rats fed a conventional diet than a semisynthetic diet and that the activity depends critically on the absorption of iron from the intestine (hoensch et al., ) . glutathione is also present throughout the entire mucosa, although in rats, cells at the tips of the villi contain less than cells located more basally, whereas related enzymes γ-glutamyl transpeptidase and glutathione-s-transferase show highest activity in the villous tip region (ogasawara et al., ) . the fact that these enzyme activities are highest in the duodenum and lowest in the terminal ileum suggests that detoxification systems for exogenous compounds are greater in the proximal small intestine. the small intestinal mucosa is constructed not only to act as an absorptive surface but also as a barrier to potentially pathogenic substances and microorganisms. although the main cell population of the epithelium is composed of absorptive cells, other major epithelial cell types, the mucous (goblet) cells, paneth cells and endocrine cells have important protective functions. in addition, specialised epithelial cells, the microfold (membranous or m) cells are located in the epithelium over peyer's patches. these cells form part of the other important protective system of the intestine, the gut associated lymphoid tissue (galt) or mucosal associated lymphoid tissue (malt). the mucosal lining is in a constant state of renewal. enteric epithelium possesses the fastest rate of turnover of any tissue exceeded only by a few rapidly growing neoplasms . in normal circumstances, the constant turnover of small bowel mucosa is maintained by equilibrium between cell production in the crypts and cell loss at the tips of the villi. there are intrinsic controls within the mucosa itself. exogeneous substances, intraluminal secretions, mechanical and neural factors as well as alterations in blood flow all possess potential to influence mucosal cell kinetics . all main epithelial cell types are believed to arise from undifferentiation columnar cells at the crypt base (cheng and leblond, ) , although mucous cells may also arise by proliferation of partly differentiated mucous cells in the crypts. as cell division is limited to crypts, the cell population in the crypts have high activities of enzymes such as thymidine kinase that are involved in nucleic acid synthesis (imondi et al., ) . the complete cell cycle lasts about - hours in rodents and at least hours in man. enteric epithelium is completely replaced within - days in mice and rats and within - days in man . after two or more divisions in the crypt cells migrate to the villus, lose ability to incorporate thymidine and differentiate into mature cells equipped with enzymes associated with nutrient absorption (imondi et al., ) . cell migration in the rat is completed more rapidly in the ileum than in the jejunum principally as a result of the lower villous height in the ileal mucosa (altman and enesco, ). migration terminates by loss of cells from the tip of the villi. surrounding the crypt is a sheath of fibroblastic cells. these cells also undergo synchronous division and migration with the epithelial cells, maintaining the intimate relationship between the epithelium and supporting tissues (parker et al., ) . mature absorptive cells are important in the active and passive transport of nutrients as well as in the endocytosis of macromolecules. they are characterised by the presence of a striated or brush border which is seen in haematoxylin and eosin-stained sections as a refractile bi-laminar band. the inner, wider lamina corresponds to the microvillous region that is associated with the presence of neutral mucosubstances in most species. the outer, thinner band corresponds to the glycocalyx, which is composed principally of acidic mucosubstances (sheahan and jarvis, ) . this outer band of the brush border shows histochemical staining predominantly for sulphomucins in most species including mouse, hamster, dog and rhesus monkey, although in the duodenum of the rats and in the entire human small bowel sialomucins predominate in this layer. electron microscopy of the absorptive cells shows that the surface of absorptive cells is covered by tightly packed and well developed microvilli approximately µm long and . µm wide. these are considered the first site of entry of food substances into the cell. the plasma membrane of microvilli is associated with fine filamentous projections, which probably represent the polysaccharide chains of the glycocalyx (bennett, ) . as the glycocalyx is composed of a network of polysaccharides, it has been suggested that it may behave like an ionexchange resin, be able to bind certain lectin-like molecules or trap substances in its matrix so providing a site for efficient intraluminal digestion (bennett, ; goldberg et al., ; king et al., ). the plasma membrane shows a trilamina structure at ultrastructural level. freeze fracture replicas from the microvilli which cleave this membrane through the plane of apposed non-polar groups of the lipid bilayer, demonstrate smooth complementary surfaces studded with small particles. these particles represent integral globular proteins of the plasma membrane. some of these intramembranous particles, mostly those of nm diameter show irregular outlines with a central pit and are believed to represent gap junctions or transport channels (yamamoto, ) . a particularly important aspect of the absorptive cell membrane is its high concentration of disaccharidases such as sucrase, maltase and lactase, related to the absorption of sugars. alkaline phosphatase activity is also abundant on the surface of absorptive cells, although its precise role here uncertain (owen and bhalla, ) . immunocytochemical demonstration of alkaline phosphatase provides a useful tool to examine the effects of xenobiotics on intrinsic membrane glycoproteins in the small intestine (hasegawa et al., ) . enterokinase, the glycoprotein enzyme, which initiates the activation of pancreatic zymogens by converting trypsinogen to trypsin, is also present in the brush border and glycocalyx of the small intestinal epithelium, both in man and animals. immunocytochemical studies have demonstrated that in man this enzyme is located in the duodenum and proximal jejunum but not ileum, colon and stomach (hermon-taylor et al., ) . these enzymes constitute integral structural proteins of the cell membrane with active sites protruding from the cell surface. they are synthesised by the absorptive cells (blok et al., ) . the lateral surfaces of absorptive cells are in direct contact with neighbouring cells and firmly attached to each other by terminal bars or junctional complexes. a terminal bar comprises an apically situated tight junction or zonula accludens, a central zone, the zonula adherens, below which is situated a desmosome or macula adherens. the junctional complexes are relatively impermeable to macromolecules. studies with labelled tracers in the rat jejunum have shown that horseradish peroxidase (molecular weight , , diameter nm) and ferri-tin (molecular weight , , diameter nm) do not penetrate junctional complexes (yamamoto, ) . below the junctional complexes, the cell membranes interdigitate and the intercellular space widens towards the cell base, a feature that may be important in the movement of electrolytes and water across the intestinal epithelium (rhodin, ) . the cytoplasm of absorptive cells contains smooth and granular endoplasmic reticulum, free ribosomes and mitochondria. the golgi apparatus is located above the nucleus. the apical part of the cytoplasm is devoid of organelles except for a tight meshwork of filaments called the terminal web. filaments of actin within the microvilli are linked to the terminal web and this is believed to be important in the movement of microvilli (moosker and tilney, ; moosker et al., ) . goblet cells are much fewer in number than absorptive cells in the small intestine but they increase in number from the duodenum to the lower ileum. they are important in the production of mucus, which remains on the surface of the mucosa as a viscous layer and acts as the first line of defence against intestinal pathogens. goblet cells are characterised by the presence of abundant mucous droplets formed by the golgi complex and which accumulate in the apical part of the cell cytoplasm. histochemical study shows that neutral mucosubstances are present in the goblet cells found in crypts and on the villi in the entire small bowel mucosa of most species including man but there is an interspecies variation in the population of sialo-and sulphomucins (sheahan and jarvis, ) . in the mouse, sulphomucins predominate but among rats considerable individual variation in the proportion of sialo-and sulphomucins is reported. in the hamster, sulphomucins are more prominent in the proximal and sialomucins in the distal small bowel. in the dog, both sulphomucins and sialomucins are found with predominance of one or other in individual animals. staining for acidic mucins is less intense in the goblet cells of the small intestine in man compared with non-human primates but sialomucins are predominant in both species. a few goblet cells in the distal ileum in man also contain sulphated mucins (sheahan and jarvis, ) . paneth cells remain located near the crypt base throughout the small intestine (sandow and whitehead, ) . they are found in rodents and man but typically not in carnivores such as dog and cat (rhodin, ; . they are characterised by the presence of numerous eosinophilic cytoplasmic secretory granules between about . and µm diameter that contain various enzymes and mucosubstances. particular care is needed in fixation and staining for optimal demonstration of paneth cells for they rapidly degranulate after death and granules are destroyed by acetic acid fixation. formalin and mercuric fixatives appear appropriate methods and they permit staining with methylene blue, lendrum's phloxine-tartrazine and masson's trichrome (lewin, ) . the apical parts of paneth cells show glucose- -phosphatase, carbonic anhydrase and monoamine oxidase activity and they have been shown to contain lysozyme and immunoglobulins, particularly iga (speece, ; riecken and pearse, ; ghoos and vantrappen, ; . staining for lysozyme appears to be the most practical immunocytochemical stain for the detection of paneth cells in formalin fixed paraffin wax embedded tissue sections. however, other immunocytochemical reagents have been employed in human and rodent tissues. these include antibodies to the cd antigen (ariza et al., ) and to a rat paneth cell zinc binding protein (sawada et al., ) as well as the use of pokeweed lectin for murine paneth cells (evans et al., ) . the paneth cell granules not only contain lysozyme but also a range of antimicrobial peptides such as secretory phospholipid a , α-defensins, also called cryptins. these are believed not only to possess antimicrobial activity but also important in the regulation of cell volume, chemotaxis, mitogenesis and inhibition of natural killer cell activity (ouellette, ) . it has been shown that these substances are released from paneth cells when germ free rats are dosed with the intestinal flora from specific pathogen free rats (satoh and vollrath, ; . however, it has been shown that paneth cells develop under germ free conditions and do not require luminal bacteria or dietary material for their development (ouellette, ) . endocrine cells are also scattered throughout the small intestinal mucosa. they are of both argentaffin and argyrophil types and are situated predominately in crypts. immunocytochemical study shows that they contain a variety of different peptides although gastrin, secretion and serotonin-containing cells have been those most extensively studied (inokuchi et al., ) . in addition to the barrier formed by mucus and epithelial cells, lymphocytes, plasma cells, macrophages, dendritic cells and mast cells also form part of the protective function of the small intestine. some lymphocytes are located within the epithelium mostly, above the basal lamina but below epithelial nuclei (pabst, ) . these lymphocytes are termed intraepithelial lymphocytes and are predominantly of t-suppresser/cytotoxic type in man and laboratory animals (selby, ; martin et al., ; pabst, ) . most lymphocytes in the lamina propria are also t cells but t-helper (cd positive) cells outnumber the t-suppresser/cytotoxic or cd positive phenotype (hirata et al., ; pabst, ; bruder et al., ) . intraepithelial lymphocytes are also mainly t cells (bruder et al., ) . many plasma cells present in the lamina propria produce iga, the major immunoglobulin of mucosal secretions (michalek et al., ) . iga represents another important component of the mucosal barrier between the gastrointestinal mucosa and intraluminal antigens. the main function of iga is to effect immune exclusion by intimate co-operation with non-specific defence mechanisms (brandtzaeg et al., ) . plasma cells in the lamina propria produce dimeric iga with two dimeric molecules joined by a joint (j) piece. a secretory component (sc), a glycoprotein expressed on the basolateral surface of epithelial cells acts as a receptor for dimeric iga and as a transport system for iga to the gut lumen where monomeric iga is secreted (brandtzaeg et al., ) . morphometric analysis of iga-containing immunocytes in the rat ileal mucosa using immunocytochemical staining has shown that the number of these cells varies with alterations in the microbiological status of intestinal contents (rodning et al., ) . a significant reduction in iga-containing lymphocytes and plasma cells was observed following microbial reduction associated with gnotobiosis, probably reflecting decreased microbial antigenic stimulation. experimental studies using labelled mesenteric lymphocytes also suggests that local microenvironments are important in the distribution of there cells in the intestinal wall (mcdermott et al., ) . peyer's patches are the most prominent aggregates of lymphoid tissue in the gastrointestinal tract and constitute important sites at which antigens from the gut lumen encounter immune competent cells which are responsible for the initiation of immune responses. peyer's patches are located on the ante-mesenteric wall of the small bowel and consist principally of collections of lymphoid follicles. in man, peyer's patches are more common in the ileum (cornes, ) but in mice they are more uniformly distributed (owen and neumanic, ) . in rats they are also more numerous in the distal than in the proximal small intestine and the number of follicles in patches usually varies from to but sometimes many more may be seen in any particular section (martin et al., ) . peyer's patches vary between rat strains. a comparative study showed that in fischer rats they are smaller than those in wistar rats (bruder et al., ) . particular care in selection and orientation of tissue blocks is therefore essential for any form of quantitative or semiquantitative assessment of peyer's patches. peyer's patches are more than simple aggregates of lymphoid follicles. they consist of lymphoid follicles surrounded by a corona of small lymphocytes principally of b-cell type. the interfollicular area contains post-capillary venules with specialised cobblestone type epithelium (yamaguchi and schoefl, ) and many t lymphocytes. beneath the epithelium, over the bulging follicles (dome area), mixtures of t and b lymphocytes, plasma cells and macrophages can be seen (pabst, ) . immunohistochemical study in rats demonstrates the presence of w / -positive t (cd ) cells in the interfollicular area. in the rat, many cells with macrophage morphology also stain with the w / (cd ) monoclonal antibody in the interfollicular zone (bland and warren, ; martin et al., ) . immunocytochemical study of the peyer's patches in the mouse has also shown considerable heterogeneity of staining patterns, particularly under the dome epithelium (ermak and owen, ) . similar patterns have been observed following immunohistochemical study of peyer's patches in man (spencer et al., ) . the epithelium overlying the peyer's patch follicles (dome area) contains specialised epithelial cells, called microfold, membranous or simply m cells. these cells have been identified in many species including rats, mice, hamsters, dogs, monkeys and man (owen and bhalla, ; wolf and bye, ) . these cells differ functionally from other enterocytes by their ability to transport large molecules such as ferritin and horseradish peroxidase and particulate matter from the lumen to the underlying lymphoid tissue (owen, ; jeurissen et al., ) . they have also been shown to be the site of penetration of reoviruses into the epithelium and they can transport vibrio cholerae and other organisms (wolf and bye, ; smith et al., ) . m cells therefore form weak points in the intestinal wall which transport intact antigen and macromolecules to the follicles where they can be processed and be transported to lymph nodes with consequent iga immune responses. this contrasts with the uptake of soluble antigens, which can be taken up by ordinary epithelial cells and transported in the circulation of the villi to be ultimately trapped in the spleen possibly to evoke an igm/ igg response (jeurissen et al., ) . understanding of these cellular and molecular characteristics is critical for the design of mucosal vaccines for pathogens that exploit this pathway (neutra, ) . no simple, specific histological markers for m cells have been identified for use in routine histological sections although many specialist techniques can be applied (reviewed by gebert et al., ) . this has hampered their study because they are still best identified on the basis of their ultrastructural characteristics or ability to capture and transport macromolecules (hamzaoui and pringault, ) . the m cell shares tight junctions and desmosomes with adjacent epithelial cells but it has fewer and shorter microvilli than absorptive cells. there is lack of a well organised terminal web. vesicles are abundant in the apical cytoplasm but lysosomes are reduced in number. attenuated cytoplasmic processes may be seen embracing lymphocytes (owen and nemanic, ; wolf and bye, ) . a cardinal feature is the presence of a intraepithelial pocket on the basal membrane which acts as an internal docking site for lymphocytes, such that they are filled by b and cd positive lymphocytes, macrophages and dendritic cells that move between underlying follicles and the epithelium (ermak et al., ; farstad et al., ) . with care, these attenuated microvilli can be seen at light microscopy in semithin plastic sections (wolf and bye, ) . cytochemical analysis has also demonstrated that they can be distinguished at light microscopic level by markedly reduced alkaline phosphatase activity on their terminal surface, in contrast to the dense reaction product produced by other enterocytes (owen and bhalla, ) . as the glycoproteins on the surface of m cells are different to those on surrounding cells, they can be selectively stained by labelled lectins, although these patterns are different in different species. for instance, mouse m cells are labelled by the fucose-specific probe ulex europaeus and to some extent anguilla anguilla (giannasca et al., ) . mucosal mast cells also appear to be involved in the immunological defence of the gastrointestinal tract. they respond by proliferation, migration and discharge of granules during nematode infestations (miller, ) . it has been shown in the rat that mucosal mast cells of the gut differ in several ways from connective tissue mast cells. these differences result in poor preservation of mast cells of the gut if the usual metachromatic staining techniques employed for the demonstration of mast cells in tissue sections (wingren and enerbäck, ) . histochemical study suggests that mucosal mast cells differ from connective tissue mast cells by a lower degree of sulphation of glycosaminoglycans and different spatial relationships of protein and glycosaminoglycans in their granules. these cross link following formalin fixation in a way, which is sufficient to prohibit cationic dye binding. wingren and enerbäck ( ) showed that these staining difficulties can be surmounted in tissues fixed in formaldehyde by staining in toluidine blue for prolonged periods of time ( - days), a procedure which allows adequate penetration of the toluidine blue molecule. optimal histopathological study of the small intestine is complicated by its length and mucosal fragility. it is important to avoid vigorous washing procedures or any form of excessive manipulation of the unfixed bowel, as artefact caused by washing may confound interpretation of changes induced by xenobiotics (roe, ) . combination of artefact due to washing, autolysis and the presence of neutrophils can produce a histological appearance that mimics in vivo damage. although careful visual inspection of the intestine and sampling of appropriate segments for histological examination is usually sufficient for routine examination, various forms of 'swiss roll' techniques are helpful for more complete study. rolling the unfixed, opened rodent intestine around a wooden stick prior to freezing or fixation is one proposed method (moolenbeck and ruitenberg, ) , although this method risks undue manipulation of the unfixed tissue. another more versatile technique applicable to rodent, large animal and human intestine can be performed after fixation. the unfixed opened bowel is pinned flat on a cork or board and fixed in a bath of formal saline. after fixation, the full thickness of rodent intestine can be rolled, transfixed by a pin and embedded in paraffin wax. likewise the mucosa of the intestine of large animal species or humans can be rolled after fixation by separating it from the muscularis externa (filipe and branfoot, ) . inflammation and ulceration of the mucosa occurs as a result of stress, infection with bacteria, viruses, and infestation by parasites or as a direct result of the effects of xenobiotics or ionising radiation. antimitotic or radiomimetic agents as well as ionising radiation are liable to adversely affect the rapidly dividing cells of the small intestine with resulting breakdown of the mucosal barrier. the ulcerogenic activity of non-steroidal anti-inflammatory drugs may also be expressed in the small bowel mucosa. different agents also act in synergy to enhance damage to the small bowel mucosa. an important example is the effect of drugs that depress the immune system and permit the development of pathological infections by microorganisms of the opportunistic type in the small intestine. the histological features of the inflammatory process in the small intestine are not usually specific for a particular agent. it is important to search for evi-dence of microbiological organisms and viral inclusions, which can indicate the cause of intestinal inflammation and ulceration. associated features in non-ulcerated mucosa such as morphology of the villi, accumulation of abnormal cells or foreign substances and changes in lymphoid cells or blood vessels are also important in the assessment of these changes. a number of organisms including those, which are normal residents of the gastrointestinal tract, can cause inflammatory changes in the intestinal mucosa of laboratory animals. with the notable exception of non-human primates, inflammatory bowel disease caused by microbiological organisms is not usually evident or of concern in most toxicity or carcinogenicity studies. however, when animals are treated with antibiotics, immunosuppressive agents or other drugs, which alter the normal intestinal flora, conditions may favour the proliferation of potentially pathogenic organisms in sufficient quantities to cause overt damage to the mucosa. certain bacterial flora may also act synergistically with intestinal protozoans to produce pathological changes (boorman et al., ) . in non-human primate colonies, gastrointestinal disease remains one of the most important causes of death . in contrast to other laboratory species, histological evidence of intestinal infectious disease is relatively common and may confound the interpretation of gastrointestinal alterations occurring in toxicity studies. although the majority of potentially pathogenic organisms affect the primate colon, a number of bacteria, protozoa and metazoa occur in the small intestine. a detailed review of the protozoa and metazoa occurring in the primate gastrointestinal tract has been published (toft, ) . owen ( ) has reviewed the parasites of laboratory animals including those of the gastrointestinal tract and the principle reference for identification of metazoa in tissue sections remains that of chitwood and lichtenfels ( ) . organisms which can cause inflammatory disease in the small bowel but which are primarily agents that produce inflammatory disease in large bowel are reviewed under 'colon' (see below). bacillus piliformis is the agent responsible for tyzzer's disease produces intestinal inflammation and ulcers in rats, mice and hamsters. susceptibility of different species and strains to experimental infection with bacillus piliformis is variable. for instance, c bl, balb mice and f rats appear more resistant to infection than outbred syrian hamsters (waggie et al., ) . lesions of variable severity usually occur in the ileum but may also extend into the caecum and colon. severe infections are characterised histologically by ulceration of the mucosa, oedema and acute inflammation of the submucosa and muscle coats. muscle may also show focal necrosis. non-ulcerated mucosa is typically infiltrated by polymorphonuclear cells and crypt abscesses form. there is blunting and fusion of villi and reactive hyperplasia and mucin depletion of the overlying epithelium . mucosal lymphoid tissue may also show reactive changes or hyperplasia. filamentous bundles of bacillus piliformis can usually be found in the cytoplasm of both epithelial cells and smooth muscle cells at the edges of necrotic zones. methylene blue, giemsa or silver impregnation techniques such as warthin-starry or levaditi stains are the best stains for the demonstration of these organisms although with care they can be visualised in haematoxylin and eosin stained sections (weisbroth, ) . they are gram negative and pas positive. intestinal infections due to salmonella species are relatively common in the mouse but also occur in the hamster and rat . salmonella typhimurium and salmonella enteritidis are regarded as the organisms typical of murine salmonellosis (weisbroth, ) . lesions occur in the ileum and may extend into the jejunum and caecum. they are characterised by the presence of ulcers covered by fibrinous exudate and associated with diffuse infiltration of the adjacent mucosa by macrophages, neutrophils and lymphocytes. intact crypt epithelium shows mucin loss and reactive proliferative changes. a characteristic feature is the presence of poorly defined granulomatous lesions composed of macrophages mainly in associated lymphoid tissue or peyer's patches. clostridia species, especially clostridia difficile which cause a pseudomembranous colitis in man and laboratory animals (especially hamsters) may also produce inflammation and ulceration in the terminal ileum with histological features similar to those found in the colon (see following). proliferative ileitis (transmissible ileal hyperplasia) is a striking lesion of hamsters affecting the distal segment of the ileum that is associated with intracellular invasion of the intestine mucosa epithelium by bacteria. the organism has not been cultivated, but has been suggested that it is a campylobacter (jacoby, ) although other organisms have been identified in this condition (fox et al., ; peace et al., ) . although it is characterised by hyperplasia of the ileal mucosa in its early stages, an inflammatory phase intervenes in which there is focal necrosis and haemorrhage of the mucosa, crypt abscesses and infiltration of the lamina propria by acute inflammatory cells and macrophages. the histological features of the associated hyperplasia are characteristic. the mucosa is covered by immature, mucin-depleted pseudostratified hyperchromatic epithelium with mitoses extending to the tips of villi and densely basophilic intracytoplasmic inclusions (jacoby, ) . helicobacter jejuni (campylobacter jejuni) is a common cause of diarrhoea in man and may be the causative agent in small intestinal inflammation in laboratory dogs and primates. campylobacter species may be more prevalent in beagle dogs and primates than commonly appreciated. it is important to recognise that dogs colonised with these agents may be susceptible to stress-induced, acute onset gastroenteritis (fox et al., ; reed and berridge, ) . in man this form of bacterial disease is characterised histologically by mucin-depletion, flat-tening and reactive changes in the small bowel epithelium, crypt abscesses, oedema and infiltration of the mucosa by neutrophils, lymphocytes and plasma cells. similar histological findings have been reported in dogs infected with this organism (prescott and monroe, ) . the organisms are gram-negative curved, slender rods, which can be visualised in tissue sections with the warthin-starry stain, a recognised technique for spiral bacteria. the carbol fuchsin technique of gimenez ( ) , first used for the identification of ricketsiae in yolk sac culture and a cresyl fast violet technique are also useful methods for the identification of campylobacter species in paraffin sections (burnett et al., ; mcmullen et al., ) . another campylobacter-like organism, helicobacter pylori (campylobacter pylori) has been identified in human patients with gastritis, gastric and duodenal ulcers. it is an aetiological or predisposing factor in these forms of gastrointestinal disease (marshall and warren, ; rouvroy et al., ; richter-dahlfors et al., ) . spironucleus muris (hexamitis muris) is also a cause of inflammation in the small bowel of rats, mice and hamsters. during overt infestation, organisms are seen extracellularly in crypts and intervillous spaces associated with blunting of intestinal villi, epithelial degeneration and mucin-depletion, reactive epithelial hyperplasia, oedema and leucocyte infiltration (boorman et al., ; wagner et al., ) . the morphological expression of damage is accompanied by decreased levels of disaccharidases such as maltase, sucrase and lactase, which may represent a direct effect of the trophozoites on the brush border enzymes (gillon et al., ) . trophozoites are characteristically elongated symmetrical flagellates approximately - µm wide, - µm long. giardia species represent marginally pathogenic flagellates, which are found in the upper gastrointestinal tract. they are opportunistic agents that can become important in both animals and man with depressed immune function. studies in mice infected experimentally with giardia muris have shown that an early response is an increased infiltration of the epithelium by lymphocytes, predominantly t cells (gillon et al., ) . this has led to the suggestion that the response to infection by these parasite is primarily a cell-mediated immune reaction similar to experimental graft versus host reaction in the small intestine of mice (mowat and furguson, ; gillon et al., ) . depression of the immune response by treatment with corticosteroids has been shown not only to increase parasite numbers in murine giardiasis but also cause recrudescence of occult infections (nair et al., ) . it has also been suggested that decreased gastric acidity can predispose to giardiasis in man (nalin et al., ) . giardia muris (lamblia muris) is sometimes found in the small intestine of rat and mouse but it is common in the hamster (fig. ) . trophozoites appear in histological sections as crescent-shaped structures on the brush border of the intestinal mucosa or in the adjacent lumen. mucosal lesions may be totally ab-sent or there may be blunting of villi, reactive epithelial hyperplasia. a typical feature is increased infiltration of the epithelium and lamina propria by mononuclear cells (boorman et al., ) . another important finding is that lactase, sucrase and maltase levels have been shown to decrease in the small intestine in mice infested with giardia muris (gillon et al., ) . giardia lamblia may colonise the small intestine of non-human primates and of man and produce similar morphological appearances to those found with infestations in rodents by giardia muris. other flagellates such as tritrichomonas muris are also be found in the small intestine of mice, rats and hamsters. the coccidian protozoan parasite cryptosporidium represents a striking example of the close relationship between some human and animal diseases. this organism was first recognised in the gastric glands of mice by tyzzer in and has since been confirmed as a cause of diarrhoea in animals and as a human pathogen. it causes mild diarrhoea in normal subjects especially children and young adults but it can produce severe intestinal disease in immunocompromised individuals (casemore et al., ) . histological examination of the small intestine of laboratory animals infested by cryptosporidium reveals the presence of organisms attached to the mucosal surface, often associated, as in man, with other parasites or infections. they are rounded, weakly basophilic structures - µm diameter in haematoxylin and eosin stained sections but are strongly basophilic following romanowsky staining. transmission electron microscopy reveals the detailed internal structure of cryptosporidium attached to the microvillous surface of the epithelial cells. the various stages in the life cycle have been visualised by light and electron microscopy. infection starts with ingestion of an oocyst containing four sporozoites, which are probably released by the action of digestive enzymes. these attach themselves to the intestinal mucosa and undertake their life cycle attached to the epithelial cells (casemore et al., ) . these organisms have been demonstrated in laboratory species including mice, hamsters, rabbits, dogs and nonhuman primates (cockrell et al., ; rehg et al., ; toft, ; fukushima and helman, ; davis and jenkins, ) . hymenolepis nana (dwarf tapeworm) and hymenolepis diminuta (rat tapeworn) are described in the intestine of rats, mice, hamsters, non-human primates and man (hsu, ) . a variety of other metazoan patients are found in the small intestine of non-human primates, see review by toft ( ) . certain viruses produce inflammatory small bowel changes in mice. mouse hepatitis virus (lethal intestinal virus of infant mice) can cause mucosal epithelial necrosis and inflammation with characteristic compensatory epithelial hyperplasia and the formation of epithelial syncytia . murine rotavirus (epidemic diarrhoea of infant mice) produces swollen enterocytes of small and large bowel with fine cytoplasmic vesiculation with little or no inflammation but dilated lymphatics and vascular congestion. cytoplasmic acidophilic inclusions, - µm diameter, are characteristic findings . electron microscopic examination shows cytoplasmic vesicles arising from the rough endoplasmic reticulum, which contain virus particles and electron dense granular material . a mouse adenovirus may also produce large basophilic intranuclear inclusions in the epithelial cells of the small intestine and caecum. k virus infection produces lesions in the jejunal and ileal mucosa of mice. histological features are characterised by mild polymorphonuclear infiltration, with ballooning of occasional endothelial cells within intestinal villi. intranuclear inclusions can be demonstrated in these endothelial cells by light microscopy using appropriate fixation (greenlee, ) . a variety of viruses have been isolated from the gastrointestinal tract of nonhuman primates including viruses of man (kalter, ) . however, they appear to be relatively infrequent causes of gastrointestinal disease and when disease is caused by these viruses it usually affects other organs. not only can non-steroidal anti-inflammatory agents such as indomethacin and phenylbutazone produce gastric ulceration but also penetrating ulcers of the small bowel of laboratory animals (shriver et al., ) . imaging studies with indium-labelled leukocytes in man have also suggested that subclinical intestinal inflammation is associated with long-term therapy with non-steroidal anti-inflammatory drugs (bjarnason et al., ) . it has been postulated that indomethacin-induced intestinal ulcers in rats and dogs are produced by a prostaglandin-independent mechanism, different from the manner in which gastric ulceration is induced (tabata and okabe, ; whittle, ) . conversely, satoh et al. ( ) have suggested that similar mechanisms are responsible for both gastric and intestinal ulceration. they showed that indomethacin-induced gastric ulceration developed in the body mucosa in fasted rats but in the antrum and small intestine in rats given indomethacin minutes after a -hour period of refeeding following a -hour fast. there was good temporal correlation between the development of intestinal ulcers and inhibition of prostaglandin synthesis (satoh et al., ) . the ulcers in the small intestine were morphologically similar to those occurring in the stomach and they were distributed mainly in the mucosa on the mesenteric aspect of the bowel wall. single-dose studies with indomethacin and ibuprofen in rats conducted by suwa et al. ( ) have demonstrated differences between pathology of induced gastric and intestinal damage. gastric damage was superficial, occurred within hours and was fully repaired weeks after dosing. ulcers in the jejunum and ileum reached a maximum area at - hours after dosing, occurred on the mesenteric border, penetrated through the muscularis mucosa and were accompanied by inflammation and oedema. ulcers were still present weeks later. rainsford ( ) has shown that potent intestinal ulcerogens such as indomethacin inhibit the incorporation of radioactive s sulphate into glycoproteins of the upper intestinal mucosa as well as the stomach of rats. this may decrease the capacity of the mucus in the intestine to act as a buffer for hydrogen ions. indomethacin given orally to dogs in doses of . mg/kg/day for - days was also shown to produce intestinal ulceration. these ulcers were deep, punchedout lesions, many of which were lying over peyer's patches (stewart et al., ) . some ulcers involved the whole circumference of the small intestine wall. histologically, the ulcers were associated with an intense inflammatory response principally of mononuclear cells, which infiltrated the bowel wall to the serosa particularly adjacent to peyer's patches. it was suggested that this distribution of ulcers was a result of an exaggerated immune response to normal intestinal antigens. these antigens may have been produced by inhibition of suppresser cells in peyer's patches, following depression of prostaglandin synthetase by indomethacin (stewart et al., ) . special dye techniques, scanning and transmission electron microscopy have also shown that non-steroidal anti-inflammatory drugs also produce adverse effects on the small intestine mucosa without overt pathological changes being evident by light microscopy (brodie et al., ; djaldetti and fishman, ) . following administration of aspirin to mice for weeks, shortening and erosion of microvilli and increased numbers of goblet cells were only demonstrated in the duodenum and jejunum by scanning and transmission electron microscopy (djaldetti and fishman, ) . morphometric studies of the intestinal mucosa of indomethacin-treated mice have also shown widespread alterations to columnar cells, goblet cells and paneth cells suggesting generalised effects on mitotic activity and crypt loss (ettarh and carr, ) . such submicroscopic findings support the idea that non-steroidal anti-inflammatory agents may induce damage to the small intestine more commonly than supposed. although non-steroidal, anti-inflammatory drugs are the best-known drugs with adverse effects on gastrointestinal mucosa, small intestinal inflammation and ulceration can also produced by other agents through different mechanisms. anticancer drugs and other therapeutic agents, which affect cell proliferation, depress the bone marrow or the immune system can also produce intestinal mucosal necrosis, haemorrhage, inflammation and opportunistic gastrointestinal overgrowth when administered to dogs or rodents in high doses (fig. ) bregman et al., ) . lymphoid infiltrates without tissue fig. . section of small intestine from a wistar rat treated with a -day course of an antiproliferative anticancer drug. the mucosa shows mucus-depletion, loss of villous architecture as well as a reparative and inflammatory response. (he, × .) damage were also reported in the small intestine of rats treated with human recombinant interleukin- (anderson and hayes, ) . agents of particular toxicological interest are cysteamine, propionitrile and their structural analogues as well as -methyl- -phenyl- , , , -tetrahydropyridine (mptp) which are capable of producing ulcers of chronic type in the duodenum of rats and mice (szabo and cho, ) . these compounds vary in their ulcerogenic capacity but they are all able to produce ulcers of chronic type with crater formation, granulation tissue and reactive changes in adjacent mucosa in the anterior and posterior wall of the proximal segment of the duodenum of rodents. although these different agents influence gastric acid secretion in different ways, structure-activity relationships suggest that they produce duodenal dysmotility, decrease bicarbonate production and reduce its delivery from the distal to proximal duodenum. these factors decrease the neutralisation of gastric acid in the first part of the duodenum and this may contribute to the development of ulceration (szabo and cho, ) . furthermore, these effects can be attenuated or prevented by dopamine agonists or their precursors whereas dopamine antagonists can potentiate their effects. this suggests that the central or peripheral dopamine-mediated actions of these agents may be involved in the pathogenesis of duodenal ulceration (szabo, ; . using appropriate fixation and staining procedures, fine granular lipid droplets can be normally visualised in the apical parts of epithelial cells covering the upper third of small intestinal villi. administration of drugs and chemicals may produce an excessive accumulation of lipid through specific effects on lipid metabolism or as part of general cellular toxicity. in the preclinical toxicity studies with , -di-tert-butylamino- -acetyl- -methylpyridine (sa h - ), an inhibitor of glucose transport intended for use as an anti-obesity drug, lipid accumulation occurred in the lamina propria of the small intestinal villi of sprague-dawley rats and guinea pigs but not in dogs or primates (visscher et al., ) . after administration of sa h - to rats, there was progressive accumulation of lipid droplets in the epithelial cells over the tips of the duodenal villi demonstrable by osmium tetroxide staining. ultrastructural examination revealed uniform electron-lucent droplets within profiles of the smooth endoplasmic reticulum and golgi apparatus. lipid droplets increased with time, accumulated and coalesced to form large droplets in the lamina propria. larger droplets were phagocytosed by macrophages in the lamina propria but there was no evidence of epithelial damage or necrosis. changes were most pronounced in the duodenum but were also noted to a lesser extent in jejunum and ileum but not in colon or stomach. sequential studies using electron microscopy showed that lipid rapidly accumulated within several hours in the profiles of smooth endoplasmic reticulum and golgi apparatus of the epithelial cells and formed droplets or chylomicra in the intercellular space. the absence of any other subcellular changes or evidence of derangement of protein synthesis suggested that sa h - altered the pathways of lipid resynthesis or transport. this was consistent with the distribution of the lipid in the upper third of the jejunal villous epithelium, a zone most active in lipid absorption, resynthesis and transport (dobbins, ) . it was suggested fatty change might have taken place because of alterations in the sugar moiety of chylomicra brought about by interference with glucose transport (visscher et al., ) . lipid droplets which stained with oil-red-o in formalin-fixed frozen sections and showed uniform electron density characteristic of neutral lipid were also observed in the epithelial cells and macrophages in the lamina propria of jejunum and duodenum and mesenteric lymph nodes in rats given a synthetic '-dodecyl glutaramide ester of erythromycin (gray et al., ) . unlike the erythromycin base, rats poorly tolerated this ester. it appeared that the ester was absorbed unhydrolyzed and converted to chylomicron-like droplets, which then accumulated in the macrophages of the lamina propria and local mesenteric lymph nodes, without overt damage to epithelial cells. accumulation of lipid in epithelial cells of intestinal villi has been observed in rats following administration of puromycin (friedman and cardell, ) and ethionine (hyams et al., ) , agents which have inhibitory effects on protein synthesis. detailed morphological study of the intestinal epithelial cell in rats treated with puromycin have shown that there is concomitant accumulation of lipid with a decrease in the quantity of rough endoplasmic reticulum and golgi membranes (friedman and cardell, ) . these changes were in keeping with the concept that lipid accumulates as a result of inhibition of the synthesis of membrane components of the golgi by the rough endoplasmic reticulum which are important for the transport of lipid. in addition to lipid droplets forming as a result of altered lipid metabolism, they may form in the epithelial cells of the intestinal mucosal as a result of a direct toxic effect of the ingested drugs on the small intestinal mucosa. in such instances atrophy of villi and degenerative changes in the epithelial cells may also be observed (see following). the small intestinal mucosa is also one of the many sites at which drug-induced accumulation of polar lipids form laminated structures (myeloid bodies) or crystalloid structures within lysosomes. this form of lipid storage disorder is produced by diverse amphiphilic cationic drugs in both man and laboratory animals probably as a result of drug interaction with polar lipids rendering them difficult to digest (lüllmann-rauch, ) . species differences in susceptibility and tissue distribution of phospholipid are probably not only related to physiochemical characteristics of the inducing drugs which influences their ability to permeate selective biomembranes and react with different lipids, but also to tissue concentrations of drugs achieved and the ability of organs to metabolise parent drug to less amphiphilic products. in general terms, this form of disorder is characterised by membrane-bound, acid phosphatase-positive cytoplasmic inclusions, which on ultra-structural study are seen as lamellated or crystalloid structures in lysosomes. these appearances are characteristically reversible on cessation of treatment with the inciting agent. an example of this phenomenon occurring in the small intestine is provided by the iodinated amphiphilic drug, amiodarone, which has been used clinically in europe for the past years in the treatment of angina and more recently in the control of supraventricular cardiac arrhythmias. its adverse effects in man are believed to be the result of accumulation of drug in lysosomes particularly in liver, skin and eye (d'amico et al., ; shepherd et al., ) . when high doses of amiodarone were administered orally to rats and beagle dogs, multilamellated lysosomal inclusion bodies accumulated first in the jejunal mucosa and mesenteric lymph nodes before becoming widely distributed in other organs particularly in the lungs (mazué et al., ) . in both rats and dogs the small intestinal lesions were characterised by the presence of foamy macrophages with pale finely vacuolated cytoplasm and condensed eccentric nuclei within the lamina propria of the jejunal villi (mazué et al., ) . mesenteric lymph nodes were also involved early after the onset of treatment. in the dog, jejunal villi were somewhat flattened and widened or showed a variable degree of villous atrophy, most marked in the proximal and middle jejunum (vic et al., ) . electron microscopy confirmed the presence of lamellated lysosomal bodies distending macrophages. the early accumulation of foam cells in the jejunal macrophages was probably a reflection of the disposition of drug following oral absorption. although similar lipidosis was seen in many organs following intravenous administration in dogs, more lipidosis was seen in the jejunum after oral dosing. moreover, there were species differences in sensitivity to these changes, baboons being relatively insensitive compared to dogs. fischer rats were very sensitive to these changes compared to sprague-dawley rats and wistar rats were almost completely resistant to lipidosis induced by amiodarone under similar conditions (mazué et al., ) . similar cytological changes have been reported in cells of some organs in patients treated with amiodarone (d'amico et al., ; shepherd et al., ) . villous shortening or stunting results when the proliferative activity of the crypt epithelium is reduced or under circumstances in which crypt cell proliferation is insufficient to compensate for increased cell loss as a result of mucosal cell damage. decreased cell proliferation can be seen segments, which are surgically bypassed, or following decreased food intake, parenteral nutrition, hypophysectomy or thyroidectomy bastie et al., ) . as adrenergic factors are important in the control of small intestinal epithelial cell division, drugs that alter α or β adrenoreceptor activity may influence the proliferative capacity of the epithelium. in mice, increased α or β receptor stimulation by appropriate agonists (e.g. phenylephrine) diminishes proliferation of crypt cells but proliferation is increased by stimulation of α receptor activity (kennedy et al., ) . yohimbine, a α -antagonist also reduces cell proliferation in the same animal model. some of the effects of these agents may be mediated by changes in splanchnic blood flow . the detailed morphological study of the small intestinal mucosa in the rat following hypophysectomy by bastie et al. ( ) has shown a reduction in the height of the small intestinal villi associated with reduction in mitoses in the crypt epithelium. the number of goblet cells was shown to fall particularly in the jejunum and the number of paneth cells increase in the ileum. ultrastructural examination showed decreased height of the microvilli of absorptive cells and a lower number of their intracytoplasmic organelles and ribosomes. there were also significant decreases in brush border enzyme activities of alkaline phosphatase, aminopeptidase, maltase and lactase reported about week following hypophysectomy. substances which reduce mitotic activity and therefore lower regenerative capacity of the intestinal epithelium also produce shortening or stunting of small intestinal villi and eventually flattening of the mucosa. a wide variety of anticancer agents and antiviral drugs with radiomimetic properties interfere with cell division in the crypts thereby reducing the number of epithelial cells produced. histologically, the effects of such agents are characterised by blunting, shortening or complete atrophy of villi. mitotic activity is reduced in the crypts and the crypts become dilated and lined by flattened cells. the overlying epithelium loses its normal regular arrangement and cells show pleomorphic nuclei with irregular chromatin patterns. increased numbers of inflammatory cells may infiltrate the lamina propria and epithelium. ulceration, haemorrhage and secondary infection of the gut wall ensue if there is overwhelming cell damage. comparison of the gastrointestinal toxicity expressed by antimitotic anticancer drugs of different classes in rodents, dogs, non-human primates and man have suggested that there is a higher degree of correspondence between effects in man and dog than between man and other species (owens, ; schein et al., ) . in studies with the antiviral agent acyclovir, a radiomimetic effect was noted in the gastrointestinal tract of dogs at high doses but not rodents (tucker et al., ) . another example is the villous atrophy described in rats following treatment with an antibacterial agent ici , . this was believed to arise as a result of both interference with cell division and a direct effect on the surface epithelial cells (murgatroyd, ) . the effects of ici , were characterised by atrophy of villi with dilatation of crypts and atypical features in the crypt epithelium suggestive of an effect on mitotic activity. in addition, vacuolated lipid-laden epithelial cells were observed over the tips of villi accompanied by reductions in the numbers of goblet cells and reduced activity of acid and alkaline phosphatase, esterase, adenosine triphosphatase, glucose- -phosphatase and succinic dehydrogenase, compatible with a direct adverse effect on superficial mature epithelial cells. a variety of factors stimulate cell proliferation in the small intestinal epithelium. these include enterectomy, increased feeding and stimulation of autonomic nerves. administration of neurotransmitters, thyroxine, growth hormone, corticosteroids, testosterone, gastrin, glucagon and recombinant epidermal growth factor may also stimulate epithelial cell proliferation breider et al., ; reindel et al., ; vinter-jensen, ) . in rats hypothalamic damage, hyperthyroidism, tube feeding, diabetes mellitus and insulin injections have been shown to produce intestinal hyperplasia (mackay et al., ; levin and smyth, ; jarvis and levin, ; forrester, ) . most causes of greater cell production lead to increased villous height and mucosal hyperplasia, although intense crypt cell proliferation as a compensatory regenerative response can be associated with villous atrophy (see previous). the compensatory response to the surgically resected or bypassed intestine has been the focus of the most detailed studies of increased cell renewal in the small intestine. partial resection in both rats and man is accompanied by increased villous height and crypt length (hanson et al., ) . this is primarily the result of hyperplasia for it has been shown that the numbers of cells per unit length of villus remains unchanged (hanson et al., ) but there is an overall increase in the cell population of villus and crypt (hanson and osborne, ) . dna/rna ratios also remain largely unaltered . no gross changes in villous shape have been reported after resection and the total number of crypts remains constant. although increased intestinal uptake of substances from the bowel lumen occurs in hypertrophied segments per unit length of bowel, disaccharide and dipeptidase activities are normal or even decreased after resection suggesting a comparative immaturity of cells in the residual mucosa. functional adaptation therefore is achieved by a larger number of cells, the individual absorptive capacity of which is not increased . increased numbers of specific goblet cell populations are also seen in hyperfunctional states. following jejuno-ileal bypass operations in rats, increased numbers of pas-positive goblet cells develop in the villi and crypts of the hyperfunctional segments of the duodenum, jejunum and ileum (olubuyide et al., ) . mucin histochemistry using the high-iron diamine and alcian blue techniques have shown that the goblet cells in the hyperfunctional segments contain increased sialomucins in the villi and crypts of the jejunum and ileum but not in the duodenum and increased sulphomucins in the distal ileal segment. sialomucin production may reflect relative cellular immaturity of the more rapidly proliferating cells under these circumstances. however, as sialic acid conveys more viscoelastic properties to mucin, it has been suggested that the goblet cells change following intestinal bypass fulfils a protective function against the increased flow of gastrointestinal contents (olubuyide et al., ) . a number of nutritional factors, particularly dietary fibre, can influence the proliferative characteristics of the small bowel mucosa. carefully controlled studies in rats given different forms of dietary fibre have shown that the prolif-erative characteristics of the small intestine can be modified by both the quantity and the quality of the fibre. one study has shown a decrease in the length of villi, crypt cell hyperplasia and shorter transit times in rats fed pectin-supplemented diet but an increase in mucosal growth without alteration in relative differences in crypt and villous length with guar supplementation compared with rats fed fibre-free diet (jacobs, ) . another rat study has shown that pectin feeding leads to increased mucosal area and height associated with an increase muscle mass (stark et al., ) . these different effects may be the result of differences in solubility, gel formation, water holding capacity, effect on transit time and ion exchange activity or bile acid adsorption of the different fibres. interactions between dietary constituents are complex. for instance, in rats the effects of % dietary cholestyramine, a non-absorbable ion exchange resin, on small intestinal histomorphology have be shown to depend on interaction of dietary factors (burkhardt et al., ) . administration of an inhibitor of cholesterol biosynthesis, α-cholest- -( )en- β-ol- -one, to rats for up to days was also shown to produce enlargement of the small intestine in a way which was morphologically similar to the changes found following intestinal bypass (smith et al., ) . the enlargement was most marked in the proximal segment of the small intestine and progressively diminished towards the ileocaecal junction, sparing the stomach, caecum and colon. histological examination and morphometric analysis revealed an increase in smooth muscle mass, lengthening of the villi as well as an increase in the depth and cellular proliferation in the crypts of lieberkuhn without evidence of cell damage or fatty change. like the changes following jejunal bypass procedures, there was also an increase in acid mucosubstances in the goblet cell population overlying the villous mucosa (smith et al., ) . the mechanism for this change in the rat was not clear, particularly as intestinal hyperplasia was not seen in baboons treated with this -ketosterol for long periods. however, it was suggested that it was an adaptive response, possibly related to inhibition of cholesterol metabolism and cholesterol absorption from the diet, particularly as the laboratory diet employed in the rat study was particularly low in cholesterol. local and systemic changes in hormones and various transmitter substances also influence the number of cells in the small intestinal epithelium. morphometric studies of the small intestinal mucosa in mice following gastrin administration have shown increases in villous area associated with decreases in microvillous area, increased number of goblet cells and paneth cells . studies in which rats were treated with the prolactin-inhibitor, ergocryptine, have shown that the total number of mucous cells and the number staining with alcian blue at ph . increase in the ileal crypts, possibly as a result of increased synthesis of sulphated mucosubstances (gona, ) . in this context, it is of interest that chronic treatment with the rauwolfia neuroleptic, reserpine, causes an increase in the sulphation of goblet cell mucin in the small intestine as demonstrated by alcian blue staining at ph . and the high iron diamine technique without changes in the goblet cell numbers (park et al., ) . agents which affect activity of the sympathetic nervous system can also alter epithelial cell proliferation in the small (and large) intestine. treatment of rats with adrenaline, isoprenaline, phenylephrine, phentolamine and yohimbine all result in decreased mitotic activity of jejunal and colonic crypt cells (tutton and helme, ; kennedy et al., ) . by contrast, administration of metaraminol, clonidine, propranolol, prazosin and labetolol as well as simultaneous injection of propranolol and adrenaline all resulted in an increased rate of crypt cell proliferation (kennedy et al., ) . these results suggest that agents that stimulate α adrenergic receptor activity and those that are α -antagonists and β-adrenergic receptor antagonists increase proliferative activity in the rodent intestinal mucosa. caffeine is also reported to produce an increase in thickness of the intestinal mucosa when administered in high doses to rats (lachance, ) . this raises the possibility that intestinal mucosal hyperplasia can be produced by phosphodiesterase inhibition and resultant increases in intracellular camp in a similar way to the hypertrophy induced in salivary tissue. this is supported by recent findings in rats treated for periods of up to months with the inotropic vasodilator, ici , , a phosphodiesterase inhibitor intended for treatment of congestive cardiac failure. administration of high doses not only produced salivary gland hypertrophy but also marked thickening of the small and large intestinal mucosa. this was characterised histologically by an increase in villous length and deepening of intestinal glands, with a relatively unchanged number of epithelial cells per unit length of gland or villus (westwood et al., ) . although prostaglandin e analogues produce most of their effects in the stomach, increased thickness of the small intestine characterised by longer villi, deeper crypts and increase in cell size have been reported in rats treated with these agents (levin, ) . focal hyperplasia, focal avillous hyperplasia, focal atypical hyperplasia, duodenal plaque, polypoid hyperplasia, polyp -mouse irregular, atypical single or multiple foci of glandular hyperplasia may be found in the small intestinal mucosa of several strains of aged, untreated mice. the lesions are usually located in the first part of the duodenum where they form discrete, raised plaques composed of elongated, irregular or branched glands which replace the normal villous structure of the mucosa (rowlatt and chesterman, ) . the glands are lined by hyperchromatic columnar cells, which show marked pseudostratification and proliferative activity. paneth cells and mucinsecreting goblet cells may also be prominent. some glands are cystic and the stroma is fibrous and infiltrated by chronic inflammatory cells. the lesions become pedunculated or polypoid in appearance and show a fibrovascular core that is infiltrated by inflammatory cells. they resemble adenomatous polyps described in man. the cause of these changes in the mouse small intestine is unknown but their prevalence can be altered by dietary fibre and panthothenic acid deficiency as well as by administration of drugs and chemicals (hare and stewart, ; seronde, ; ito et al., ) . in their study of dba mice, hare and stewart ( ) considered that the lesions were not genuine neoplasms since they were composed of a mixture of cell types, which normally populate the mucosa. furthermore, they suggested that the presence of an inflammatory component in the stroma and the fact that the prevalence of these lesions was increased in mice fed a high roughage diet were consistent with the concept that they represent an inflammatory adenomatoid hyperplasia. seronde ( seronde ( , reported these lesions in mice fed purified diets, particularly when deficient in panthothenic acid. panthothenic acid deficiency was also associated with inflammation and deep penetrating chronic ulcers of the duodenum in affected mice, compatible with an inflammatory aetiology of the lesions. an increase in the prevalence of these duodenal changes was described in cd- mice treated with the synthetic prostaglandin e analogue, misoprostol for months . these authors suggested that the findings posed no real concerns for the safety of patients treated with misoprostol on the grounds that the mouse was unique in this aspect of the response to misoprostol because the mouse had a particular liability to develop such changes in the small intestine. the proliferative lesions were found in a few control cd- mice in the same study. in addition, it was also argued that the lesions were neither neoplastic nor preneoplastic in nature . they were not seen in rats treated with misoprostol for years . lesions characterised by such intense proliferative activity may be difficult to distinguish from neoplastic lesion. indeed chronic administration of hydrogen peroxide to c bl/ j mice in drinking water was not only shown to potentiate the development of a similar type of duodenal hyperplasia but also to produce frankly invasive adenocarcinomas (ito et al., ) . anatomically the large intestine is broadly similar in man and laboratory animals but there are significant functional differences. the rat colon is probably one of the best studied of the laboratory animal species because the rat is widely used for experimental work on colon carcinogenesis . as the canine model is popular for oral dosage-form testing, differences in colonic physiology between dog and man may be better understood than between man and many other species (dressman, ) . in man, as well as in the non-human primates, the large intestine can be divided anatomically into caecum, appendix, ascending colon, transverse colon, sigmoid colon rectum and anal canal. like the small bowel, the colon comprises mucosa, submucosa, muscularis mucosa and serosa. mucosal plicae are only found in the rectum although plicae semilumaris, formed by folds of the entire thickness of the bowel wall, are found in the colon. the large intestine of the dog resembles that of man more than that of most other domestic species. it is a simplified tubular structure only slightly larger in diameter than the small intestine. the colon of the dog is divided anatomically into ascending, transverse and descending parts, but there is no well-defined sigmoid segment. the caecum in dog is a small diverticulum, similar to that found in other carnivorous species and it communicates directly with the colon. the colon of the rat and mouse is shaped like an inverted v that can be divided into ascending and descending segments. there is no clearly defined transverse colon. a characteristic feature in both rat and mouse is the presence of a curved kidney-shaped caecum. its size is intermediate between the large and anatomically complex caecum of herbivores such as the rabbit and the small caecum of carnivorous species. this probably reflects the omnivorous nature and flexibility of the rat and mouse in their dietary habits, particularly their ability to breakdown cellulose (rerat, ) . the caecum of the rat and mouse is a blind pouch from which the colon and ileum exit in close proximity and in which antiperistaltic movements occur. this structure and the presence of bacteria undoubtedly contribute to its ability to function as a fermentation organ in which breakdown of substances can occur in a reasonably controlled milieu (snipes, ) . in rat and mouse, the caecum is the site of absorption of many substances including calcium, magnesium, water and electrolytes vitamin k and fatty acids (snipes, ) . caecectomy has been shown to decrease digestion of carbohydrates and protein and increase loss of faecal water in these species (ambuhl et al., ) . the activity of intestinal microflora in the metabolism of both endogenous and exogenous substances has been demonstrated in the rodent caecum rowland, ) . the usual stock diets for rodents contain abundant plant fibre which provides bulk and fermentable carbohydrate for the microbial population in the caecum. rats fed stock diets have been shown to possess high levels of reductive and hydrolytic enzyme activity (e.g. azoreductase, nitroreductase, nitrate reductase, β-glucosidase and β-glucuronidase) in their caecal contents compared with rats fed purified fibre-free diets . intestines of germ free animals have thinner lamina propria, lower cell turnover, enlarged caecum, altered metabolism of cholesterol, bilirubin and bile salts and larger amounts of mucin in faeces compared with animals possessing normal gastrointestinal microflora (midtvedt, ) . species differences in microflora are also reported. comparative studies with human and rat intestinal microflora have suggested that each population of organisms possesses a degree of autonomous self-regulation and capable of responding quite differently to dietary changes (mallett et al., ) . comparative studies have shown large differences in the numbers of facultative anaerobic gram-negative bacteria in the gastro-intestinal tract of mice from three, major specific pathogen free units in australian laboratories. it was shown that these differences could influence the immune system, susceptibility to infection and experimental results (o'rourke et al., ) . the colon and caecum in man and laboratory animals are lined by a fairly uniform mucosa devoid of villi. columnar cells of two main types cover the surface epithelium. these are absorptive and mucous cells similar to those found in the small intestine. intestinal glands or crypts of lieberkuhn extend downwards from the surface generally as simple, unbranched tubules lined principally by mucous cells with smaller populations of absorptive, endocrine and undifferentiated cells. the mucosa in man and laboratory animals is not entirely flat but shows a slightly corrugated or uneven pattern, which varies with the particular site within the colon. in histological sections of the colon in man, this corrugated pattern is seen as an anthemion-like structure of crypts reminiscent of a greek architectural feature (filipe and branfoot, ) . this is also seen in larger laboratory animal species. in rats and mice the crypts of the caecal mucosa are wider near the lumen than in the crypt base and crypts may be branched, features which may be related to the absorptive function of this zone (snipes, ) . the proliferative zone in the large bowel is found in the lower part of the gland and mitotic figures are normally limited to this zone. as in the small bowel, multipotent, undifferentiated stem cells situated in the gland base give rise to the principle cell types which migrate to the cell surface with subsequent differentiation and alteration of their enzyme activities and morphological features (chang and leblond, ). in studies with mouse aggregation chimaeras in which mosaic cell populations of the intestinal epithelium were localised immunocytochemically, it was demonstrated that the entire epithelium of each adult gland descended from a single progenitor cell (ponder et al., ) . the single progenitor may itself give rise to several stem cells responsible for the cell renewal in the complete crypt. absorptive cells are found most commonly in the surface epithelium but also to a lesser extent in the glandular epithelium. they are morphologically similar to those in the small intestine each possessing apical plasma membranes with uniform microvilli and a well-formed glycocalyx. microvilli of absorptive cells have been shown by electron microscopic study to become longer and denser with increasing distance distally in the gastro-intestinal tract. thus, they are longer and more dense in the ileum than in the caecum and least dense and shortest in the colon, perhaps reflecting their relative absorptive capacity (snipes, ) . this is reflected at light microscopic level by the less conspicuous brush border in the large intestine compared with that in the small intestine. there are species and regional differences in the glycoconjugates found in the brush border of the large intestine, although they generally contain predominantly acidic mucosubstances. in the mouse and rat, sialomucins with some neutral mucins are found. in hamsters, dogs, non-human primates and man both sulphomucins and sialomucins may be seen in the brush border (sheahan and jervis, ) . the glycocalyx is important in the protective function of the colonic mucosa for its disruption by agents such as salicylates has been shown to increase absorption of xenobiotics from the rat rectal mucosa (sithigorngul et al., ) . although there has been some debate about the precise nature of the mucous cell populations based on structural studies in mice and rats (chang, ; , for practical purposes two principle types of mucous cell can be defined. one of these is the typical goblet cell with cytoplasmic mucous droplets forming a goblet shape, which is found both in glandular and surface epithelium. the other type, the so-called vacuolated cell or mucous cell, is found only in the crypts (chang and leblond, ; thomopoulos et al., ) . these vacuolated or mucous cells show empty-appearing vacuoles in the cytoplasm which rather than being empty contain abundant sialomucins of a type different from those in goblet cells (spicer, ; wetzel et al., ) . detailed structural studies and cytochemistry using lectin probes have suggested that these vacuolated cells are able to differentiate into absorptive cells with the cellular apparatus to produce the cell surface glycoconjugates of the glycocalyx (thomopoulos et al., ) . sulphomucins, as demonstrated by the high-iron diamine technique (table ) , generally predominate in the distal colonic segment. in both rat and mouse, goblet cells of the proximal colonic mucosa contain largely sialomucins in the lower parts of the crypts with sulphomucins predominating in the upper parts of the crypt. the distal colon contains largely sulphomucins. the only difference between rat and mouse appears to be the fact that the mouse caecum contains almost exclusively sulphomucins but sulphomucins and sialomucins are found in the rat caecal mucosa. in hamsters, the entire colon contains predominantly sulphomucins. neutral mucins and sulphomucins predominate throughout the dog colon with occasional goblet cells containing sialomucin. in non-human primates, neutral mucins, sialomucins and sulphomucins are seen throughout the colon with sialomucins generally more prominent in the proximal colon and sulphomucins in the distal segment. in man, neutral mucins are found mostly in the caecum. in the caecum and ascending colon, sulphomucins are found in the upper crypts and sialomucins in the crypt base. the converse occurs in the distal colon where sulphomucins predominate in the lower two-thirds of the glands and sialomucins in the upper third of the glands and in the surface epithelium. lectin-labelling (table ) shows even greater heterogeneity of mucins in the colonic mucosal cell population, probably reflecting differentiation patterns and changes in glycosyltransferase activity as cells migrate upwards (freeman et al., ; thomopoulos et al., ) . it has been suggested by jass and roberton ( ) that the two principle changes in pathological processes in the human colonic mucins are loss of oacylation substituents at sialic acid c and c , , and increased sialylation, neither being specific for neoplasia. the colon, like many other tissues also possess drug metabolising activity, although less than in the liver. it has been shown that the activity of cytochromes p involved in hydroxylation of benzo[a]pyrene in microsomes prepared from the colons of sprague-dawley rats retain their activity and responsiveness to inducers better than those in the liver with advancing age (sun and strobel, ) . the lamina propria of the large bowel is arranged in a similar way to that of the small bowel. by virtue of the presence of lymphocytes, plasma cells, macrophages and dendritic cells as well as scattered small lymphoid aggregates or patches, it forms an integral and important part in the mucosal immune defence system. most of the lymphocytes in the lamina propria of the human colonic mucosa, like that of the ileum, have been shown to be t cells with helper t cells out numbering the t-suppresser phenotype (hirata et al., ; pabst, ) . this contrasts with intra-epithelial lymphocytes of the human colonic mucosa which are also t cells but more than % of which possess characteristics of the suppresser/cytotoxic phenotype and only - % being helper t cells. this distribution of lymphocyte subsets is seen immunocytochemically in the rat colon using the monoclonal antibodies w / , w / and mrc ox (see haemopoietic and lymphatic systems, chapter iii). the pan t-cell marker w / shows the presence of t lymphocytes in the lamina propria and most of these are labelled by w / demonstrating their cd helper phenotype (bland and warren, ) . mrc ox demonstrates that few lymphocytes in the lamina propria are of suppresser/cytotoxic (cd ) type, which contrasts, with the high proportion of mrc ox positive lymphocytes in the colonic epithelium (bland and warren, ) . the monoclonal antibodies mrc ox and mrc ox , specific for the rat ia antigen, also label numerous cells with macrophage and dendritic cell morphology in the large bowel of the rat (bland and warren, ; martin et al., ) . mature, small b lymphocytes are relatively uncommon in the colonic lamina propria of man and laboratory animals. however, the lamina propria contains large numbers of plasma cells, which are mainly of iga type, followed by smaller numbers of igm an igg subtypes (pabst, ) . a feature of the colonic mucosa is the presence of lymphoid aggregates also called lymphoid nodules, patches, lymphoid-glandular complexes or microbursa. these are similar to peyer's patches of the small intestine as they are composed principally of lymphoid cells of the b-cell series arranged in follicles with germinal centres with interfollicular and perifollicular zones composed of t cells (pabst, ) . they are distributed along the entire length of the colonic mucosa although they are generally smaller than peyer's patches. in sprague-dawley rats, lymphoid aggregates are usually about mm diameter except in the distal colon where they attain sizes of up to mm in maximum diameter (martin et al., ) . unlike peyer's patches which are characteristically not associated with crypts or villi, the colonic lymphoid aggregates frequently contain irregular atypical mucosal glands which may enter deeply in the lymphoid tissue and penetrate below the muscularis mucosa both in man and laboratory animals scott, ; martin et al., ) . in some strains of rat, cells in these glands express the ia antigen, unlike the other parts of the colonic epithelium (martin et al., ) . these glandular structures, which are intimately associated with lymphoid tissue, may be important in the immune protection of the colonic mucosa, perhaps by acting as a special local receptor for antigens . it has been proposed that these glandular structures represent sites of predilection for the spread of inflammatory disease to the submucosa by allowing microorganisms to pass through the muscularis mucosa (scott, ) . it has also been suggested that they constitute physical weak points in the bowel wall and may play a part in the pathogenesis of diverticular disease of the colon in man . colonic carcinomas induced by dimethylhydrazine in the rat also appear to develop more commonly in the lymphoid aggregates than in other zones (martin et al., ) . m cells have been described over the lymphoid aggregates in the caecum of the mouse (owen and nemanic, ) , see small intestine. although microorganisms are important causes of inflammatory disease in the large intestine of man and animals, among laboratory animals they are usually only significant problems in non-human primates and hamsters. in the strains of rats and mice and in beagle dogs commonly employed in drug safety evaluation, spontaneous disease of the colon as a result of infectious agents is uncommon. nevertheless, treatment with some therapeutic agents may alter the normal bacterial flora to permit overgrowth of pathogenic organisms or disturb the normal balance between antigens in the lumen or control mechanism to evoke inflammation. inflammation induced by organisms may also confound the histological assessment of drug-induced changes in the colon. ulceration and inflammation of the colon as a direct result of administration of potential therapeutic agents is reported in humans although less commonly that in the small intestine. it has been suggested from studies of the effects of anticancer compounds on neoplastic colonic cells that intestinal cells may possess inherent protective properties in the form of an accelerated efflux pump which can serve to protect them from potentially damaging agents (klohs and steinkampf, ) . ulceration and inflammation can be induced by the local application of drugs and vehicles to the rectal mucosa. assessment of these effects in an appropriate animal model is important in the safety evaluation of preparations designed for use in man as rectal suppositories. although inflammatory conditions of the large bowel may possess morphological features typical for some inducing agents, inflammation of the large intestinal mucosa is usually characterised by non-specific histological features. in early or mild inflammation, the surface and glandular mucosa remains intact but shows mucin depletion. this is characterised by reduction in the mucus in goblet cells and increased cytoplasmic basophilia. scattered neutrophils may be seen in the epithelium and adjacent lamina propria. in more severe cases, crypts become filled or distended with acute inflammatory cells (crypt abscesses). the lamina propria is variably hyperaemic and congested and contains increased numbers of mononuclear cells. severe changes are characterised by attenuation or frank erosion of the epithelium and the formation of penetrating ulcers filled with fibrinous exudate and surrounded by intense inflammation, granulation tissue and eventually fibrosis. residual glands may be dilated and lined by flattened epithelium or show reactive changes and mitotic activity. regenerative hyperplasia, which can become florid in chronic ulcerative conditions, is characterised by lengthening, irregularity and cystic dilatation of glands which are often lined by hyperplastic epithelial cells and goblet cells distended with mucin. where ulcerative damage has destroyed glands and supporting stroma, regeneration of glands may not occur in the normal regular fashion and branching of crypts may be evident. clostridium difficile may cause inflammatory changes in the colon of laboratory animals, particularly hamsters, and this may extend into the distal ileum. as in man this form of colitis, often referred to as pseudomembranous colitis, is usually associated with antibiotic therapy. in man it was originally associated with lincomycin and clindamycin therapy but other antibiotics have been implicated. it has been shown that both in man and the hamster experimental model that the enteritis is the result of the toxin produced by clostridium difficile (bartlett et al., ; milligan and kelly, ) . in man this condition is histologically characterised by the presence of plaques or pseudomembranes on the colonic mucosal surface. the pseudomembrane is composed of mucus, fibrin, blood cells, inflammatory cells and cell debris, which has an appearance of streaming from the underlying mucosa. the mucosa may be partly necrotic or mucosal glands are dilated and lined by flattened or hyperplastic cells. the ileal mucosa may show similar changes (milligan and kelly, ) . similar features are observed in the antibiotic-treated hamster although the pseudomembrane is less prominent and it may be distributed more proximally with involvement of the terminal ileum (rehg, ) . in the hamster, the condition is characterised histologically by erosion of the colonic epithelium and the variable presence of a pseudomembranous plaque of mucin and cell debris. intact but affected mucosa is thickened with reactive changes accompanied by mucin loss in the epithelium and infiltration of a hyperaemic and oedematous lamina propria and submucosa by polymorphonuclear cells (rehg, ) . although most instances of this form of clostridia colitis in the hamster have been associated with antibacterial therapy, it has also been reported in untreated hamsters (rehg and lu, ) and those treated with antineoplastic drugs (cudmore et al., ) . similar changes have been reported in antibiotic-treated guinea pigs and rabbits (rehg and lu, ; rehg and pakes, ) . guinea pigs are particularly sensitive to antibiotics especially those active against gram-positive organisms (young et al., ) . as in man, these drugs are believed to alter the intestinal flora, permitting overgrowth of clostridium difficile as well as gram-negative organisms, resulting in a severe and frequently fatal enterocolitis. a study of the disposition of ampicillin administered parenterally to guinea pigs showed that this drug was rapidly eliminated from the systemic circulation and excreted in urine and bile, possibly favouring this effect on flora in the colon (young et al., ) . citrobacter freundii, a gram-negative, short, plump rod and member of the family of enterobacteriaceae, is the causative agent of naturally occurring transmissible colonic hyperplasia of mice. this agent usually produces a mild or even asymptomatic enteritis in susceptible mouse populations, although it is a cause of rectal prolapse in mice (ediger et al., ) . marked strain differences have been noted in mice infected with this organism. nih swiss mice show the most severe histological changes and c bl/ j mice appear the least affected (barthold et al., ) . rats and hamsters seem to be unaffected by citrobacter freundii (barthold et al., ) . microscopic changes are found primarily in the descending colon, although proximal segments of the colon and the caecum may also be affected. an important morphological feature is epithelial hyperplasia, which occurs maximally - weeks after experimental inoculation with citrobacter freundii (barthold et al., ) . the colonic glands are elongated and lined by cells that show mucin depletion or loss of goblet cells, considerable immaturity and mitotic activity. the surface epithelium may be covered with numerous coccobacilli, which can be visualised in routine haematoxylin, and eosin stained sections. crypt abscesses, inflammatory cells in the lamina propria, mucosal erosions and ulceration are also features (barthold et al., ; . in regressing lesions there is a rebound increase in goblet cells, which are often distended with mucin. the colonic glands may be branched or irregular (barthold et al., ) . most laboratory animals are naturally resistant to shigella infections but this is not the case for most non-human primates (takeuchi, ) . in infections with shigella, the colon shows a superficial acute inflammatory reaction comprising oedema, congestion, haemorrhage and infiltration by acute inflammatory cells. the surface epithelium shows mucin loss and formation of microulcers where total destruction of the epithelium has occurred. ulcers can extend into the lamina propria but in general terms the inflammatory process remains relatively superficial (takeuchi, ) . organisms are also located predominantly in the superficial epithelium. another bacterial infection of the gastrointestinal tract, which affects the colon in primates, is that produced by non-tuberculous mycobacteria . large intestinal lesions are characterised by massive accumulation of epitheloid macrophages in the lamina propria, which may extend into the submucosa and muscular layers and along lymphatics to involve mesenteric lymph nodes. small intestinal lesions may also occur, characterised by the presence of similar large macrophages in the lamina propria of villus tips. superficial ulcers may occur in severely affected segments of intestine . acid-fast bacteria are typically found within macrophages. other organs, including spleen, liver, bone marrow and lungs, may also be involved by focal accumulations of bacteria-laden macrophages or occasionally discrete granulomas with multinucleated giant cells. numerous protozoa and metazoa have been described as inhabitants of the caecum and colon of the non-human primate (toft, ) . far fewer are observed in the usual laboratory rodents and beagle dogs. amoebiasis caused by entamoeba histolytica is a widespread disease among non-human primates. it is characterised histologically by the presence of necrotizing ulcers, which reach the muscularis mucosa to form typical flank-shaped ulcers containing or surrounded by trophozoites. extensive haemorrhage may be seen as well as an inflammatory infiltrate composed of neutrophils and mononuclear cells (toft, ) . the ciliate, balantidium coli, can also cause an ulcerative process in the colon of primates, characterised by ulcers which extend down to the muscularis mucosa accompanied by lymphocytic infiltrate and balantidium coli trophozoites of up to µm in greatest diameter (toft, ) . a variety of metazoan parasites can be observed in the primate colon and usually can be reasonably well identified in tissue sections (see review by chitwood and lichtenfels, ) . the nematode of species strongyloides is an important parasite, which may be observed in the intestinal mucosa of primates. oxyurids commonly known as pinworms are essentially innocuous parasites seen in man, non-human primates and rodents. enterobius vermicularis is found in the large intestine and appendix of man and non-human primates, syphacia muris and syphacia obvelata in rodents. oesophagostomum species (nodular worms) are especially common nematode parasites of non-human primates forming characteristic nodules up to mm diameter most frequently on the serosal surface of the large intestine and caecum and adjoining mesentery as well as in other sites in the peritoneal cavity. histologically, the nodules are composed of parasite cell debris surrounded by fibrous tissue and a variable mantle of chronic inflammatory cells and occasional foreign-body giant cells. they are frequently found in close proximity to small arteries and arterioles in the submucosa and subserosa of the colon and may be associated with a local granulomatous arteritis (lumb et al., ) . the inflammatory process may spread to surrounding or draining tissues, particularly if nodules rupture. mild periportal hepatic chronic inflammation is sometimes associated with the presence of this parasite in the mesentery, which may confound interpretation of drug-induced hepatic changes in the non-human primate. although the stomach and to a certain extent the small intestine remain the primary sites of predilection for the ulcerogenic action of non-steroidal anti-inflammatory, the colonic mucosa may become involved under certain conditions. less common complications of non-steroidal anti-inflammatory drugs and potassium chloride therapy in humans are colonic strictures. it appears that nonsteroidal anti-inflammatory drugs produce local inflammation followed by focal scarring of the submucosa with constriction and formation of a mucosal diaphragm whereas potassium causes segmental full thickness scarring and constriction (fellows et al., ; haque et al., ; van velzen et al., ; wolfe et al., ) . another form of induced colon damage has been reported in children with cystic fibrosis, the majority of who take high strength pancreatic-enzyme supplements to control malabsorption (smyth et al., ; fitzsimmons et al., ) . this condition has distinctive pathological features. there is involvement a long segment of ascending colon by a fusiform stenosis primarily as a result of submucosal thickening by deposition of mature collagen. the mucosa appears relatively spared but shows some ulceration and reparative changes (van velzen et al., ) . although it has been suggested that the changes may have been linked to the methylacrylate copolymer used for enteric coating of the high-strength preparations, a case-control study showed a strong relation between high daily doses of the enzyme supplements, accentuated by more recent availability of high dose forms (fitzsimmons et al., ) . in view of their usage for over years, preclinical data on this material is scarce. colonic damage can be induced experimentally by administration of therapeutic agents. dogs administered . mg/kg indomethecin orally each day for periods of up to days developed not only gastric and small intestinal ulceration but also scattered haemorrhagic erosions in the colon and rectum. histologically, these lesions were characterised by loss of superficial epithelial cells, mucus-depletion of glandular epithelium, crypt abscesses, frequently with acute inflammation in adjacent lymphoid aggregates in the submucosa (stewart et al., ) . an example of chemically induced colitis of relevance to safety assessment of therapeutic agents is that induced by degraded carrageenans or synthetic sulphated dextrans. carrageenans are a heterogeneous group of sulphated polysaccharides composed mainly of long chains of d-galactose subunits (d-galactan) derived from red seaweed species which are widely used as food emulsifiers, stabilisers, thickeners and gelling agents (ishioka et al., ) . when carrageenans are degraded by acid hydrolysis into smaller molecular weight fragments of about , - , and administered orally in high doses (e.g. % of diet) to rats, mice, guinea pigs, rabbits and rhesus monkeys, colitis results (sharratt et al., ; marcus and watt, ; benitz et al., ; fath et al., ; kitano et al., ) . similarly, colitis has been induced in rats following administration of a % dietary admixture of dextran sulphate sodium, a sulphated polymer of glucose (a d-glucose) of molecular weight of , (hirono et al., ) and a very high molecular weight d-glucan, amylopectin sulphate (ishioka et al., ) . although histological features of this form of induced colitis vary between study, species and strain, the colitis is generally characterised mucosal ulceration mainly in the caecum but also in the distal ileum, distal colon and rectum. there is mucus-depletion with variable acute inflammatory infiltrate of the in-tact epithelium, crypt abscesses, inflammatory infiltrate of the lamina propria with oedema, hyperaemia and even vascular thrombosis in the submucosa (hirono et al., ; fath et al., ) . increased proliferative activity of the mucosa is confirmed by an increase in the tritiated thymidine index compared with controls (fath et al., ) . in the caecum of rats, ulcers are linear but often circulating the entire circumference of the intestinal wall with subsequent scarring and stricture formation (oohashi et al., ) . ulcerating lesions in the rectum and at the anal margin are associated with squamous metaplasia. both the squamous metaplasia and the regenerative hyperplasia of the columnar epithelium have been shown to progress even after cessation of treatment (oohashi et al., ) . foamy macrophages containing metachromatic material, presumably polysaccharide, are also seen in the lamina propria, submucosa, regional lymph nodes, liver and spleen (hirono et al., ; oohashi et al., ) . the cause of this colitis is unclear. low dose levels, which may be expected to mimic human exposure, do not produce colitis. dextrans, carrageenans and other polysaccharides of molecular weights outside the range , - , tend not to incite colitis. an exception to this is the agent amylopectin sulphate, which has a far higher molecular weight. however, amylopectin is composed of polysaccharide chains, which can be degraded by amylase, and therefore smaller molecular weight fragments may be formed in vivo (ishioka et al., ) . it has been suggested that colonic disease produced by these agents is in some way linked to induced changes in intestinal microflora (marcus and watt, ) although the evidence for this is conflicting (ishioka et al., ) . a recent study in guinea pigs and rats using permeability markers of different molecular weights has suggested that degraded carrageenans enhance intestinal permeability in the absence of overt ulceration (delahunty et al., ) . it was therefore proposed that carrageenan-induced colitis could be the result of increased intestinal permeability to antigenic or inflammatory substances normally resident in the large intestine. moreover, long-term administration of high doses of these agents to rats leads to the development of colorectal cancer despite their being devoid of any mutagenic potential (see below). the only obvious features, which are common to a number of these non-genotoxic agents, is chronic inflammation and increased proliferative activity. the rectal administration of therapeutic agents and surfactants may also induce similar ulcerative and inflammatory changes. chemical colitis resembling pseudomembranous colitis has been reported in man as a result of chemical cleaning agents accidentally induced by endoscopic examination (jonas et al., ) . cellular degeneration, with loss of mitotic activity and mucin depletion can also occur in the colon following treatment with antimitotic drugs. lymphoid infiltrates without tissue damage were reported in the large bowel of rats treated with human recombinant interleukin- (anderson & hayes ) . melanosis coli is a well-described phenomenon in man associated with chronic ingestion of anthraquinone purgatives. it is considered to be due to the excessive accumulation of lipofuscin-like pigment in the macrophages of the colonic lamina propria (schrodt, ; ghadially and parry, ; steer and colin-jones, ) . this pigment probably originates from organelles of epithelial cells or macrophages, which are damaged by treatment. similar morphological changes have been induced in laboratory animals (guinea pigs) by treatment with anthraquinones (walker et al., ) . as a result of these animal studies, walker et al. ( ) suggested that the primary process is a treatment-induced increase in apoptotic bodies in the surface colonic epithelium that are phagocytosed by intraepithelial macrophages and transported to the lamina propria. lipofuscin and iron pigment is occasionally observed in the lamina propria of untreated rodents, notably hamsters, presumably a result of ageing, previous inflammatory processes and haemorrhage. as in other glandular epithelial tissues, hyperplasia may be focal or diffuse with or without atypical cellular features. the term used for hyperplasia with atypical features is atypical hyperplasia in the iarc classification (mohr, ) although others use the term dysplasia. like small intestine, cell proliferation in the large intestinal mucosa can be stimulated by a variety of different factors although these functional adaptive responses have been less well studied. physical stimulation by distension or increased dietary bulk is sufficient to initiate hyperplasia including thickening of the muscle coats (dowling et al., ; stragand and hagemann, ) . one of the most clearly documented forms of compensatory hyperplasia is that which occurs as a response to surgical resection or bypass of a segment of the colon. following resection of a segment of colon in rats, barkla and tutton ( ) showed that the remaining proximal segment of the right side of the colon showed an increase in the thickness of the mucosa and the muscularis externa as well as enlargement of lymphoid aggregates. histologically, the mucosa of the right side of the colon was uniformly thickened showing accentuated folds, elongated mucosal glands with increased height of the surface columnar cells. the changes were most marked up to days following surgery but were less pronounced after days. there was also a significant increase in the mitotic index in the proximal segment at days although at days and later the mitotic index had returned to normal. the distal, down-stream segment showed little or no morphological change but rather a long-lived increase in mitotic activity. it was suggested that these differences were related to the different embryological origin of the segments (barkla and tutton, ) . a similar form of uniform colonic hyperplasia affecting principally the caecal and right-sided colonic mucosa also occurs in rats following oral administration of sulphated dextrans of molecular weight of approximately , (figs and ). oral administration of a wide range of compounds such as raw and chemically modified starches, various dietary fibres, caramels, sugar alcohols (lactitol, sorbitol, mannitol, xylilol), lactose, a synthetic polydextrose, polyethylene glycol and magnesium sulphate to rats or hamsters has also been linked to an increase caecal size and colonic mucosal hyperplasia (leegwater et al., ; roe and bär, ; newberne et al., ; stark et al., ) . the characteristic histological appearance of the caecum following administration of these agents is lengthening of the mucosal glands which are lined by epithelium composed of increased numbers of enlarged epithelial cells (i.e. hypertrophy and hyperplasia) showing increased proliferative activity and more rapid incorporation of tritiated thymidine (newberne et al., ) . in addition, mucosal and submucosal oedema has been reported in association with the administration of lactose and increased mucosal lymphoid aggregates following lactose or xylitol feeding (newberne et al., ) . changes in the colon due to fibre are complex. morphometric analysis has shown that changes to the mucosa depend on the fibre type (stark et al., ) . there may also be an interaction between dietary fibre content and colonic microflora that influences mucosal growth, although the mechanism is unclear (whiteley et al., ) . hypertrophy of the muscularis external is also reported in rats fed high fibre diets (stark et al., ) . as the increase in caecal size and mucosal hypertrophy appears generally related to the osmotic activity of the caecal contents in rodents treated with these agents, it has been postulated that the changes represent a physiological adaptation to increased osmotic forces, irrespective of the contributing compounds (leegwater et al., ) . treatment of rodents with antibiotics also causes caecal enlargement or dilatation without significant histopathological changes, probably as a result of changes in caecal microflora. it has been suggested that the enlargement relates to accumulation of urea as a result of inhibition of bacterial ureases (juhr and ladeburg, ) . however, histochemical studies of the intestinal mucosa of rats treated with neomycin have also shown treatment-related reductions in activities of nad tetrazolium reductase, succinate dehydrogenase, esterase, alkaline and acid phosphatase in the distal ileum, suggesting that some antibiotics also posses the potential to directly influence absorption and metabolic functions of mucosal cells (van leeuwen et al., ) . long-term administration of , -dimethyl prostaglandin e to rats also produced thickening of the proximal colonic mucosa, although this was less fig. . similar area of colonic mucosa to that seen in fig. at the same magnification but from a rat treated with % dextran (molecular weight , ) in the diet for weeks. this shows diffuse hyperplasia of the mucosa with elongation of colonic glands that are lined by relatively normal epithelial cells with abundant mucin and prominent vesicular nuclei. (he, × .) marked than in the stomach and duodenum (reinhart et al., ) and similar changes have been reported in rats treated with other prostaglandin e analogues (levin, ) . as in the small intestine, administration of epidermal growth factor to rats and cynomolgus monkeys induces hyperplasia of the colonic mucosa characterised histologically by hyperplasia and increased mitotic activity of crypt cells and reduction in goblet cell numbers with an increase in crypt depth and a slight increase in the numbers of infiltrating neutrophils (breider et al., ; reindel et al., ) . in common with other epithelial surfaces, atypical hyperplasia is associated with the development of colonic cancer in both man and laboratory animals. the early alterations observed in rats treated with colonic carcinogens are similar to those found in the immediate vicinity of human colorectal carcinomas. the changes are characterised by lengthening, dilatation and branching of glands. the epithelium lining these glands shows mucous cell hyperplasia (goblet cell hyperplasia, see fig. ), goblet cells containing predominantly sialomucin instead of the normal sulphomucin (filipe and branfoot, ; filipe, ; olubuyide et al., ) . despite mucin alterations, activities of glucose- -phosphatase, glucose- -phosphate dehydrogenase and gly- fig. . section from the colon of an aged hamster from a colony that developed intestinal inflammation and neoplasia spontaneously. this shows focal mucous cell hyperplasia characterised by enlargement and lengthening of the colonic glands with lining cells replete with mucins. (he, × .) ceraldehyde- -phosphate dehydrogenase were shown to be normal in this epithelium in rats treated with , -dimethylhydrazine (mayer et al., ) . in man, this form of hyperplastic mucosa associated with cancer, has been termed 'transitional mucosa' (filipe and branfoot, ) . as lesions become more atypical, these dilated, branched glands become more complex and lined by epithelium that shows increasing pseudostratification and vesicular cell nuclei. for example in rats treated with the carcinogens azoxymethane or , -dimethylhydrazine, crypts show diminution of mucus secretion, increased cytoplasmic basophilia, prominent, rounded or enlarged nuclei which show variable degrees of pseudostratification and which eventually develop into frankly invasive glands . in contrast to goblet cell hyperplasia, these atypical zones show increased activity of glucose- -phosphate, glucose- -phosphate dehydrogenase and glyceraldehyde- -phosphate dehydrogenase (mayer et al., ) . described similar alterations in rats treated with azoxymethane and the non-genotoxic agent dextran sulphate. these authors also demonstrated that these atypical foci could be identified by low power microscopy as aberrant crypt foci by translumination of the whole mounts of the fixed mucosa stained with methylene blue. note: some compounds may induce qualitative and quantitative changes in mucin content in the colonic mucosa without marked morphological alterations. an example of this phenomenon was described in rats treated with reserpine for days. the colonic mucosa showed an increase in sulphomucin (high-iron diamine positive) containing goblet cells in the surface epithelium (park et al., ) . adenomas and adenocarcinomas of the small and large intestine are infrequent spontaneous neoplasms in laboratory animals compared with man where colorectal carcinoma is one of the most prevalent neoplasms in the western world. adenomas and adenocarcinomas probably occur spontaneously in older dogs more than in any other animal species and as in man these are located most frequently in the distal colon and rectum (lingeman and garner, ) . in nonhuman primates glandular neoplasms of the intestine occur with increasing age in the ileum and in the colon with a predilection for the zones near the ileocaecal valve (depaoli and mcclure, ) . in rats and mice, spontaneous intestinal neoplasms are uncommon although adenocarcinomas are occasionally observed in the ileum or colon in mice and rats used in chronic toxicity studies and carcinogenicity bioassays (burn et al., ; wells, ; maeda et al., ; greaves and faccini, ; zwicker et al., ) . most of these arise in the small intestine and appear to originate in the distal part of the small intestine, caecum and right side of the colon. they may produce metastases, mostly to liver and lungs. in a review of spontaneous adeno-carcinomas developing over a -year period in wistar rats, vandenberghe et al. ( ) identified adenocarcinomas, all in ascending colon. in of these cases there appeared to be an intimate relationship with campylobacter-like organisms together with diverticulae and chronic inflammation. these authors suggested that the organisms and the associated inflammation were involved in the pathogenesis of these cancers. in view of the importance of colon cancer in humans, a number of new genetic mouse models predisposed to colon cancer have been developed over recent years (heyer et al., ) . some hamster colonies, liable to develop inflammatory bowel disease (see above), also have a high incidence of small and large intestinal polyps, adenomas and adenocarcinomas (fortner, ; van hoosier et al., ; personal observations) . poorly differentiated carcinomas may infiltrate local lymph nodes and it may be difficult to locate the primary neoplasm. polyps are predominantly adenomatous in nature although inflammatory or regenerative polyps are observed (van hoosier et al., ) . adenocarcinomas are induced experimentally in the rodent intestine by the carcinogens , -dimethylhydrazine and azoxymethane. the histogenesis of these induced carcinomas has been extensively studied and it is generally accepted that they resemble human colorectal cancer (ward, ; freeman, ) . however, there are differences between reported studies. some have shown that these experimental carcinomas arise from pre-existing adenomas consistent with the 'adenoma-carcinoma sequence' theory (ward, ; ward et al., ) . others suggest that they arise 'de novo' from altered mucosa as microinvasive carcinomas (sunter et al., ; maskens and dujardin-loits, ; rubio et al., ) . these differences may be partly the result of different dosage schedules. rubio et al. ( ) have shown that a single dose of , -dimethyhydrazine produces non-polypoid, micro-invasive carcinomas, particularly in the mucosa overlying lymphoid aggregates, whereas in their earlier studies using multiple doses in the same strain of rat, an adenoma-carcinoma sequence was more evident. in addition, there are undoubtedly species and strain differences in the response to these agents. teague et al. ( ) demonstrated clear differences in the distribution and both macroscopic and histological types of adenomas and adenocarcinomas between three different inbred strains of rat given a similar dosage regimen of , -dimethylhydrazine. in general, many of these carcinomas develop in the distal colonic segments similar to the distribution of human colorectal cancer, although tumours also develop in the proximal colon and in the ileum in rats treated with this agent (ward, ; teague et al., ) . neoplasms occurring in the rat colon following administration of high doses of degraded carrageenans and sulphated dextran also commonly occur in the distal colon and rectum and are commonly polypoid adenomas and adenocarcinomas (hirono et al., ; oohashi et al., ; ishioka et al., ) . however, in these models adenomas and adenocarcinomas also occur in the caecum and proximal colon and squamous carcinomas are sometimes seen in association with squamous metaplasia at the colorectal junction (oohashi et al., ) . the pathogenesis of neoplasms induced by carrageenans and dextrans remains unexplained. although they are biologically active compounds, they are non-mutagenic in the usual short-term tests (ishioka et al., ) . it has been suggested that carrageenans act as tumour promoters as they potentiate the appearance of carcinomas in rats treated with standard intestinal carcinogens hirono et al., ) . conversely it has been proposed that these agents are tumour initiators based on the development of carcinomas in rats treated with degraded carrageenans for only months (oohashi et al., ) . however, despite only a short period of treatment, inflammation, regenerative changes and squamous metaplasia persisted throughout a period of months after treatment was withdrawn before development of cancer in these rats. the only consistent association of carrageenans with development of carcinoma in rats is that of chronic inflammation and increased cell proliferation. although dose levels needed to produce inflammation are far higher than any exposure likely to be achieved in man, interpretation of this inflammation-cancer sequence in rats remains a challenge in safety assessment for similar xenobiotics. this situation is interest in view of the unquestionable risk of carcinogenesis in ulcerative colitis in man (riddell et al., ) . a similar range of neoplasms can be defined histologically in both human and experimental pathology. it is appropriate, to use the same classification for all species including man. lingeman and garner ( ) who reviewed a range of tumours from domestic and laboratory animals were able to employ the classification for human gastrointestinal neoplasms. a similar approach has been used in the iarc classification of rat intestinal tumours (mohr, ) . this classification can be summarised as follows: these represent localised, sessile or polypoid neoplasms composed of proliferating tubular glands, which show varying degrees of nuclear hyperchromatism, pseudostratification and cellular pleomorphism. a useful scheme for grading the carcinogenic potential of hyperplastic mucosa and adenomatous polyps in man based on the degree of epithelial pseudostratification has been proposed by kozuka ( ) . although experimental neoplasms may not always show the full spectrum of these changes reported in man, this grading provides a useful baseline concept for the assessment of these non-invasive proliferative lesions. with increased nuclear pseudostratification and atypical branching of the glandular structures of these polyps becomes more prominent. if neoplastic cells or glands are seen in the stroma of the stalk or base the diagnosis of carcinoma is made. villous adenoma is a form of adenoma in which the epithelial proliferation takes the form of elongated villi with a sparse fibrovascular stroma. they can be graded in a similar way to other adenomas. these are glandular neoplasms of variable differentiation, sometimes originating in 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the safety of drugs for the long-term treatment of peptic ulcers the mechanism of mitrazepam-induced drooling and aspiration blood vessels of the peyer's patch in the mouse. iii high endothelial venules ultrastructural basis of intestinal absorption the effects of omeprazole and famotidine on mucin and pge release in the rat stomach forestomach ulcers in crj:b c (c bl/ ncrj × c h/hencrj) f mice forestomach ulcers in crj:b c (c bl/ ncrj × c h/hencrj) f mice an evaluation of ampicillin pharmacokinetics and toxicity in guinea pigs drug-induced gastro-intestinal disease naturally occurring intestinal neoplasms in aged crl:cd ® br rats key: cord- - m ch ls authors: ford, james d.; berrang-ford, lea; king, malcolm; furgal, chris title: vulnerability of aboriginal health systems in canada to climate change date: - - journal: glob environ change doi: . /j.gloenvcha. . . sha: doc_id: cord_uid: m ch ls climate change has been identified as potentially the biggest health threat of the st century. canada in general has a well developed public health system and low burden of health which will moderate vulnerability. however, there is significant heterogeneity in health outcomes, and health inequality is particularly pronounced among aboriginal canadians. intervention is needed to prevent, prepare for, and manage climate change effects on aboriginal health but is constrained by a limited understanding of vulnerability and its determinants. despite limited research on climate change and aboriginal health, however, there is a well established literature on aboriginal health outcomes, determinants, and trends in canada; characteristics that will determine vulnerability to climate change. in this paper we systematically review this literature, using a vulnerability framework to identify the broad level factors constraining adaptive capacity and increasing sensitivity to climate change. determinants identified include: poverty, technological capacity constraints, socio-political values and inequality, institutional capacity challenges, and information deficit. the magnitude and nature of these determinants will be distributed unevenly within and between aboriginal populations necessitating place-based and regional level studies to examine how these broad factors will affect vulnerability at lower levels. the study also supports the need for collaboration across all sectors and levels of government, open and meaningful dialogue between policy makers, scientists, health professionals, and aboriginal communities, and capacity building at a local level, to plan for climate change. ultimately, however, efforts to reduce the vulnerability of aboriginal canadians to climate change and intervene to prevent, reduce, and manage climate-sensitive health outcomes, will fail unless the broader determinants of socio-economic and health inequality are addressed. climate change has been identified as potentially the biggest health threat of the st century (costello et al., ) . addressing the challenge will be a defining question for public policy (campbell-lendrum et al., ; costello et al., ; lim et al., ; parry et al., ) . while reducing greenhouse gas emissions responsible for climate change is essential (i.e. mitigation), health systems will have to adapt to climate change through interventions to manage climate-sensitive health outcomes, many of which are now unavoidable ebi and semenza, ; frumkin et al., ; furgal and prowse, ). without such intervention, the burden of climate-sensitive health outcomes will increase confalonierei et al., ) . international assessments have highlighted that developed nations have a high capacity to adapt to the health effects of climate change (confalonierei et al., ; costello et al., ). on the whole, this is true: individuals, communities, and governments have access to significant resources and engage in a range of actions to manage and control climate-sensitive health outcomes (ebi, ; ebi et al., ; watson et al., ) . this assumption, however, does not adequately consider the persistence of withincountry economic and health inequities and their implications for vulnerable populations. this is particularly pertinent for developed nations with indigenous populations living in what has been referred to as the 'fourth world' (o'neill, ) . indigenous peoples in canada, new zealand, united states, and australia all experience a disproportionate burden of morbidity and mortality, with many social, economic, and health indicators similar to those in middleincome nations (cooke et al., ; green et al., ; stephens et al., ) . the existing burden of ill-health increases the sensitivity of indigenous peoples to the adverse impacts of climate change, which combined with a proportionally higher dependence of many indigenous climate change health adaptation vulnerability aboriginal inuit mé tis first nations canada social determinants of health inequality indigenous a b s t r a c t climate change has been identified as potentially the biggest health threat of the st century. canada in general has a well developed public health system and low burden of health which will moderate vulnerability. however, there is significant heterogeneity in health outcomes, and health inequality is particularly pronounced among aboriginal canadians. intervention is needed to prevent, prepare for, and manage climate change effects on aboriginal health but is constrained by a limited understanding of vulnerability and its determinants. despite limited research on climate change and aboriginal health, however, there is a well established literature on aboriginal health outcomes, determinants, and trends in canada; characteristics that will determine vulnerability to climate change. in this paper we systematically review this literature, using a vulnerability framework to identify the broad level factors constraining adaptive capacity and increasing sensitivity to climate change. determinants identified include: poverty, technological capacity constraints, socio-political values and inequality, institutional capacity challenges, and information deficit. the magnitude and nature of these determinants will be distributed unevenly within and between aboriginal populations necessitating place-based and regional level studies to examine how these broad factors will affect vulnerability at lower levels. the study also supports the need for collaboration across all sectors and levels of government, open and meaningful dialogue between policy makers, scientists, health professionals, and aboriginal communities, and capacity building at a local level, to plan for climate change. ultimately, however, efforts to reduce the vulnerability of aboriginal canadians to climate change and intervene to prevent, reduce, and manage climate-sensitive health outcomes, will fail unless the broader determinants of socio-economic and health inequality are addressed. ß elsevier ltd. all rights reserved. livelihoods on the environment, spiritual and cultural ties to the land, demographic trends, and experience of marginalization, makes indigenous peoples particularly vulnerable (furgal, ; furgal and seguin, ; green et al., ) . public health interventions focused on indigenous peoples are needed to prevent, prepare for, and manage climate change risks (i.e. adaptation). assessing vulnerability is essential to this end, locating at-risk populations, identifying vulnerability determinants that need to be addressed, and directing attention to the most appropriate level for intervention (ipcc, b) . canada has been a leader in vulnerability assessment in public health (berrang-ford, ; berrang-ford and noble, ; berrang ford et al., ; charron, charron, , charron et al., ; doyon et al., ; health canada, a,c; ogden et al., ; thomas et al., ) . natural resources canada's from impacts to adaptation: canada in a changing climate , for example, assesses the current state of knowledge on climate change for six regions of canada, and health canada's human health in a changing climate (seguin, ) specifically focuses on climate change and health. however, there remains a significant deficit in information required to inform and guide adaptation among aboriginal peoples-part of what we broadly term an indigenous peoples 'vulnerability deficit.' major canadian assessments of climate change, for example, identify research on aboriginal health a priority for action , and while furgal and prowse ( ) focus on the health of mostly inuit inhabitants in the north, other aboriginal populations have been less studied (healey and meadows, ; wilson and young, ) . this lack of information leaves aboriginal health systems insufficiently prepared to identify the risks of climate change and develop adaptations (seguin, ) . the vulnerability deficit is further evident in the peer-reviewed literature. a review of pubmed ( pubmed ( - , for example, reveals only articles referring to a combination of ''indigenous or aboriginal'' and ''health'' and ''climate change'' and ''canada.'' of this literature, only article formally assesses vulnerability with a focus on the north (furgal and seguin, ) , and only are explicitly concerned with aspects of health in a changing climate. for comparison, articles match the search criteria for canada if ''indigenous or aboriginal'' are excluded. addressing the vulnerability deficit should be a priority for aboriginal health research and planning on climate change (furgal, ; green et al., ; seguin, ) . a common approach to examining health vulnerability begins with the question, 'given projected climate changes, how will this affect health systems?' the long range future is the focus of interest here (often or ), with assessment starting by modeling how health exposures will be affected by climate projections. an alternative approach starts with the question 'what predisposes health systems to be negatively affected by climate change.' the first step here is to characterize the current structure and functioning of health systems, and identify the processes and conditions which affect how health risks are experienced and managed: these characteristics in turn will determine sensitivity and adaptive capacity to climate change (smit and wandel, ) . given that the second approach focuses on current capacity and vulnerability of the health system, we can use existing literature to characterize sensitivity and adaptive capacity to climate changes. this is important given the volume of research on all aspects of aboriginal health: articles using search terms ''indigenous or aboriginal'' and ''health'' and ''canada'' in pubmed ( - ) . in this paper we systematically review the literature on aboriginal health in canada to identify health outcomes, determinants, and trends, and use a vulnerability framework to synthesize how these factors will affect the vulnerability (and resilience) of aboriginal health systems to climate change. on this basis we identify strategic priorities for policy intervention and research needs. aboriginal peoples in canada are constitutionally defined as north american indian (commonly referred to as first nations), mé tis, and inuit (table ). in , aboriginal peoples accounted for % of canada's population, numbering approximately . million, and are the fastest growing segment of the population (table ). in many instances, indicators of economic, social and health well-being among aboriginal canadians compare unfavourably with the canadian population in general (table ) (adelson, ; cooke et al., ; phac, ; stephens et al., ) . aboriginal peoples experience lower life expectancy, higher incidence of chronic diseases (e.g. diabetes), higher rates of infectious diseases (in particular tuberculosis and childhood rsv bronchiolitis), and higher rates of substance abuse, suicide, and addiction, than canadians in general (table ) (adelson, ; banerji et al., ; clark and cameron, ; frohlich et al., ; macdonald et al., ; macmillan et al., ; mcdonald and trenholm, ) . canada's aboriginal population is diverse, including people living on reserves, in the territories, and in rural communitiesthese groups account for approximately half the aboriginal population. we collectively refer to this segment of the population as the 'remote aboriginal population': they are generally remote and rural in character, maintain a close and intimate relationship with the environment, with many still practicing aspects of traditional lifestyles (table and fig. ) (furgal and seguin, ; richmond and ross, ). this remoteness, combined with close links to environmental conditions for livelihoods, creates particular sensitivity to climate change. the remainder of the aboriginal population lives in urban areas, and this population is growing rapidly with migration from remote communities, particularly among young people seeking education and employment (siggner and costa, ; statistics canada, a) . urban aboriginal peoples, in many respects, are engaged in livelihood activities similar to non-aboriginal people and are often disconnected from land based activities. nevertheless, many retain connections with their traditional home (inac, ) . we recognize that focusing on all aboriginal subpopulations risks overgeneralization as there is significant heterogeneity in health outcomes and determinants of health between urban and remote and rural aboriginal populations, and also between and within first nations, inuit, and mé tis. table characteristics of aboriginal habitation. reserves are tracts of land, the legal title to which is held by the crown, set apart for the use and benefit of first nations with constitutional responsibility resting with the federal government. there are approximately reserves in canada. the territories are administrative subdivisions of canada which, unlike provinces, derive their mandate and powers from the federal government. a significant proportion of the population of territories is aboriginal, residing in approximately communities. over half of the aboriginal population lives in urban centres. winnipeg, edmonton, saskatoon, and regina are major urban areas where aboriginal people make up > of the population. aboriginal people also reside in rural settlements outside of the territories and not on reserves, data on this segment of the aboriginal population is lacking. there are, however, shared experiences of health inequalities which result in a unique vulnerability to climate change. the focus in this paper on canada's aboriginal population reflects the serious risks posed by climate change to aboriginal health. canada has already experienced disproportionate warming with climate change, with average temperatures in some northern regions increasing beyond c (acia, ; barber et al., ; ford, a; ipcc, a; prowse et al., b,c) . implications for aboriginal health have already documented, particularly in the arctic (ford, b; ford et al., a; furgal, ; furgal and prowse, ; furgal and seguin, ) . projections of relevance to aboriginal health include: changing temperature and precipitation regimes will increase the probability and severity of extreme events including heatwaves, storms, floods, drought, and wildfire with implications for asthma, chronic respiratory disease, water quality, cardiovascular disease, and the health effects of dislocation and displacement ( fig. ) (charron et al., ; doyon et al., ; hess et al., ; thomas et al., ) . sea level rise and associated coastal erosion are already threatening the viability of some aboriginal settlements . remote aboriginal settlements are particularly sensitive to these impacts, with many dependent on water delivery and sewage collection by truck, basic water treatment facilities, and some settlements located on marginal and hazardous locations, a consequence of government relocation in the s and s (furgal and prowse, ; lemmen et al., ) . warmer, wetter summers have the potential to increase the incidence of water and insect borne disease (e.g. entamoeba histolytica, giardia lamblia, cryptosporidium parvum) (hennessy et al., ; martin et al., ) , while rising temperatures are expected to increase the incidence of temperature-dependant food-borne diseases, including salmonella as well as toxins produced by staphylococcus aureus and clostridium botulinm (hess et al., ; parkinson et al., ) . these are important in an aboriginal context given traditional food cultures, which includes the consumption of raw meats, in which small changes in storage and transport temperatures can significantly increase the risk of food-borne diseases. there will be a number of indirect pathways through which climate change will affect health involving second, third, or even fourth order indirect impacts. these are generally less researched and understood but could be more extensive and disruptive (hess et al., ; mcmichael, ) changing temperatures, for example, are likely to impact the distribution and availability of animal populations important in aboriginal subsistence hunting with implications for community health, nutrition, and well-being ( fig. ) (ford, b; furgal and seguin, ) . climate change could result in increased migration to urban centres as traditional activities and livelihoods are compromised, with implications for disease transmission and diagnosis (parkinson et al., ) . changes to the incidence and prevalence of some infectious diseases will also be indirect, stemming for example from climate impacts in other countries from which diseases may be introduced into canada, or changes in the distribution and densities of vector habitat or animal hosts of zoonotic and vectorborne diseases (berrang-ford, ; berrang ford et al., ). there might also be positive health implications associated with increased economic opportunity with improved transportation in areas currently inaccessible (e.g. by melting sea ice). we structure the literature review using the concept of vulnerability-a concept that underpins much of the research in the human dimensions of climate change (hdcc) field. vulnerability is a measure of the susceptibility to harm in a system in response to a stimulus or stimuli, and can essentially be thought of as the 'capacity to be wounded,' (smit and wandel, ) . in this paper we are interested in aboriginal health systems, defined collectively as organizations, institutions (formal and informal) and resources whose primary purpose is aboriginal health. this includes frontline health personnel, community and territorial health authorities, federal agencies, aboriginal organizations, research bodies, and also individuals and households who are an important informal component of health care provision and advice in many aboriginal communities. the stimulus or stimuli are health risks linked directly or indirectly to climate change. a general model of vulnerability has emerged in climate change scholarship that conceptualizes vulnerability as a function of exposure and sensitivity to climate change and adaptive capacity (ebi et al., ; ipcc, b; smit and wandel, ) . in a health context, exposure refers to the nature of climate-related (direct or indirect) health outcomes. sensitivity concerns the organization and structure of health systems relative to the climate-related health outcomes and determines the pathways through which exposure is manifest. adaptive capacity reflects the ability of health systems to address, plan for, or adapt to adverse climate-related health outcomes and take advantage of new opportunities (ebi and burton, ; ebi et al., ; ebi and semenza, ; ford and smit, ) . exposure, sensitivity, and adaptive capacity are not mutually exclusive, with interaction between these components potentially moderating or exacerbating vulnerability. the recognition of the role of adaptive capacity and sensitivity is important, directing attention to health systems themselves and the nonclimatic factors operating at multiple spatial-temporal scales that determine how climate change will be experienced and responded to (ebi and burton, ; ebi et al., ; ford and smit, ; ford et al., b ). this general model of vulnerability has been applied essentially in two main ways in vulnerability research (burton et al., ; o'brien et al., ; ford et al., b) . firstly, 'end point' approaches begin by projecting climate change impacts and then estimate potential vulnerabilities to future conditions, and have historically dominated the hdcc literature in general. the first step towards tackling the vulnerability deficit for aboriginal health using this approach would be to develop climate scenarios to model changing exposure. this is a time consuming and intensive process, however, and is particularly problematic in a canadian aboriginal context where baseline data that is needed for health modelling is lacking . more generally, 'end point' assessments have been criticized for neglecting the complex dynamics that shape how climate change is experienced and responded to, focus on future conditions and risks as opposed to current stresses that are relevant to the people being affected, neglect of indirect health risks that cannot easily be modeled, and failure to capture the dynamic nature of vulnerability (brooks et al., ; ford and smit, ; o'brien et al., o'brien et al., , smit and wandel, ) . conversely, 'starting point' approaches begin with the system of interest, examining the factors that determine sensitivity and adaptive capacity to climate related risks and change (burton et al., ; o'brien et al., ) . common in the hdcc literature in general (adger, ; burton et al., ; o'brien et al., ; smit and wandel, ) and increasingly in a health context (ebi and burton, ; ebi et al., ; ebi and semenza, ; furgal and seguin, ) , vulnerability here is viewed as a state or condition, not an outcome, continually evolving and changing. the 'starting point' approach seeks, therefore, not to identify vulnerability independently attributable to climate change, but to understand the conditions and processes that predispose a system to negative effects. in the context of the vulnerability deficit this approach directs attention to aboriginal health systems themselves, a topic which has been the focus of significant research. recurring themes in this literature concern the social, economic, political conditions that affect aboriginal health outcomes. this work has renewed importance for understanding climate change vulnerability, and we use a 'staring point' approach to identify and examine the broad characteristics of aboriginal health systems that influence their sensitivity and adaptive capacity. we used keyword combinations to search for peer reviewed articles on aboriginal health published from to july using pubmed database. keywords used included: ''aboriginal '' or ''indigenous'' or ''first nations'' or ''inuit'' or ''health problems,'' and ''social determinants of health.'' information was also obtained from other sources, including aboriginal organization websites, government reports about the status and health of aboriginal peoples and health of canadians in general. our search was limited to publications in english. once all relevant sources were identified and retrieved, pertinent information on aboriginal health outcomes, determinants, and trends, was extracted, categorized, and analyzed using the vulnerability framework described above. in this section we examine the broad characteristics of aboriginal health systems that will determine vulnerability to climate change. these determinants do not exist in isolation and fig. captures important interactions and highlights how these broad scale or underlying factors provide the context within which local to regional health systems will experience and respond to [ ( f i g . _ ) t d $ f i g ] table provides an overview of the determinants of vulnerability. canada is a wealthy nation consistently placed near the top of the united nations develop programs human development index, and as such is expected to be less vulnerable to climate change (cooke et al., ; o'brien et al., ) . national level indicators, however, hide significant disparities, with aboriginal peoples at substantively higher risk of living in poverty and experiencing housing and food insecurity (adelson, ; mcdonald and trenholm, ; phac, ; raphael et al., ) . these gaps continue to grow despite policy intervention, and are particularly pronounced among the remote aboriginal population (table ) . a complex interplay of factors has been identified to contribute towards high rates of poverty including low labour force participation, lack of employment opportunities, low educational attainment, loss of land and sovereignty, high cost of living in remote areas, job market discrimination, and the burden of illhealth (rcap, ; adelson, ; phac, ) . poverty influences climate vulnerability at two main levels: individual/household level and institutional level. firstly, at an individual and household level, poverty translates to negative health outcomes through material conditions and associated behavioural factors (frohlich et al., ; woolf, ) increasing the sensitivity of aboriginal canadians to climate risks. poverty forces many to live in suboptimal conditions, engage in dangerous livelihood activities, live in areas at high risk, and increases the risk of engaging in unhealthy behaviours (e.g smoking, drinking). overcrowding in inadequate housing and food and water insecurity, for example, are chronic poverty-related problems facing aboriginal people across canada (boult, ; dunn et al., ; egeland et al., ; ford and berrang-ford, ; hamelin et al., hamelin et al., , harvey, , ; shaw, ) . those who are nutritionally challenged will be particularly vulnerable to changing access, availability, and quality of traditional foods with climate change, and susceptible to increasing incidence of climate-sensitive infectious diseases (ford, b; furgal and seguin, ) . similarly, house overcrowding and high rates of tobacco use increase the risk of person-to-person spread of infectious diseases, favor transmission of respiratory and gastrointestinal diseases, and increase susceptibly to heat stress; health outcomes with a strong link to climate and expected to increase in prevalence with warming temperatures and changing precipitation regimes (furgal and seguin, ; orr et al., ; parkinson et al., ) . the sensitivity of aboriginal canadians will result in health impacts of climate change occurring faster, sooner, and of a greater magnitude than for non-aboriginal people. this will challenge the ability of health systems to adequately invest time and resources in prevention, preparedness, and response seguin, ) . adaptive capacity at an individual and household level is also negatively affected by poverty, with many adaptations exceeding financial means. poverty has already been noted as a major constraint to adaptation to climate change related disruptions to subsistence hunting and fishing in arctic regions with implications for food security (furgal, ; furgal and seguin, ; turner and clifton, ; wolfe et al., ) . a number of behavioral and psychological conditions associated with poverty including substance abuse, addiction, stress, family disruption, alienation, and compromised education are also strongly associated with limited capacity to identify and respond to risks phac, ; tanner and mithcell, ) . research has identified how social networks which increase uptake of adaptive measures and are essential to well-being and managing climate change can be undermined by addictive behaviour (chan et al., ; beaumier and ford, in press; ford et al., a; pearce et al., ). notwithstanding, a critical factor in moderating the effects of climate change on aboriginal health lies in the adaptability and the resilience of aboriginal peoples (ford, a; ford and furgal, ; furgal, ; furgal and seguin, ) . aboriginal peoples have a deep attachment to their lands and their cultures, and this forms part of identity, which is a determinant of health . however, aboriginal cultures and identity are not static or fixed in time. aboriginal peoples are adaptable if they are allowed to, and current rates of poverty, and associated health impacts challenge this capacity. secondly, poverty is a major constraint on institutional capacity to respond to climate change. institutions with jurisdiction over reserve and territory-based aboriginal populations are challenged by human and financial resources, and exacerbated by the challenge of poverty (adelson, ; . substantial shortfalls remain in meeting basic needs, and planning for future health problems frequently ranks behind other existing challenges (mathias et al., ) . even in nunavut, which is globally believed to be a climate change 'hot spot' and where awareness of climate change is high, health systems at a local and territorial level have not had the financial or human resources to assess or plan for climate change health impacts; poverty related issues are more pressing (boyle and dowlatabadi, in press; ford et al., ) . to compound these difficulties, it has also been argued by some policy makers that resources and attention directed to climate change should be invested in poverty alleviation, with climate change perceived as a distant, diffuse, and uncertain threat (mathias et al., ) . this is ill-advised on a number of levels yet is challenging institutional response to climate change. firstly, climate change has the potential to exacerbate poverty, further increasing health vulnerability to climate change. secondly, climate change projections for canada in the ipcc (ipcc, c) and lemmen et al. ( ) are now widely believed to be conservative, with significantly greater impacts projected (barber et al., ; schellnhuber, ) . finally, addressing climate change and poverty need not be a zero sum game: climate policy has significant potential for co-benefits and can be mainstreamed into ongoing health activities and planning (campbell-ledrum and woodruff, ; ebi and burton, ; ebi and semenza, ; patz et al., ) . ameliorating poverty is a key challenge to improving aboriginal health, reducing vulnerability to the health effects of climate change, and creating an enabling environment for adaptation. as long as aboriginal people experience a higher prevalence of poverty than canadians in general they will bear a disproportionate vulnerability to climate change. reversing the current state of poverty is a significant undertaking requiring long term strategic investments in sustainable economic development, education, infrastructure, health care, the settlement of outstanding land claims, and greater involvement of aboriginal communities and organizations in decision making (rcap, ) . there is evidence that the cycle of poverty is being broken in some communities and regions, offering hope for the future. the assembly of first nations, for example, has recently launched a strategic plan to decrease poverty through creating opportunities, building on community assets and structural change for management of resources (assembly of first nations (afn), ) while the northern territories are trying to harness resource development to provide a basis for creating new and lasting economic opportunities. technological capacity refers to the ability of health systems to identify, respond to, and manage health risks, including those associated with climate change, through the application of appropriate technical strategies or interventions in the areas of diagnosis, treatment, surveillance, early warning, and planning. the canadian health system generally has a high technological capacity: geographical information systems are widely utilized to project future health burden and optimize planning, advanced treatments and preventive care are universally available, a strong educational and scientific base underpins a vigorous health research sector, and effective surveillance and early warning systems are in development or operation (davidson, ; gosselin et al., ) . this capacity reduces vulnerability to climate change and provides a strong basis for adaptive planning ipcc, b; phac, ) . aboriginal canadians enjoy many of the health benefits of the technologically sophisticated canadian health system. diagnosis and treatments, for example, are provided to aboriginal peoples through canada's universal and comprehensive health care system (madore, ) . however, technological capacity to address health outcomes for aboriginal peoples and plan for future risks is constrained by the accessibility of health services and availability of technology to health systems, contributing to unequal health burden (romanow, ) . this increases the sensitivity of aboriginal health systems to climate-related health outcomes and compromises adaptive capacity. firstly, canada's remote aboriginal population frequently note barriers to accessing health services, ranging from wait times, a shortage of doctors/nurses in the area, limited access to specialty and emergency services, the cost of transportation to health centres, to complaints that services provided are inadequate or not culturally sensitive (mackinnon, ; minore et al., b; muttitt et al., ; nti, ; wardman et al., ) . this is partly a function of geography. servicing small communities located in remote regions, many only accessible by air, is difficult and poses significant strain on health budgets. frontline health care in communities therefore often only involves basic diagnostic and treatment services, with other services provided in regional centres which may require significant travel and associated family disruption and financial stress (anderson et al., ; muttitt et al., ; wardman et al., ) . challenges of geography are compounded by the cross-cultural context of health provision. in particular, high turn-over of frontline health personnel, lack of training on working in aboriginal contexts for health professionals, language, and history of oppression through the medical system, have been noted to create reluctance among aboriginal people to seek health advice (adelson, ; bird et al., ; minore et al., b; tester and irniq, ) . urban aboriginal populations generally face fewer problems with health care services widely available. physical availability of services however, does not ensure health services are accessed or considered accessible; in many ways challenges associated with cultural sensitivity are more pronounced among urban aboriginal populations as they leave community health networks to the anonymity of urban health systems (adelson, ) . improving access and reducing inequalities to health services is a major challenge and has significant importance for climate change vulnerability. preventing, reducing, and managing the health burden of climate change requires individuals having access to timely and effective information, diagnosis, and treatment (berrang-ford, ; kovats and haines, ) . while some barriers to health service access reflect the reality of living in remote areas and others will only be overcome over time (e.g. trust in health system), some of the more egregious determinants can be addressed. entry points suggested in the literature include: collaboration between aboriginal stakeholders, policy makers, and frontline health workers to improve cultural sensitivity, training of health practitioners in both traditional and western health systems, and improved use of multi-media technology to communicate health messages (abonyi and jeffery, ; minore et al., b; muttitt et al., ; . secondly, effective surveillance and early warning systems are critical components of efforts to anticipate and respond to the effects of climate change and other risks on health (ebi and semenza, ; harrell and baker, ; parkinson et al., ) . surveillance involves the systematic collection of information on health determinants and outcomes necessary to determine the occurrence and spread of health risks, identify the emergence of new risks, and disseminate information to relevant actors. early warning systems provide timely information to populations and frontline health personnel when a threat is expected. current surveillance and early warning capacity for canada's remote aboriginal population, is underdeveloped (furgal, ; furgal and prowse, ; parkinson et al., ) . the fundamental challenge, as note, is the inadequacy of health data for planning: data sources that do exist are often inconsistent, sometimes based on faulty calculation methods, and are of limited coverage, baseline data do not exist for some health conditions and universally accepted measures, collection methods and techniques vary over time limiting the possibility for longitudinal analyses, surveillance among small populations limits analytical capacity for identifying significant changes and thresholds, and culturally and locally specific indicators have not been developed. these problems are compounded by challenges to institutional memory and high staff turnover in aboriginal health systems noted in sections . and . . early warning and surveillance capacity in canada's urban centres is generally more advanced, and information on a range of emerging health risks is available to urban aboriginal populations as part of broader health initiatives. nevertheless, problems surrounding culturally specific communication, surveillance of aboriginal-specific health sensitivities, and lack of baseline data on health outcomes, have also been noted in urban contexts (health canada, b; tudiver et al., ) . for remote aboriginal populations, existing surveillance is insufficient to detect the occurrence and spread of climate change related health risks, and early warning systems are insufficient to deliver projections in a timely and effective manner (kondro, ; , increasing sensitivity and reducing adaptive capacity to climate-related risks negative health outcomes. investments are needed to increase surveillance and early warning capacity, including the identification and monitoring of culturally specific and locally relevant health indicators in a systematic manner, examination of the potential to use sentinal health events as indicators, identification of indicators to monitor emerging climate change impacts and vulnerabilities, and development of infrastructure to link indicators to early warning (eyles and furgal, ; furgal and gosselin, ) . several aboriginal groups have initiated projects to develop indicators for surveillance, including climate change indicators, and offer insights for the development of national level systems (abonyi and jeffrey, ) . researchers have also started to work with communities to develop innovative surveillance approaches (martin et al., ; tremblay et al., ) . continuation and expansion of these initiatives requires addi-tional financial and human resource commitments at all levels of government. climate change will result in the emergence of health risks which cross borders, extend over multiple spatial-temporal scales, and span jurisdictions of government departments (campbell-lendrum and woodruff, ) . addressing these risks will require new governance structures, including increased participation of vulnerable peoples in decision making, increased accountability, and financial commitments (costello et al., ) . aboriginal canadians, however, face unique political challenges to achieving a range of social, economic, environmental, and health goals, with implications for sensitivity and adaptive capacity to climate change effects on health. as damman et al. ( ) note, government policies and actions often do not sufficiently address aboriginal interests, culture and lifestyle, specifically socioeconomic and spiritual connections to the land. inequality is evident in the neglect of aboriginal rights which are -in theoryprotected by the canadian constitution and international human rights obligations of the canadian state (damman et al., ; nilsson, ; raphael et al., ) . overcrowded living conditions, food and water insecurity, discrimination, and outstanding land claims are a few examples of this neglect, earning canada rebukes from the united nations (inac, ; statistics canada, b; united nations human rights council, ). marginalization of aboriginal peoples has been compounded by refusal of the canadian government to ratify international treaties which establish obligations for states towards indigenous peoples including the un declaration on the rights of indigenous peoples (un, ) , and domestic initiatives including the kelowna accord which aimed to substantively invest in programs to address aboriginal inequality (government of canada, november - , ; patterson, ) . political inequality links to climate change vulnerability in a number of ways. firstly, political inequality has been linked to a range of negative social, economic and health outcomes which increase sensitivity to climate-related health outcomes (adelson, ; richmond and ross, ). secondly, concerning adaptive capacity, inequality reduces the political power of aboriginal peoples to draw attention to pressing issues and develop interventions to manage emerging threats including climate change. this is evident in the reluctance of successive federal governments to advocate aboriginal rights on an international stage and develop effective policy domestically, for example with regards food and water security, and health inequality (budreau and mcbean, ) . this is also evident with climate policy. canada has made limited progress towards meeting its ratified commitments under the un framework convention on climate change (fccc), specifically its mitigation commitments to ''stabili [ze] greenhouse gas concentrations in the atmosphere at a level that would prevent dangerous anthropogenic interference with the climate system,'' (fccc, ) ; indeed emissions have rapidly increased since the signing of kyoto (weaver, ) . the fact that this commitment is unmet has particular relevance for aboriginal canadians who's livelihoods are sensitive to climate change, particularly in arctic regions where a strong climate change signal has already been detected (cbc, ; prowse and furgal, ; prowse et al., a) . the canadian state nevertheless has obligations to aboriginal peoples through the constitution and human rights treaties. while these obligations have often been overlooked, increasing public sympathy with aboriginal issues, visibility of inequality, and increasing political power of aboriginal peoples as reflected in self government for some regions, provides a basis for renewed political lobbying. the recent apology by the federal government to aboriginal canadians for past abuses of the state also potentially signals willingness for a new era of cooperation. this will be important with climate change which has the potential to compromise the basic human rights of many aboriginal peoples in absence of intervention, particularly in remote and northern regions (crump, ; ford, a; lukovich and mcbean, ). open and meaningful dialogue will be essential to ensure that policies respect, protect and fulfill the rights of aboriginal peoples. federal departments (e.g. health canada, indian and northern affairs canada, public health agency) are also working with aboriginal communities in vulnerable, mostly arctic, regions to develop programs and measures to facilitate adaptation. aboriginal canadians are also uniquely sensitive to climate change deriving from the cultural and spiritual relationship to ''the land.'' the ability to engage in traditional activities (e.g. hunting and fishing) and environmental stewardship remain central to aboriginal identify and culture and are closely linked to health, even for many urban aboriginal populations who no longer live on traditional lands or regularly engage in traditional activities. climate change threatens this: changing ice regimes are limiting access and availability of culturally and nutritionally important animal species, warming temperatures are increasing incidence of abnormalities and certain diseases in animals with implication for meat consumption, coastal erosion is threatening important cultural sites, and changing weather and wind patterns are challenging traditional environmental knowledge lemmen et al., ) . policy intervention can help manage some of these changes but some predicted impacts will result in irreplaceable cultural loss. in arctic regions, for example, climate change will make some communities uninhabitable and necessitate relocation, in other instances changing access to traditional areas and loss of livelihood may precipitate increased migration away from aboriginal communities (parkinson and berner, ). relocation could lead to an exacerbation of loss of culture and disconnection from the land, with implications for depression, anxiety, and substance abuse (berry et al., ; hess et al., ; kvernmo and heyerdahl, ) . these socio-cultural impacts present limits to adaptive capacity, where a limit implies an absolute barrier to adapting (adger et al., ) and indicates the continued importance of mitigation. health systems are composed of multiple institutions, including government health departments at municipal to federal levels, frontline health provision, research institutions, international donors, and informal community wellness groups. high income countries including canada are generally believed to have well developed institutional capacity underpinning the ability to identify, recognize, evaluate, anticipate and respond to health risks and learn from past mistakes. recent experiences in canada and developed nations more generally, however, including severe acute respiratory syndrome (sars), h n , and hurricane katrina, have challenged this complacency (ebi, ; ebi and semenza, ) . the institutional capacity of aboriginal health systems in remote areas is a particular concern, and is constrained by two key challenges: jurisdictional conflict over health care provision and human and financial resource limits. firstly, health care provision in canada for first nations and inuit falls under two jurisdictions, federal and provincial. all canadians are entitled to comprehensive, accessible, portable and universal health care as stated in the canada health act, , with provinces responsible for providing and financing the majority of health care services. however, provincial jurisdiction on health care in most instances does not extend to reserves which, along with the territories, fall directly under the jurisdiction of the federal government. through the first nations and inuit health branch, the federal government is mandated to provide a range of services to aboriginal people not covered by provincial jurisdiction, including primary health care and mental health (madore, ) . more recently, health transfer initiatives have sought to empower aboriginal communities and regions directly to manage their own health care with varying degrees of success. many services remain federally directed however. while in theory this model should cover aboriginal health needs, the reality is often much different. as documented in the literature, jurisdictional responsibility is not always clear and in some instances is shared, the status of health as a treaty right has not been settled with implications for local level health provision, decision making is often fragmented, and disputes within and between federal and provincial agencies regarding a range of health services for which there are pressing needs impedes health care access (adelson, ; mackinnon, ; minore et al., b) . urban aboriginal populations receive health care through provincial health care systems and do not generally face the same jurisdictional challenges. the consequences of this jurisdictional predicament are fragmented delivery of health care for aboriginal peoples on reserves and in the territories, uncoordinated management, constrained and short-term policy development, and a bureaucratic maze that constrains health provision and compromises health kirby, ; romanow, ) . these problems have implications for climate change vulnerability. indirectly, institutional constraints increase sensitivity and reduce adaptive capacity to climate change and act as a barrier to adaptation by limiting access to health services, constraining technology transfer and development, and posing barriers to developing baseline information on health vulnerabilities. directly, institutions characterized by jurisdictional conflicts, limited accountability, and complex bureaucracy are less likely to be able to identify, prepare for, and manage emerging risks like climate change, and learn from past mistakes (adger, ; berkes et al., ; gunderson and holling, ; keskitalo and kulyasova, ) . secondly, institutional capacity, particularly in remote areas, is constrained by human and financial resources. this has direct implications for sensitivity and adaptive capacity to climate change because local capacity is important for identifying and managing risks: evidence from multiple contexts has shown that well developed local health capacity increases the likelihood that policies and actions will be appropriate, effective and acceptable (blas et al., ; ford et al., ) . difficulty in recruiting and retaining human health resources has been widely noted (bird, ; boyle and dowlatabadi, in press; marrone, ; minore et al., a) . high staff turnover presents barriers to developing relationships with community members and stakeholders and creates problems for basic service delivery with employees often overworked and fatigued, key positions vacant, and inexperienced personnel undertaking responsibilities for which they do not have the necessary training or expertise. in this context, action on climate change is often undermined by other priorities, dependant on personnel, and subject to sudden change. if the health of aboriginal peoples is to be improved, sensitivity to climate change reduced and adaptive capacity enhanced, there is a need to build health systems that work well, provide universal care, ensure enough staff, and provide adequate health education (adelson, ) . policy discussion has focused on numerous ways to address institutional problems including resolving aboriginal land claims, consolidating fragmented funding, adapting health programs to local priorities, giving aboriginal people a direct voice in health planning, making aboriginal health the responsibility of provincial and territorial governments, and increasing self reliance (mackinnon, ; phac, ; richards, ; romanow, ) . as romanow ( ) reports, this will require a new structured approach that cuts across jurisdictional barriers and develops a new ethos of coordination and cooperation among government levels. the inadequacy of attempts to address the institutional determinants of poor health that have been widely recognized since the s is discouraging. however, increased self government, self determination, community level health initiatives, and the recent apology to aboriginal peoples by the federal government provide a strong basis from which to build. as fankhuaser and tol ( ) argue, ''successful adaptation requires a recognition of the necessity to adapt, knowledge about available options, the capacity to assess them, and the ability to implement most suitable ones.'' without this information, health systems are less likely to develop anticipatory adaptation interventions, leaving them sensitive to climate change risks and constraining adaptive capacity. vulnerability assessment is an important first step for providing the necessary information for adaptation. as noted in the introduction, however, information on aboriginal health system vulnerability is limited. this paper goes some way towards addressing this deficit, developing an understanding of the broad level or underlying determinants of vulnerability (fig. ) . this needs to be complimented by in-depth vulnerability assessments at local to regional scales to examine how broader determinants interact and shape local level factors, and specifically identify high risk regions and groups, characterize sensitivity to important health outcomes, evaluate current health planning in light of current impacts and future projections, and assess and prioritize response options (fig. ) . such initiatives require close collaboration with aboriginal communities, organizations, and policy makers, integrating local expertise, knowledge, and understanding of health determinants (adelson, ; green et al., ; pearce et al., pearce et al., , raphael et al., ; richmond and ross, ) . there is also need for a comprehensive assessment of the various pathways through which climate change will affect the incidence and prevalence of various outcomes affecting aboriginal health systems, many of which will be unique influenced by livelihoods, living conditions, and traditional food cultures. uncertainly about climate change impacts on aboriginal health characterises much current scientific understanding, and is magnified at regional and local levels where risks are largely unknown among policy makers and communities. this constrains effective risk assessment and planning. improved scientific understanding needs to be complimented by more effective partnerships with aboriginal communities and organizations and culturally relevant knowledge translation. the significant body of literature on aboriginal health provides considerable insights on the broad level determinants of vulnerability which will shape the extent to which aboriginal health systems are able to prevent, prepare for, and manage the effects of climate change. a number of key trends are highlighted in the review: material conditions and behaviours associated with poverty will increase sensitivity and constrain adaptive capacity to climate change. surveillance and early warning capacity for those living in remote regions is underdeveloped for identifying emerging risks and vulnerable populations. comprehensive, reliable, and culturally specific health assessment measures from which to assess climate change impacts are absent. access to health information, diagnosis, and treatment is insufficient for timely and effective intervention to manage climate-sensitive health outcomes. the special rights and needs of aboriginal peoples have often been neglected, resulting in continued and persistent inequality which exacerbates climate change health vulnerability. institutions responsible for aboriginal health are challenged by jurisdictional conflict and resource constraints, limiting the ability to identify and prepare for future risks and address inequalities. these underlying determinants provide the context within which health system vulnerability at regional to local levels will be influenced. how they influence vulnerability at lower scales, however, will not be uniform. inuit will probably experience the greatest vulnerability to climate change on account of the sensitivity of arctic environments to climate change, magnitude of projected changes, remoteness of communities, dependence on the environment, burden of poor health, and limited institutional capacity (furgal and prowse, ; furgal and prowse, ). however, the settlement of outstanding land claims with inuit could provide the basis for progress in addressing inequalities that lie at the heart of many health problems (tester and irniq, ) . the challenges facing first nations and mé tis will be similarly diverse, reflecting colonial history, extent of environmental dispossession, existence of outstanding land claims, and local institutional capacity and accountability. geography also matters. aboriginal peoples living in remote areas generally face more challenges and enhanced climate vulnerability than urban populations on account of their remoteness, close links to the land, and more pronounced socio-economic-political marginalization. access to informal health networks (e.g. through family, community wellness initiatives), however, remain strong in remote settlements and more is known about the burden of ill health among remote aboriginal populations than urban-based populations (adelson, ; wilson and young, ) . the factors that are identified here as creating aboriginal health system vulnerability in canada are also evident among indigenous populations in other 'fourth world' contexts. similarly, many adaptation challenges noted here are comparable to those documented by other studies in mostly middle-and some lowincome nations, challenging the generalization that developed nations have a high capacity to adapt to climate change. yet despite this, there are few examples of systematic attempts to assess the vulnerability of indigenous peoples, identify adaptation needs, or initiate adaptation action. as has been argued in the general climate change and health literature, a new public health movement is required to promote adaptation to the health effects of climate change (costello et al., ) . for indigenous peoples in developed nations this movement needs: interdisciplinary scientific research to characterize climate vulnerabilities specifically at local and regional levels and identify and prioritize opportunities for adaptation. health sector leadership to integrate climate change into health planning and provide guidance on the risks of climate change and importance of adaptation. effective communication from frontline health professionals on actions that can reduce sensitivity and increase adaptive capacity to climate change effects on health. government action to uphold the rights of indigenous peoples, tackle inequality, coordinate climate change adaptation planning across departments, and provide financial and human resources. institutional reform to improve health care access and delivery. international cooperation to transfer experience and knowledge from other contexts. co-ordination and collaboration between clinicians, public health officials, scientists and policy makers. partnership with indigenous peoples and organizations is imperative to identify health needs, generate knowledge, prioritize adaptations, and improve the functioning of health systems. moving population and public health knowledge into action developing a community health tool kit with indigenous health organizations: moving population and public health knowledge into action arctic climate impacts assessment the 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research in the social sciences: current trends and future directions from isotopes to tk interviews: towards interdisciplinary research in fort resolution and the slave river delta, northwest territories future health consequences of the current decline in us household income this project was supported by the social sciences and humanities research council of canada, the canadian institutes of health research, arcticnet, and the international polar year caviar project. two anonymous reviewers provided detailed and constrcutive feedback. thanks to adam bonnycastle for figures and . key: cord- -duxm u v authors: sweileh, waleed m. title: bibliometric analysis of peer-reviewed literature on climate change and human health with an emphasis on infectious diseases date: - - journal: global health doi: . /s - - - sha: doc_id: cord_uid: duxm u v background: assessing research activity is important for planning future protective and adaptive policies. the objective of the current study was to assess research activity on climate change and health with an emphasis on infectious diseases. method: a bibliometric method was applied using sciverse scopus. documents on climate change and human health were called “health-related literature” while documents on climate change and infectious diseases were called “infection-related literature”. the study period was from to . results: the search query found documents in the health-related literature and in the infection-related literature. the growth of publications showed a steep increase after . there were four research themes in the health-related literature: ( ) climate change and infectious diseases; ( ) climate change, public health and food security; ( ) heat waves, mortality, and non-communicable diseases; and ( ) climate change, air pollution, allergy, and respiratory health. the most frequently encountered pathogens/infectious diseases in the infection-related literature were malaria and dengue. documents in infection-related literature had a higher h-index than documents in the health-related literature. the top-cited documents in the health-related literature focused on food security, public health, and infectious diseases while those in infection-related literature focused on water-, vector-, and mosquito-borne diseases. the european region had the highest contribution in health-related literature (n = ; . %) and infection-related literature (n = ; . %). the usa led with ( . %) documents in health-related literature and ( . %) documents in infection-related literature. the australian national university ranked first in the health-related literature while the london school of hygiene & tropical medicine ranked first in the infection-related literature. international research collaboration was inadequate. documents published in the environmental health perspectives journal received the highest citations per document. a total of ( . %) documents in the health-related literature were funded while ( . %) documents in the infection-related literature were funded. conclusion: research on climate change and human health is on the rise with research on infection-related issues making a good share. international research collaboration should be funded and supported. future research needs to focus on the impact of climate change on psychosocial, mental, innovations, policies, and preparedness of health systems. climate change refers to long-term statistical shifts of the earth's climate system that result in new climate patterns [ ] . over the past century, industrial activities have led to long-term changes in the climate system that included global warming, flooding, and drought [ ] . the paris agreement, an agreement within the united nations framework convention on climate change (unfccc) signed in , represents an opportunity for all countries to implement measures to reduce, combat, and adapt to climate change [ ] [ ] [ ] [ ] . combating and reducing climate change is an important goal of the sustainable development goals (sdgs) which states "take urgent action to combat climate change and its impacts" [ ] . the implications of climate change on human health have led the world health organization (who) to declare climate change as one of the top ten global health threats in [ ] . climate change is negatively affecting human lives by changing the quality of air, water, and food supply [ ] [ ] [ ] . it is estimated that between and , climate change will cause approximately , additional deaths per year and - billion usd loss per year by [ ] . these devastating economic and health consequences require national and international planning to slow down climate change and to build resilient health systems that can tackle these changes [ ] . the effects of climate change are global and diverse [ , , ] . however, the impact on developing countries with limited resources and weak health systems will be more obvious [ ] [ ] [ ] [ ] [ ] . climate change has affected the epidemiology and pattern of both communicable and non-communicable diseases [ ] . for example, changes in temperature have serious adverse effects on the pattern and incidence of infectious diseases [ ] . global warming favors the survival and transmission of causative pathogens or vectors of the causative agent [ ] [ ] [ ] [ ] [ ] . climate change influences the dynamics of vector-borne, water-borne, foodborne, rodent-borne, and air-borne infectious diseases [ , ] . furthermore, a recent study predicted that climate change might worsen antimicrobial resistance [ ] . the study indicated that a spike in temperature of c was linked with a . % increase in antibiotic resistance to e.coli, which can trigger serious food poisoning; a . % increase in staphylococcus aureus, which can cause skin infections and food poisoning; and a . % increase in klebsiella pneumoniae, which can cause pneumonia. the spread of antimicrobial resistance is believed to have a serious negative global impact on human health [ ] . a study predicted that if antimicrobial resistance is not addressed, then by , million people will die because of antimicrobial resistance [ ] . many recent studies predicted that serious and emerging infectious diseases could appear or get worsened by climate change [ , ] . it is expected that the epidemiology and geography of many infectious diseases will change due to climate variability [ ] . for example, climate change will be an important factor for the spread of lassa virus in western africa [ ] . droughts are expected to increase the epidemics of west nile virus globally [ ] . higher incidence of cases of chikungunya and zika virus infections in brazil have been attributed to areas with more frequent rainfall and severe droughts [ ] . climate change and increased global temperatures have been associated with an increase in the probability of rift valley fever, cholera and malaria [ ] . the expected rapid spread of infectious diseases with climate change in the presence of antimicrobial resistance might cause global mass fatalities [ ] [ ] [ ] . the who considers climate change as a new threat to global health. this threat is compounded by globalization and modernization which can allow novel diseases to travel rapidly as what happened in the case of covid- [ ] . actually, the emergence of entirely novel diseases, like covid- , reintroduced the discussion of the impact of climate change on infectious diseases carried by wild animals or mosquitos and transmitted to humans. assessing research activity on climate change helps identify the national and international contribution to this field, the hot themes discussed by researchers, and research gaps in the field. climate change is a broad scientific topic and assessing research activity on climate change, in general, might not be very helpful. therefore, in the current study, the research activity of climate change on human health with an emphasis on infectious diseases was investigated. emphasis on infections was made due to suspected serious global outbreaks of infectious diseases such as dengue, ebola, and others [ ] [ ] [ ] [ ] [ ] . second, investigating research activity on climate change will help understand the type of infections mostly affected by climate change. third, research on climate change helps in developing appropriate protective measures and preparedness plans for certain infectious diseases in certain geographical areas. fourth, research on climate change comes as a response to calls made by international organizations such as the who on the importance of the impact of climate change on health and infectious diseases. based on the argument mentioned above and based on calls for papers made by certain specialized and prestigious journals in the field of public health and infectious diseases, the current study was undertaken to analyze the research aspects and research activity of climate change and human health with an emphasis on infectious diseases. the method used to display the research pattern and research activity on a certain topic is the bibliometric analysis which has been commonly used recently in various health topics [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . climate change has diverse effects that include aquatic organisms, forests, animals, and humans. the use of a bibliometric analysis is a suitable methodology to identify the volume and growth pattern of literature focusing on humans for further analysis related to health and infectious diseases. furthermore, bibliometric analysis is a suitable methodology to spot important research themes and active researchers and research institutions for future funding and planning. a literature search using well-known databases and search engines such as scopus database and google scholar revealed that there were at least ten bibliometric studies on climate change and its effects on various aspects on ecology or agriculture or adaptation [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . however, no bibliometric research papers were published on climate change and health or climate change and infectious diseases. therefore, the current study will establish the first baseline data on this topic for future comparisons and for policymakers to draw plans on climate change and human health with an emphasis on infectious diseases. the first step in any bibliometric study is to decide on the appropriate database to be used to retrieve the relevant documents. in the current study, sciverse scopus was used to accomplish the objective of the study. scopus is larger than web of science and has more than , indexed journals in all scientific fields [ ] . scopus is % inclusive of medline and therefore, it is far better than medline. furthermore, the export of data from scopus to other programs is easy to perform. scopus offers two methods of search; a basic and an advanced search in which complex and long search queries can be made to accomplish the objective with high validity. scopus allows for search using terms in titles or titles/abstracts or name of the journal or name of the author or affiliation. the second challenge in any bibliometric study is to build a valid search query that will retrieve as many documents as possible but with minimum irrelevant (false-positive) results. in the field of climate change, many keywords could be used. however, in the current study, the authors reviewed many articles published as "systematic reviews" or "bibliometric analyses" to build a search query for climate change [ , [ ] [ ] [ ] [ ] [ ] . the keywords used included, but not restricted to, the followings "climat* chang*" or "greenhouse effect" or "changing climate" or "global warming" or "extreme weather" or "climate variability" or "greenhouse gas" or "rising temperature" or "heat waves". other non-specific keywords were also used but under certain constraints. for example, keywords such as flood, drought, temperature, warm*, rain*, and "air pollution" were used under the condition that a phrase related to "climat* chang*" was also present in the title/abstract of the same document. the keyword "air pollution/air pollutant*" was used with restrictions because air pollution and climate change are closely related but are not the same. therefore, documents on air pollution within the context of climate change were included [ ] . actually, in systematic review studies on climate change, the keyword air pollution was not included [ , ] . similarly, in previously published blibliometric studies on climate change, air pollution was not included in the search terms [ ] . in scopus, the quotation marks were used to retrieve the exact words while the asterisk was used as a wild card. in the current study, the authors developed an extensive and comprehensive search query to retrieve all potential documents focusing on climate change and human health. the keywords used to retrieve healthrelated documents included, but not restricted to, health, respirat*, mood, cardiac, heart, hunger, "food *security", pregnancy, asthma, infect*, "infectious", "vector-borne disease", "water-borne disease", and many others. the search query was built mainly on title search to make sure that the retrieved documents are obviously and directly related to human health. additional file included all keywords and steps used to retrieve documents on climate change and health. documents retrieved from the search query on human health and climate change were called "health-related literature". for documents related to infectious diseases, the authors used the same search query stated above but with all possible keywords related to infection/infectious diseases, pathogens, and vectors transmitting pathogens to humans. documents retrieved for infectious diseases were called "infection-related document". details on the search query are shown in additional file . validation of the search queries was based on two approaches. in the first approach, the top cited documents in the health-and infection-related literature were reviewed to make sure that they fit within the scope of climate change and health or climate change and infectious diseases. this approach was adopted to eliminate false-positive results by excluding documents focusing on the impact of climate change on certain plants or animals or any document irrelevant to human health. the second approach was based on comparing the actual number of articles for each author, obtained from his/her personal scopus profile, with the number of articles obtained by the search query for active authors. the comparison was made using the pearson correlation test. a significant and strong correlation is indicative of a high validity of the search query and the absence of missing results. this approach was previously used in several bibliometric studies [ ] . data in the retrieved literature was exported to microsoft excel. the exported data included annual growth of publications, types of documents, languages, countries, authors, institutions, journals, citations, and funding agencies. the retrieved literature was also exported to vosviewer program [ ] to create network visualization maps. the strength of international research collaboration was presented as total link strength (tls) which is automatically given by vosviewer upon mapping research activity of selected countries. the tls is proportional to the extent of international research collaboration where higher tls value indicates greater collaboration. bibliometric indicators were presented as top ten active ones. for annual growth, statistical package for social sciences (spss statistics for windows, version . . armonk, ny: ibm corp.) was used to draw the annual growth of publications. for geographical distribution of documents, the who regional classification was used: the region of the americas (amro), the european region (euro), the western pacific region (wpro), the eastern mediterranean region (emro), the south-eastern asia region (searo), and african region (afro). the quality of publications was measured by the number of citations and h-index [ ] while the quality or impact of the journal was measured using the quartile ranking of journals obtained from scimago journal rank [ ] . journals in the q rank are considered to have the highest impact. the study period was from to . all citation analysis and data export were carried out on the same day (april , ) to avoid misinterpretation. the search query found documents on healthrelated literature and documents on infectionrelated literature. therefore, infection-related literature constituted . % of the health-related literature. retrieved documents were of different types (table ). research articles constituted . % (n = ) of the health-related literature and % (n = ) of the infection-related literature. there was a larger percentage of editorials (n = ; . %) in the health-related literature compared with that in the infection-related literature (n = ; . %). the annual growth of publications in the health-related literature was low in the s and s but showed a steep increase after . the annual growth pattern of documents in the infection-related literature followed the same pattern. however, the annual growth of documents in infectionrelated literature was relatively faster than that of the health-related literature. figure shows the growth of publications in the health-and infection-related literature depicted in dual-axis for easy comparison. mapping the most frequent terms in title/abstract fields of documents in the health-related literature with a minimum occurrence of gave terms distributed in four clusters representing four main research themes ( fig. ): . the first cluster (red) included items and focused on the following topics arranged alphabetically: adaptation, climate change, food production, food security, public health, health policy, healthcare system, and psychology. analysis of author keywords in infection-related literature indicated that malaria ( occurrences), dengue ( occurrences), and arboviruses (arthropod-borne viruses) ( occurrences) were the most frequent infectious diseases/pathogens encountered ( (table ). there was a significant and strong correlation between the percentage contribution of each region to health-and infection-related literature (p < . , r = . ). the top ten cited documents in health-related literature focused on the impact of climate change on food security, public health, and infections [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in the infection-related literature, the top ten cited documents discussed water-, vector-, and mosquito-borne diseases as well as general effects of climate change on infectious diseases, particularly malaria and dengue [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the list of top active institutions/organizations for the health-related literature was dominated by australian and american institutions with australian national university ranking first with ( . %) documents ( for the health-related literature, international journal of environmental research and public health was the leading journal with ( . %) documents. in the list of active journals, two journals were in the field of infection while the remaining were in the field of public health, environment, and general medicine ( table ) . table shows the top ten active authors of health-and infection-related literature. researchers from europe, north america, australia, and china dominated both lists. the top active author for the health-related literature was from the usa while the top active author for the infection-related literature was from china. in total, author names participated in publishing the infection-related literature, an average of . authors per cholera salmonella west nile virus plasmodium falciparum influenza tick-borne encephalitis ross river virus zika rift valley fever leishmaniasis japanese encephalitis campylobacter cercariae hantaviruses a this list is not % inclusive of all pathogens or infections present in the retrieved literature. please see result section and fig. for more details document. one hundred and thirty-nine ( . %) documents were single-authored. for health-related literature, a total of ( . %) documents were funded. the national natural science foundation in china was the most active funding agency (n = ; . %) followed by the national institutes of health (nih; usa) and national science foundation (nsf; usa) ( table ) . for the infection-related literature, analysis showed that ( . %) documents were funded. the nih (usa) was the most active in funding (n = ; . %) followed by nsf (usa) (n = ; . %). the who was listed as one of the top ten active funding agencies infection-related literature. the current study was carried out to give a snap shot of research on climate change on human health with an emphasis on infectious diseases. the current study showed an increasing number of publications on climate change and health in the past decade. the gradual increase in the number of publications was parallel to international warning signals since the early s about the impact of climate change on human health. the first major international conference on the greenhouse effect at villach, austria, warned that greenhouse gases will cause a rise of global mean temperature which is greater than any in man's history [ ] . in , the united nations (un) created the intergovernmental panel on climate change (ipcc) to analyze and report on scientific findings. in , climate change convention, agrees to [ ] . in , the kyoto protocol calls for cutting emissions from industrialized nations [ ] . however, due to political and economic reasons in industrialized nations, the koyoto protocol did not come into force until . the ipcc fourth report issued in called for all countries to take adaptive measures to face climate changes [ ] . the steep rise in the number of publications on climate change and human health coincided with the release of the fourth ipcc report which blamed humankind activities for the irreversible climate changes. in the face of increasing evidence of the impact of climate change on human health, the who took an active role in developing policies to minimize the impact of climate change on health. in , the who executive board endorsed a new work plan on climate change and health that included raising awareness, endorse science and research on climate change, and support public health adaptive measures for climate change [ ] . the timeline history, debate, negotiations, and conventions at the international levels affected both the volume and pattern of research on climate change and its impact on human health. the current study indicated that there were four research themes on climate change and human health. these research themes were closely related. of particular interest in the current study was the research theme focusing on climate change and infectious disease. however, there was a small cluster representing the interplay of climate change and air pollution in the context of human health. air pollution is a complex subject and has fundamental effects on human health. in the current study, we focused on air pollution within the context of climate change and the resultant effect on human health. that is why the air pollution research theme was the smallest research theme as shown in the map. both climate change and air pollution are global environmental problems that are closely related and considered as twins but they are not the same thing. climate change is the global variation of the earth's climate which is accelerated by greenhouse gases caused by human activity. carbon dioxide is the main gas contributing to climate change, but it is not harmful to human health. air pollution is defined as the presence, in the air, of substances or particles that imply danger, damage or disturbance for humans, flora or fauna. the main sources of atmospheric contamination are gases that result mainly from emissions caused by the burning of fossil fuels emissions generated by transport, industrial processes, burning of forests, aerosol use, and radiation. both climate change and air pollution are worsened by the burning of fuel, increasing the co emissions which cause global warming. meanwhile, the generation of other pollutants, such as nitrogen oxides (no and no ), sulfur oxides (so and so ) and particulate matter, is the main reason the air is contaminated [ , ] . climate variations affect air quality; air pollution can worsen climate change and both can directly or indirectly affect health [ ] . the major and obvious health effect of climate change and air pollution is on respiratory health where both can exacerbate allergies and bronchial asthma [ ] . the complex interactions between climate change and air quality is a new area of research that requires further investigation [ , ] . the current study indicated that infections constituted a major theme of research on climate change and human health. climate change and temperature rise affect the transmission and spread of many pathogens [ ] . the current study showed that documents about malaria and dengue were among the top ten cited documents. malaria was the most frequently encountered infectious disease affected by climate change. malaria is a vectorborne disease that is sensitive to long-term climate change. for example, malaria epidemic risk increases around five-fold in the year after an el niño event in india [ ] . researchers have developed mathematical models to forecast future climatic influences on infectious diseases. the model aims to apply the statistical equations to future climate scenarios in order to predict the actual distribution of the disease. these models have been applied to malaria and dengue fever [ , , [ ] [ ] [ ] [ ] [ ] [ ] . the case incidence of dengue fever has multiplied fold since the s [ ] . according to the who estimation, . - . % of the world's population ( . - . billion) is living in areas where dengue viruses can be transmitted [ ] . in ranking, two equally active journals were given similar ranks and one position in the rank was skipped. c/d = number of citations per document. q = quartile infection-related literature: documents on climate change and infectious diseases. health-related literature: documents on climate change and health the current study showed that more than two-thirds of the global publications came from the amro and euro regions. there are many reasons for the leading role of these two regions. the presence of the us cdc, euro cdc, and many other governmental and nongovernmental research and academic institutions the field of public health and infectious diseases helped these two regions to make this tremendous and significant contribution. second, the main funding agencies are located in these two regions. third, infections have no borders and pathogens could travel with human migration waves which made europe and north american regions in a critical geographic position to any infectious disease outbreak. fourth, both regions have a great responsibility toward climate change since many of these changes were made by industrial activities. the current study showed that china was among the top ten active countries. the contribution of china might be underestimated because it is possible that most publications from china were published in national chinese journals that are not indexed in scopus. the same argument could be applied to other regions and countries with a limited number of peer-reviewed journals indexed in scopus such as russia or certain countries in south america. the current study showed that afro region made a greater contribution than either emro or searo region. a possible reason for the relatively higher contribution of the afro region is the strong research collaboration between certain african countries and the usa and the uk. climate change in the afro region increased the number of people in africa who are at risk of malaria [ , ] . the increase in the number of mosquitoes increased the opportunity for both plasmodium falciparum and plasmodium vivax parasites to proliferate and place more people at risk of contracting malaria [ , ] . aside from malaria, the afro region is expected to suffer from hunger and food insecurity due to climate change [ , ] . the climate change in the afro region is worsened by the weak economies, lack of resilient health systems, and lack of political stability in certain african regions. the current study showed that the emro region had the least contribution despite that the region is expected to suffer from serious climate variations [ ] . a systematic review on climate change and health in the emro region identified many knowledge and research gaps with research scarcity in this field [ ] . the authors of the systematic review concluded that the impact of climate change on health is not recognized as a priority area by health researchers, health professionals and policymakers in the emro region. international research collaboration and funding are important for countries in the emro, afro, searo regions where effects of climate change are expected to be beyond their economic and research capabilities. the current study showed that retrieved publications received a high number of citations suggestive of a large number of researchers who are interested in the topic. this could be attributed to the following reasons: first, the topic of climate change has caught the attention of scientists all over the world since the early s with many national and international warning reports about the climate change. second, the climate change is a multidisciplinary subject and therefore, researchers and scientists in public health, infectious diseases, nutrition, environmental health, ecology, and others were highly keen to investigate the subject and to have an input in this evolving topic. third, the fact that the top ten active journals in publishing documents were influential in their field gave credibility and attracted a larger number of citations. fourth, the leading role of the who as an international health agency played a positive role in raising the number of citations. finally, the number of authors played a positive role in increasing the number of citations [ ] . the current study indicated that infection-related documents received a higher number of citations and a higher h-index than documents in the health-related literature. this finding suggests that of the diverse health effects of climate change, its impact on the epidemiology and emerging infections receives the greatest scientific attention. this is due to the high and immediate risks of emerging and re-merging infections on global health. the h-index of the infectionrelated literature was higher than that reported for strongyloidiasis literature [ ] , epidermal parasitic skin diseases [ ] , antimalarial drug resistance [ ] , but lower than that on campylobacter or carbapenem resistance [ , ] . the current study emphasizes the importance of certain future research directions in the field of climate change and human health. the current study has a few limitations. the literature investigated has been retrieved from journals indexed in scopus while grey literature and publications in nonindexed journals have not been analyzed. therefore, journals from non-english speaking countries might be underestimated. this has further consequences on the top ten active countries, institutions and authors. the second limitation was the method for counting the number of documents for each country or author or institution. scopus makes all analysis based on the number of different affiliations in the documents. therefore, a document with several authors having the same country affiliation was counted once for that country. however, a document with two authors having two different country affiliations were counted once for each country. this has increased the research output of certain countries with greater international research collaboration even if the authors from that country was not the main or corresponding author. the citation analysis did not take into consideration the self-citations which could create a bias in the number of citations for countries, journals, and authors. finally, the search query was built to focus on climate change and human health. the definition and scope of human health and climate change are broad and complex. therefore, it is difficult to ensure a % inclusion of literature on both topics. however, the author did his best to include all relevant literature with minimum irrelevant documents. the final point is the inclusion of air pollution and air pollutants in the search query. the author included these keywords with restrictions to keep the manuscript focused on climate change. the purpose of including these terms was to retrieve documents discussing air pollution and health within the context of climate change. therefore, the number of documents retrieved in this topic was presented by research theme (cluster ) which was the smallest cluster. inclusion of air pollution in the search query without restriction will retrieve large volume of irrelevant documents on pollution that were irrelevant to climate change. this was the first bibliometric study on climate change and health or infection-related literature. key players, research themes, and research gaps were identified. the current study provided researchers and policymakers with baseline data in this field. the current study emphasized the importance of climate change on the epidemiology and geography of infectious diseases. adaptive national and international measures to combat climate change should include plans to contain the expected increase in vector-borne diseases particularly malaria and dengue. the current study showed 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interests.received: january accepted: may key: cord- - s ok uw authors: nan title: abstracts of the th annual symposium of the protein society date: - - journal: protein science doi: . /pro. sha: doc_id: cord_uid: s ok uw nan c-terminus glutamine-rich sequence deleted) to elucidate the role of metalloprotein hpn-like by fluorescence resonance energy transfer (fret) (figure ) [ ] . we found the selective coordination of ni(ii) and zn(ii) to the purified sensors and in e. coli cells. surprisingly, specific interaction between the fret sensors and bi(iii) was observed. our fret analysis confirmed the role of hpnl for ni(ii) storage and revealed the potential association of hpnl with bi-based antiulcer drugs in cells. pb- rna fate is controlled by highly-regulated rna binding proteins molecular mechanism that links the mrna-degradation pathway with extracellular signaling networks through the reversible unfolding of a rna binding domain (rbd). rna binding is also controlled by ph conditions. this finding becomes relevant for rbps such as t-cell intracellular antigen (tia- ), which shuttles between two cellular compartments (nucleus and cytoplasm) with slightly different ph values. in fact, rna binding by tia- is modulated by slight environmental ph changes due to the protonation/deprotonation of tia- histidine residues [ , ] . the ph dependence of the tia- /rna interaction provides a new insight into the function of tia- in recognizing new rna targets [ ] , like the ' terminal oligopyrimidine tracts ( tops) of translationally-repressed mrnas. along with tia- , the rbp hu antigen r (hur) is involved in the assembly/disassembly of cytoplasmic stress granules (sg), which arise as a protective mechanism by preventing mrna decay under stress situations. despite wide acceptance that rbps harboring aggregation-promoting prion related domains (prds), such as tia- , stimulate rapid self-association and formation of sgs, we propose that scaffolding sgs may be driven by rbds, since prd-lacking rbps, like hur, often form oligomers [ , , ] and are included in sgs. under continuous stress, the transition from the physiological to pathological aggregation of rbps in sgs may depend on post-translational modifications of rbds. rna-binding proteinopathies, characterized by the nucleation of irreversible sgs, are often found in neurodegenerative diseases. altogether, resulting insights into rna biology suggest that highly-regulated rbps determine mrna fate from synthesis to decay. a threat to million people in underdeveloped nations around the world, african trypanosomiasis (sleeping sickness) is a neglected tropical disease (ntd) caused by the protozoan parasite trypanosoma brucei (t. brucei). t. brucei is transmitted to humans via the tsetse fly, and replicates in the blood before crossing into the brain, causing death for the infected individual. current treatments that are available for african sleeping sickness are highly toxic and usually difficult to administer past the blood-brain barrier. it is our belief that coupling less toxic compounds with efficient drug delivery systems will contribute to the development of the most effective drug against african sleeping sickness. our goal was to determine a novel and effective chemical inhibitor with the potential to prevent the replication of t. brucei in the human body. the enzyme target for inhibition studied in this research was -phosphogluconate dehydrogenase ( pgdh), a cytosolic enzyme in the pentose phosphate pathway (ppp) of t. brucei. pgdh is essential in the ppp due to its ability to oxidize -phosphogluconate into ribulose- -phosphate, which is essential for the formation of nucleotides. primer overlap extension polymerase chain reaction (pcr) was used to synthesize the coding dna sequence of the pgdh gene, which was then cloned into a pnic-bsa inducible expression plasmid with an n-terminal histidine tag, by way of ligation independent cloning. the protein was then expressed in bl (de ) escherichia coli (e. coli) cells and purified via nickel column affinity and size exclusion fast protein liquid chromatography (fplc) to perform inhibition assays. through virtual screening, various ligands obtained from the chembridge library and nih clinical collection) were docked into the active site of the crystal structure of tb pgdh (pubchem identification pgj) using gold molecular docking software. the top scoring compounds were selected by utilizing parameters such as hydrophobic interactions, hydrogen bonds, and van der waals forces. the compounds with the best scores that also satisfied lipinski's rule of criteria for druggability were then tested in spectrophotometric enzyme inhibition assays monitoring the absorbance of nadph at nm. compounds that show inhibitory activity in the assays will be taken to higher levels of testing to determine their effect on t. brucei in other organisms. nmr studies of the structural influence of phosphopantetheinylation in nonribosomal peptide synthetase carrier proteins and impact on binding affinities andrew goodrich , dominique frueh nonribosomal peptide synthetases (nrpss) are modular enzymatic systems responsible for the production of complex secondary metabolites in bacteria and fungi. each module is comprised of (at least) three core domains whose combined action leads to the selection, activation, and incorporation of a single small molecule into a growing peptide. central to each module is the carrier protein (cp), which is first primed via attachment of a '-phosphopantetheine moiety (ppant arm) to a conserved serine to generate the active holo form. an adenylation (a) domain then covalently attaches an amino or aryl abstract acid onto the ppant arm via formation of a thioester. the cp then shuttles activated monomers and growing peptides between the active sites of catalytic domains in both the same and adjacent modules. during cp priming and peptide elongation, a cp thus exists in multiple different post-translational states and interacts with numerous catalytic domains. understanding how nrpss are able to efficiently orchestrate this series of sequential protein-protein interactions between a cp and its partner catalytic domains is key to unraveling the molecular mechanism of nrp synthesis. using a combination of isothermal titration calorimetry and nuclear magnetic resonance (nmr) titrations, we found that converting a cp from the apo to holo form alters its affinity for its partner a domain. this change in binding suggests a means by which directionality in protein-protein interactions is achieved in nrpss. however, we also found that a domain binding affects the same subset of residues in both the apo and holo forms. in order to identify the molecular features underpinning this difference in affinity, we solved the nmr solution structures of the apo and holo forms of the cp. here, we present the solution structures of an apo and holo cp and discuss them in light of their differential binding to an a domain. functional analysis of of conditional analog-sensitive alleles of essential protein kinases in the fission yeast schizosaccharomyces pombe. juraj gregan , mfpl/imp, the genome of the fission yeast schizosaccharomyces pombe encodes for protein kinases that are essential for viability. studies of the essential kinases often require the use of mutant strains carrying conditional alleles. to inactivate these kinases conditionally, we applied a recently developed chemical genetic strategy. the mutation of a single residue in the atp-binding pocket confers sensitivity to small-molecule inhibitors, allowing for specific inactivation of the modified kinase. using this approach, we constructed conditional analog-sensitive alleles of essential protein kinases in the fission yeast s. pombe. i will present the functional analysis of these mutants during meiosis. peptide conjugates: from self-assembly towards applications in biomedicine ian hamley university of reading, dept of chemistry self-assembling peptides and their conjugates offer exceptional potential in nanomedicine. i will present some of our recent work on nanoscale assembled peptides and their conjugates, focussing on lipopeptides [ , ] and peg-peptide conjugates [ ] . pegylation is an important technique in the development of conjugates for applications in therapeutics. it is found to greatly influence self-assembly of peptides and proteins -one example from our own work is a peptide which itself forms twisted fibrils but when peg is attached, self-assembly of the conjugate leads to spherical micelles [ ] . the conjugate can be enzymatically degraded using alpha-chymotrypsin, releasing the peptide. this nanocontainer delivery and release system could be useful in therapeutic applications. thermoresponsive telechelic peg/peptides with hydrophobic dipeptide end groups (di-tyrosine or di-phenylalanine) were developed, one of which shows a de-gelation transition near body temperature and which may be useful in bioresponsive delivery systems [ ] . examples from our recent work on self-assembling lipopeptides will also be outlined. our focus is to investigate potential relationships between self-assembly and bioactivity, in particular in the fields of regenerative medicine [ ] [ ] [ ] [ ] [ ] , antimicrobial systems [ , ] and immune therapies [ ] . been shown to become derivatized with argpyrimidine, a prominent nem that occurs on arginine residues [ ] , in certain human cancer tissues and cell lines [ , ] . this nem was linked to the elevated antiapoptotic activity of the protein [ , ] , whereby modification of arg- appeared to be of particular significance [ ] . in this work, hsp homogeneously modified with argpyrimidine at position is generated for the first time. using expressed protein ligation [ ] , the first semisynthesis of the unmodified protein is achieved as well. our approach, which combines organic chemistry, peptide synthesis and protein synthesis, enables complete control over protein composition and thus can provide previously unattainable insight into the properties of this vital chaperone following nonenzymatic modification. the synthesis of argpyrimidine-modified hsp and the progress towards structural and functional characterization of the protein will be presented herein. kunitz-type protease inhibitors belong to a widespread protein family present in many plant species and play an important role in plant defense against insect pests and pathogens. members of this family are typically inhibitors of proteases of serine class. interestingly, a few members were identified as inhibitors of proteases of cysteine class, however, they have not been functionally and structurally characterized. our study is focused on kunitz-type inhibitors of cysteine proteases (pcpis) from potato (solanum tuberosum). a series of kda pcpis was purified using a multi-step chromatographical protocol, and two most abundant and effective isoinhibitors named pci - and pci were characterized in detail. they were screened against a broad panel of model cysteine proteases and digestive cysteine proteases from herbivorous insects. pci - and pci exhibit different inhibitory specificity pattern and potency up to the nanomolar range. both isoinhibitors were crystallized and their spatial structures were solved and refined at . Å (pci - ) and . Å (pci ) resolutions. a position of reactive sites against cysteine proteases on the conserved b-trefoil fold scaffold was proposed. the work provides the first analysis of pcpis with respect to the structure-function relationships and evolution within the kunitz-type inhibitor family. role of the abcc transporter in the mode of action of the bacillus thuringiensis cry ac toxin in the diamond back moth plutella xylostella protonation pattern influence actively properties of molecules and play an essential role in biochemical mechanisms. for an accurate determination of the protonation equilibria, the absolute proton solvation free energy needs to be known. the determination of this energy represents one of the most challenging problems in physical chemistry. this is particularly difficult for protons solvated in water, where the solvation is dynamically performed by different water clusters and the proton is not attached to a single solvent molecule. the proton solvation is notably important in order to quantify mechanisms of proton transfer and such processes have been investigated for a long time based on different approaches, often leading to contradictory conclusions. a rigorous and accurate protocol for computing proton solvation in solvents of different nature is of prime importance for applied (pharmaceutical and material science) and fundamental sciences. in this study, proton affinities, electrostatic energies of solvation and pka values of a reference set of organic molecules are computed in protic and aprotic solvents. proportional to the free energy of proton dissociation, the pka value calculation is therefore strongly dependent on the free energy of proton solvation. such energy is then determined in acetonitrile (acn), methanol (met), water and dimethyl sulfoxide (dmso) in order to obtain the best possible match between measured and computed pka values. the computation of these values is based on a combination of quantum chemical (qc) and electrostatic approaches by using a thermodynamic cycle connecting gas-phase and solvent-phase of proton dissociation. the computed proton solvation energies in acn, met, water and dmso of the present study are very precise (rmsd much lower than ph value). they will be a basis for better understanding of proton solvation and help to predict pka values of organic compounds in different solvents more precise. biochemical characterization of two evolutionary distant ten-eleven translocation enzymes and their utility in -methylcytosine sequencing in the genomes at single-base resolution subtypes leading to an inability to perceive pain and painful neuropathies, respectively. however, as nav ion channels are intimately involved in almost all aspects of physiology, only the most selective inhibitors would be suitable as drug leads. disulfide-rich venom derived mini-proteins from cone snails and spiders are being actively pursued as novel therapeutics for pain, because of their high selectivity and potency at human ion channels, including sodium channels (nav). two main strategies of inhibition have been identified; blocking the pore and interacting with the voltage-sensor domains (vsd) surrounding the pore. the ion-conducting pore is highly conserved between all sodium channel subtypes whereas the voltage-sensor domain binding sites are less conserved. therefore, inhibition of a specific nav isoform is more achievable using inhibitors that modulate vsds than with pore blockers. gating modifier toxins from spider and cone snail venom inhibit nav . and nav . by interacting with the vsd. they appear to reach their target by partitioning into the lipid membrane surrounding the ion channel, thus enabling access to the vsd. toxin pharmacology may therefore not only be driven by the peptide-ion channel interactions, but also including the lipids surrounding the channel protein, a feature that is very much under explored. it is therefore apparent that peptide-lipid interactions in combination with peptide-channel interactions need to be considered when designing potent inhibitors. using a range of biophysical techniques, including surface plasmon resonance and nuclear magnetic resonance, we are studying the interactions underpinning the mechanism of action between toxins and membranes and toxins and ion channels. initial results show that the lipid composition surrounding ion channels play a major role in terms of toxin:lipid interaction and that these interactions can be used in combination with traditional structure-activity relationship studies to design selective and potent nav inhibitors, which will be discussed. we believe that our studies will ultimately delineate what drives toxin pharmacology and nav subtype selectivity and will lead to improve rationally engineering of novel therapeutics for the treatment of pain. micelles promote aß assembly into pore-forming oligomers montserrat serra-batiste , mariam bayoumi , margarida gair ı , mart ı ninot-pedrosa , giovanni maglia , nat alia carulla institute for research in biomedicine (irb barcelona), biochemistry, molecular and structural biology section, university of leuven, the formation of amyloid-b peptide (ab) oligomers at the cellular membrane is considered to be a crucial process underlying neurotoxicity in alzheime rs disease (ad). - therefore, it is important to understand how oligomers form within a membrane environment. using solution nuclear magnetic resonance (nmr) spectroscopy, combined with size exclusion chromatography (sec), we have studied the two major ab variants-ab and ab , the latter having a more prominent role in ad than the former-under carefully selected micelle conditions intended to mimic a membrane environment. our results indicate that after an incubation period, ab , but not ab , assembles into oligomers with specific structural properties, which we have named stabilized micelle oligomers (smos). smo complexes incorporate into lipid bilayers as well-defined pores, a feature linked to neurotoxicity. these results have important implications in the ad field as they provide a new perspective on how ab oligomers cause neurotoxicity. indeed, our findings constitute a first step towards the establishment of a new therapeutic target for ad. dimer formation. it should be noted that this nb peptide contains the autophosphorylatable ser- associated with phk activation, and phosphorylated nb; peptide was considerably less effective in promoting b-dimer formation than non-phosphorylated peptide. these results suggest a role for ser- autophosphorylation in mediating homodimeric b subunit interactions within the phk complex, and augment previous studies on the activation of phk by phosphorylation in which changes at the nterminus of b are critical in the activation of the catalytic g subunit. summing these results leads to a new model of activation. in this model, in the inactive state, the nonphosphorylated n-terminus of b interacts directly or indirectly with the regulatory c-terminal domain of the g subunit, inhibiting catalytic activity. upon phosphorylation of the n-terminus of b, three important events occur: ) the interaction between b and g is disrupted, ) the b subunits of the holoenzyme self-associate, and ) the catalytic domain is activated. thus, we envision that the n-terminus of b acts as an allosteric switch, with activation triggered by phosphorylation of this region, causing disruption of its previously inhibiting interactions with g and promotion of b b dimerization to stabilize the activated conformation of g . the research was supported financially by the university of kansas medical center biomedical research training program and nih grant dk . pb- hssb is involved in the cellular response to oxidative dna damage christine touma , nicolas paquet , derek j. richard , roland gamsjaeger , , liza cubeddu , school of science and health, university of western sydney, queensland university of te chnology, school of molecular bioscience, university of sydney cellular dna is subject to oxidative damage in the presence of reactive oxygen species. the , -dihydro- -oxoguanine ( -oxog) adduct is the most common form of oxidative damage and results in g:c to t:a transversions; these lesions are normally processed by the base excision repair (ber) pathway. singlestranded binding (ssb) proteins of the oligonucleotide binding domain family are heavily involved in dna repair processes, which involve the detection of dna damage and recruitment of repair proteins to the site of damage. using immunofluorescence we demonstrate that hssb (a novel human ssb) levels increase in response to oxidative damage (h ). cells depleted of hssb are hypersensitive to oxidative damage and are also unable to efficiently remove -oxog adducts. we show that hssb forms dimers and tetramers under oxidative conditions and that this oligomerisation is likely mediated by inter-domain disulfide bond formation. furthermore, using surface plasmon resonance, we also show that oxidised hssb binds to -oxo-g damaged ssdna with higher affinity than non-damaged ssdna, indicating a direct role for oxidised hssb in the recognition of -oxo-g lesions. as oxidative stress is associated with aging, cancer and alzheimer's disease, understanding the molecular mechanisms of how cells repair oxidative dna damage will be crucial in the development of potential therapeutic treatments. epidemic typhus, which is caused by the bacterial pathogen rickettsia prowazekii, is a menacing disease world wide that the nih lists as one of america's greatest biological weapons threats. this research seeks to find novel inhibitors of b-ketoacyl-acp-reductase (fabg), an enzyme that catalyzes one of the reactions in the fatty acid synthesis type ii system in bacteria. this pathway is essential for survival in bacteria. the fabg enzyme uses nadph as a substrate, which facilitates the binding of the second substrate, acetoacetyl-acp into the active site. the acetoacetyl-acp is subsequently reduced into b-hydroxyacyl-acp. the coding dna sequence for the rpfabg protein was cloned into a pnic vector and transformed into e.coli bl (de ), then the protein was expressed and purified using metal affinity and size exclusion chromatography methods. high throughput molecular docking software (gold) was used to screen a commercial library of ligands against the acetoacetyl-acp region of the active site. the ligands with the best gold scores were selected to be tested in vitro. spectrophotometric enzyme inhibition assays were performed to determine whether the drugs could inhibit rpfabg activity. chlorogenic acid, a previously known inhibitor of homologous fabgs, was tested along with the other potential drugs, and was determined to have moderate inhibitory effects on rpfabg. loop modeling using icm software was performed in order to create a prediction of the complete rpfabg structure, including the disordered loops that are not a part of the f i pdb structure. co-crystallization of rpfabg with both substrates was carried out in order to obtain a structure, but only nondiffracting crystals resulted. further inhibition assays and crystallography trials are being performed in order to continue the search for a novel inhibitor of rpfabg and ultimately a treatment for epidemic typhus. the university of hong kong bioconjugation of proteins has emerged as a useful tool in the study of biological systems. there is an increasing need to develop new synthetic technologies for the bioconjugation reaction of proteins, and metal-catalyzed site-selective modification of proteins has attracted considerable interest in recent years. we have developed a ruthenium glycosylated porphyrin-catalyzed carbenoid transfer reaction for the site-selective modification of proteins. we firstly applied the catalysis to the selective modification of the n-terminus of peptides. by using ruthenium glycosylated porphyrin as catalyst, the n-terminus of a number of peptides can be modified through carbenoid n-h bond insertion in aqueous media with moderate to excellent conversion. the reaction is highly selective, for example, the reaction with ytsssknvvr, which contains various types of oxygenhydrogen and nitrogen-hydrogen bonds possibly available for carbenoid insertion, catalyzed by the ruthenium glycosylated porphyrin gave the n-terminal-modified product with > % conversion and without the formation of other modified peptides including doubly modified and oxygenhydrogen bond insertion products. we next extended the n-terminal modification method to proteins. eventually success was attained in the modification of rnase a and insulin. the reaction of rnase a with a diazoacetate mediated by ruthenium glycosylated porphyrin gave corresponding n-terminal-modified protein with % conversion. we also achieved a bioconjugation to ubiquitin via ruthenium glycosylated porphyrin-catalyzed alkene cyclopropanation in aqueous solution in two steps: ( ) incorporation of an alkenic group by the reaction of n-hydroxysuccinimide ester with ubiquitin and ( ) cyclopropanation of the alkene-tethered lys ubiquitin with the fluorescent labeled diazoacetate in the presence of a catalytic amount of ruthenium glycosylated porphyrin. the corresponding cyclopropanation product was obtained with % conversion based on maldi-tof mass spectrometry. in conclusion, we developed a ruthenium porphyrin-catalyzed siteselective modification of peptides and proteins in aqueous media. the method provides an entry to new bioconjugation reactions for protein modifications using metalloporphyrins as catalysts. uridine monophosphate synthase: architecture versatility in the service of late blight control francisco tenjo castaño , , manuel garavito , , leonor garc ıa , , silvia restrepo , barbara zimmermann biochemistry and molecular biology research group, universidad de los andes., mycology and plan pathology laboratory, universidad de los andes uridine monophosphate synthase (umpase), a bifunctional enzyme in the de novo pyrimidine biosynthetic pathway, is a protein comprised of orotate phosphoribosyl transferase (oprtase) and orotidine monophosphate decarboxylase (odcase). different fusion orders of the two domains have been documented to exist in nature. in some organisms oprtase and odcase are monofunctional proteins, and act as a complex. here, umpase from solanum tuberosum (potato) and from phytophthora infestans (an oomycete) were examined. p. infestans causes late blight disease in s. tuberosum, destroying crops and increasing production costs. since pyrimidines are fundamental cellular components, we have proposed that umpase could serve as a target to control p. infestans infection. the enzymes from p. infestans and s. tuberosum differ in their fusion order of oprt and odc. the study of these two umpase could facilitate the design of species-specific inhibitors, and might shed light on the effect of fusing umpase domains in one order or the other. to this end we carried out bioinformatic and biochemical characterization of the enzymes. sequence analyses showed residue differences among the p. infestans umpase sequences from three strains: , and t - . strain t - was found to have a duplicated umpase, but neither sequence corresponded to the ones predicted previously from the genome. a recombinant umpase from strain was expressed in bacteria and purified but it showed low solubility and was inactive in vitro. the recombinant umpase from the strain complemented both oprtase and odcase deficient e. coli strains. a soluble, active, recombinant protein was expressed and purified in the presence of high salt and the product ump (specific activity . lmol min- mg- ). the sequence skq was found at the c-terminus of the p. infestans umpase sequences and resembles a peroxisome signal peptide (skl). the predicted hydrophobicity of this umpase and its architecture (oprt at the c-terminus and odc at the n-terminus) resembles that of the umpase from leishmania donovani, which has been localized to the peroxisome. we suggest that p. infestans umps could also be located in this organelle. in contrast to the oomycete enzyme, s. tuberosum umpase is highly soluble, and has a higher specific activity (vmax . lmol min- mg- ). we measured the kinetic parameters km(orotate) . lm, km(prpp) . lm, and found that it exhibited product inhibition by pyrophosphate. in conclusion, the different architectures of the two umps might be related to distinct biochemical characteristics, further supporting this protein as a good candidate for p. infestans control. we present computer simulation studies of three different antimicrobial peptides we have been studying by md computer simulation in collaboration with experimentalists. the first is daptomycin, a potent lipopeptide currently licensed to treat infections caused by multi-drug-resistent bacteria. the mechanism of action of daptomycin is currently not completely understood. we have solved the nmr structure of this molecule, and attempted to determine the size of its oligomer by small angle neutron scattering (sans) supported by computer simulation. feglymycin is a -amino-acid peptide with a high percentage of unusual amino acids such as -hydroxyphenylglycine and , -dihydroxyphenylglycine. feglymicin inhibits mura and murc enzymes which are involved in bacterial peptidoglycan synthesis, while also displaying anti-hiv activity by interaction with the viral envelope protein gp . a previous x-ray structure shows the molecule forming a dimer. here, the molecule was studied by nmr in water and dmso. in water, the molecule is clearly at least a dimer, while in dmso it is a monomer. we have performed noe refinement simulations in order to elucidate a structure, however, due to a lack of long-range noe contacts, a unique structure cannot be determined. labyrinthopeptin a is a lantibiotic that contains labionin, a unique carbacyclic posttranslationally modified amino acid that links the protein backbone in three different locations. labyrinthopeptin a has shown promising activity as a pain killer. starting from the x-ray structure, we present results from the first md simulation studies of this unique peptide. because of the extensive cross-linking, this peptide is observed to be highly rigid in its native form. simulation results of mutants are also presented. antibiotics with new mechanism of action are urgently required to combat the growing health threat posed by resistant pathogenic microorganisms. here we report the discovery of a new peptidomimetic antibiotic (l - ), which is active with a minimum inhibitory concentration (mic) in the low nanomolar range, only against pseudomonas sp., and with a non-membrane-lytic mechanism of action. a drug target identified both in a forward genetic screen for resistance determinants and by photoaffinity labeling is the ß-barrel protein lptd, which plays an important role in lps transport and the outer membrane biogenesis. the x-ray structure of lptd in complex with lpte from shigella flexneri shows a stranded b-barrel linked to a periplasmatic n-terminal jelly-roll domain. interestingly the homology model structure for lptd from pseudomonas shows a significant difference: an insertion of around amino acids in the n-terminal domain. the results of our attempts to purify and characterize this large outer membrane protein and to determine the binding site of the peptidomimetic antibiotic will be shown. the theory of how life on earth begun still remains unclear. nevertheless, according to some theories, at the beginning level proteins did not emerge as a complex globular forms as know today. at the times, when solely rna molecules stored both genetic information and catalyzed the chemical reactions in primitive cells, peptides acted as a proteins nowadays [ , ] . literature postulate that the possible role of primordial short peptides was to catalyze reactions in rna-world, as they possess an excellent ability to self-assemble into well-ordered nanostructures [ , ] . elementary functional loops (efls) can be considered as a small structures (blocks) having specific signatures and providing functional residues important for binding/activation as well as principal chemical transformation steps of the enzymatic reaction [ ] . p-loop efl is a widespread structure across vast majority of protein families such as motor domains, aaa , reca, pepck and many others. sequential alignment of these protein families reveals existence of a conserved p-loop motif, that is able to bind atp molecule. we investigated the structure and atpase activity of peptides, which sequences possessed strongly conserved gxgk[t/s] motif from ploop. the goal of our work was to check if peptides corresponding to the most conserved p-loop motif fragment are able to bind and hydrolyze atp molecule. all peptides under study were chemically synthesized and their structures was investigated by nmr spectroscopy. the ability to bind atp molecules was analyzed by using hplc chromatography. results of our study show, that peptides with conserved p-loop motif have a suitable structures to promote binding of the molecules with phosphate group, but cannot accelerate pyrophosphate hydrolysis process. conference participation for w. _ z. supported by the fp project mobi health (grant agreement no ). computational resources were provided by the informatics center of the metropolitan academic network (ic man task) in gdansk, poland. ck is a ubiquitous serine/threonine protein kinase, being one of the most pleiotropic of all protein kinases . ck plays a key role in cell growth, differentiation, cell death and survival, and become the therapeutic target in cancer treatment, since its level is significantly increased in cancer cells . halogenated ligands have been widely developed as potent inhibitors of protein kinases. among them , , , -tetrabromobenzoteriazole (tbbt) is one of the first potent and selective inhibitor of ck a, directed towards the conserved atp binding site . to assess contribution of electrostatic interactions to the specificity and strength of binding of multi halogenated inhibitors by a protein kinase, we have studied interaction between ck a and nine benzotriazole derivatives, representing all possible patterns of halogenation on the benzene ring. herein, we present results that support existence of two alternative regions that are involved in ligand binding. aspartic acid is known for its function in coordination of a mg ion, which is required for atp binding . asp has been identified in crystal structure of ck :tbbt complex (pdb j , fig. ) as the charged residue closest to tbbt. there is also lys proximal to tbbt, interaction with which may favor anionic form of ligands (pk for tbbt < ), however it is involved in the intramolecular salt bridge, and thus its mutation may significantly change stability of the protein. crystal structure of tbbt complexed with ck (pdb: j ). residues with a distance to tbbt (magenta) shorter than a are shown. red residue is negatively charged, blue ones are protonated. abstract comparison of kdiss values determined for ligands at ph and at ph shows that strength of the complex significantly varies upon deprotonation of the triazole ring. this confirms former hypothesis that a negatively charged ligands cluster at the atp binding site region proximal to lys , which is beneficial both to the specificity and to strength of the binding. we have also observed for the tested ligands variations in their binding to either wild type protein and its d n mutant (with less negative charge distributed over atp binding site). all ligands displaying higher pka for dissociation of the triazole proton bind to the mutant visibly weaker than to the wild-type protein. altogether reveals the predominance electrostatic intermolecular interactions. although, negatively charged ligands most probably cluster at the atpbinding site proximal to lys , beneficial for the strength of binding, the less dissociated forms are favored due to unfavorable interactions of the anionic form of ligands with asp . there are many virulence factors produced by these strains, many of which are encoded on mobile genetic elements. psms are of specific interest because these virulence factors are encoded on the core genome of the bacteria and therefore all strains of staphylococci bacteria produce some variation of psms with a variety of biological functions. the specific mechanism by which psms act as virulence factors has been poorly understood until recently. biological functions of psms include cell lysis, biofilm formation and the ability to kill neutrophils after phagocystosis. these toxins are of special interest to our research group due to their genetic similarities to certain bacteriocins, namely leaderless bacteriocins. both groups of peptides are ribosomally synthesized with a n-terminal formyl methionine and secreted from the bacteria by atp-binding cassette (abc) transporters without any leader sequence or signal peptide. abc transporters may also play a role in immunity towards psms and leaderless bacteriocins. these similarities led our group to investigate the solution structure of these peptides through nuclear magnetic resonance (nmr). isolating psms from the producer organisim, s. aureus, typically involves lengthy extractions and low yields. for these reasons, we opted to chemically synthesize the desired peptides using solid phase peptide synthesis (spps). utilizing a variety of spps techniques, psm a and psm a were successfully synthesized, however, due to the hydrophobic nature of psm b , an alternate genetic approach was devised to isolate psm b . formation of a fusion protein between psm b and the small ubiquitin like modifier (sumo) protein allowed for heterologous expression. upon cleavage of the fusion protein with sumo protease, and subsequent purification and isolation of the cut peptide, psm b was obtained. as previously reported, the psms were found to be alpha-helical in structure inducing solvents. a series of dimensional ( d) nmr experiments were ran to determine chemical shift assignments and to obtain noe data. importing the chemical shift assignments and noe data into the structure calculating software, cyana, we were able to elucidate the solution structure of psm a and psm a and we are currently working towards the elucidation of psm b . the synthesis, isolation, characterization and solution structures of the aforementioned psms will be discussed here. transition metals are critical for enzyme function and protein folding, but their excess can mediate neurotoxic oxidative processes [ ] . as, energy production involves oxidative phosphorylation, a process requiring a continuous flow of electrons, mitochondria are particularly vulnerable to oxidative damage [ ] . as such, mitochondria are the major sites of reactive oxygen species (ros) generation, which are produced as byproducts of the electron transport chain. since free iron and certain ros can engage into potentially deleterious processes such as fenton reaction, mitochondrial iron homeostasis must be tightly controlled, and dysregulation of iron metabolism in this organelle has been associated with various diseases, including friedichs ataxia (fa), alzheimer's, and other neurodegenerative disorders [ ] . engineering an efficient mitochondriatargeting, cell-permeable vector is a challenge due to the fact that mitochondrion is impermeable to a wide range of molecules. the development of delivery vectors has been made possible by a greater understanding of mitochondrial structure and chemical features of molecules that selectively localize within this organelle. from these findings, two generalized requirements for mitochondrial localization are delocalized positive charge and lipophilicity [ , ] . targeting iron in this organelle is proposed as a means to ameliorate fa symptoms. desferrioxamine (dfo) is a bacterial siderophore with high affinity for iron, but low cell penetration. we prepared conjugates of dfo with mitochondria penetrating peptides and studied their iron-binding characteristics in vitro. the lipophilic and charged peptides tat - (h-arg-lys-lys-arg-arg-gln-arg-arg-arg-oh) [ ] , a (h-cha-arg-cha-lys-cha-arg-cha-lys-nh ) [ ] , ss- (h-dmt-arg-phe-lys-nh ) [ ] and ss- (h-phe-arg-phe-lys-nh ) [ ] , are known to permeate cytosolic and mitochondrial membranes. they were prepared and conjugated to dfo in solid-phase [ ] , an alternative synthetic route. once detached from the resin, fully deprotected, purified and characterized by means of lc/ms and aminoacid analysis, it was observed that the dfo-conjugated peptides displayed iron-binding abilities identical to the free chelator dfo. dfo-conjugated peptides were also able to quench the iron-catalysed oxidation of ascorbate (a model of oxidative stress in plasma of iron-overloaded patients), as probed by a high throughput fluorimetric method [ , ] . these results indicate that our synthesis and conjugation strategy were successful in preserving the iron-binding moiety and the antioxidant ability of the free chelator dfo. the proteolytic activity and oligomerization status of the human htra protease functioning as a tumor suppressor of an n-terminal domain not required for proteolytic activity, a central serine protease domain and a cterminal pdz domain. the latter serves as a substrate or regulator binding domain and may participate in oligomerization. htra s, its short natural isoform, lacks the pdz domain which is substituted by a stretch of c-terminal amino acid residues, unique for this isoform. down-regulation of htra in tumors, shown by other groups and us, suggests htra s involvement in oncogenesis [ ] . htra acts as a proapoptotic protein and is suggested to function as a tumor suppressor. it promotes cytotoxicity of etoposide and cisplatin in lung cancer cell lines [ , ] . to date, htra has been poorly characterized from the biochemical point of view, mainly due to the fact that it is difficult to purify recombinant htra . we were able to express in bacterial system and purify htra in quantities sufficient to perform structural studies. the aim of this study was to characterize and compare the proteolytic properties and quaternary structure of the htra isoforms. both studied isoforms lacked the n-terminal domain. htra with the pdz domain removed (htra -dpdz) and htra s (htra s) were fully active at a wide range of temperatures and their substrate affinity was not impaired. this indicates that the pdz domain is dispensable for htra activity. as determined by size exclusion chromatography, htra formed stable trimers while both htra -dpdz and htra s were monomeric. this suggests that the presence of the pdz domain, unlike in other human htras (htra and htra ), influences htra trimer formation. the unique c-terminal sequence of dn-htra s appeared to have little effect on activity and oligomerization [ ] . cyclodextrins (cds) are cyclic oligosaccharides that have been recognized as useful pharmaceutical excipients. in aqueous solution cds are capable to form complexes with various ligands, hosting inside their cavity either a whole molecule, or part of a ligand. inclusion complexes with cds offers a variety of physicochemical advantages over the biologically active ligands, including the improved aqueous solubility, solution stability or an increase of bioavailability. ck is an ubiquitous, highly pleiotropic and constitutively active ser/thr protein kinase. halogenated benzotriazoles have been developed as potent and selective inhibitors of this enzyme. the interaction of the catalytic domain of human protein kinase ck with a series of brominated ligands, which represent all possible patterns of halogen substitutions to the benzene ring of benzotriazole, was previously studied by microscale thermophoresis (mst) [ ] . this method alloweddetermination of binding affinities for seven ligands, all of which were found consistent with the values determined independently by isothermal titration calorimetry (itc). however, a very limited aqueous solubility of some brominated benzotriazoles may decrease their bioavability, thus affectingtheir apparent activity [ ] . to overcome this limitation, the aqueous solubility of halogenated benzotriazoles in the presence of cyclodextrins has been tested. the formation of inclusion complexes with b-cyclodextrin (b-cd), hydroxypropylb-cyclodextrin (hp-b-cd) and g-cyclodextrin (g-cd) in aqueous solutions, followed by uv-vis spectroscopy, substantially improved the solubility of tbbt and its derivatives. the interaction between protein kinase ck and cyclodextrins, and also with their inclusion complexes with halogenated benzotriazoles, was followed with the aid of the microscale thermophoresis. the results obtained clearly demonstrate that the binding of halogenated benzotriazoles by ck is only moderately affected by cyclodextrins. oligonucleotide-based molecular circuits offer the exciting possibility to introduce autonomous signal processing in biomedicine, synthetic biology, and molecular diagnostics. here we introduce bivalent peptide-dna conjugates as generic, noncovalent, and easily applicable molecular locks that allow the control of antibody activity using toeholdmediated strand displacement reactions. employing yeast as a cellular model system, reversible control of antibody targeting is demonstrated with low nm concentrations of peptide-dna locks and oligonucleotide displacer strands. introduction of two different toehold strands on the peptide-dna lock allowed signal integration of two different inputs, yielding logic orand and-gates. the range of molecular inputs could be further extended to protein-based triggers by using proteinbinding aptamers. insights of a novel kind of cell wall binding domain that cleaves the peptidoglycan muropeptide: the cw_ motif noem ı bustamante , , manuel iglesias, noella silva-mart ın, isabel uson, pedro garc ıa, juan hermoso, marta bruix, margarita men endez institute of physical-chemistry 'rocasolano', csic, institute of physical-chemistry 'rocasolano', csic, ciber of respiratory diseases (ciberes), center of biological research (cib), csic, enzybiotics constitute a hopeful alternative to current treatments to fight against bacterial infections. phage endolysins are consider as enzybiotics due to their capacity to cleave the peptidoglycan (pg) of gram-positive bacteria in a generally species-specific manner and kill bacteria when exogenously added ( , ) . the cpl- endolysin, a lysozyme encoded by the pneumococcal cp- bacteriophage, is a remarkable exception among all the pg hydrolases produced by streptococcus pneumoniae and its bacteriophages due to its capacity of degrading pneumococcal cell walls containing either choline or ethanolamine ( , ) . this fact confers to cpl- the advantage of displaying a broader microbicide spectrum comparing to choline binding proteins ( ) . this behavior results from the acquisition of a cell wall binding module (cwbm) made of three identical repeats of amino acids each (cw_ motifs), with unknown specificity and totally unrelated with the choline-binding motives present in pneumococcal hydrolases. interestingly, cw_ repeats have been identified in many putative proteins potentially involved in cell wall metabolism (pfam entry: pf ) from different species of gram positive and gram negative bacteria, and some bacteriophages ( ) . preliminary studies of thermal stability in presence of a small cell wall structural-analogue (glcnac-murnac-l-ala-d-isogln) point to the muropeptide as the cell wall target recognized by cw_ motifs ( ) . in this communication we have gone in depth in the characterization of cw_ repeats. we present the first crystal structure of the cw_ motif, which reveals a three-helical bundle folding. using std_nmr spectroscopy the epitope of binding of the disacharide dipeptide to this repeats has been identified. interestingly, the b anomer of the murnac moiety, the form present in the peptidoglycan, seems to be preferentially recognized with respect to the a anomer. finally, a docking model of the complex cw_ /gmdp compatible with std results was built allowing to identify the major contacts between the protein and the muropeptide and to propose the relevant role of a conserved arginine residue in this interaction. energy-dependent aaa proteases carry out regulated proteolysis to ensure protein quality control and post-translational regulation of many cellular processes. control of proteolysis occurs primarily at the level of substrate recognition, which can be modulated by adaptor proteins. the clps adaptor protein enhances and inhibits degradation of different classes of substrates, and thus triggers a specificity switch in clpa. whereas the mechanism for substrate delivery by clps has been described in detail, the inhibition mechanism is poorly understood. we show that clps inhibits ssra substrate recognition and processing, instead of simply preventing substrate binding. we demonstrate that clpa engagement of the clps n-terminal extension (nte) is necessary, and may even be sufficient, for inhibition. in addition, we find that inhibition of substrate processing requires a longer nte, as compared to inhibition of substrate recognition. interestingly, the nte length required for inhibiting substrate processing is also necessary for suppression of the clpa atpase rate. furthermore, preliminary data suggests that clps slows down substrate translocation. these results support a model where there is an ssra•clpa•clps inhibitory complex in which the clpa pore engages the clps nte. this engagement of the nte causes suppression of atpase activity, and therefore slower substrate translocation and processing. this model illustrates how an adaptor protein can inhibit recognition of one type of substrate while efficiently promoting degradation of a different substrate. single-molecule assay development for studying human rna polymerase ii promoter-proximal pausing rna polymerase ii (polii) pausing has been shown to play a significant role in transcription regulation of elongating polii complexes in a large number of metazoan and mammalian genes ( ) . the traditional understanding of transcription regulation in mammals involved controlling polii recruitment to promoters and controlling initial steps at the promoter, including pre-initiation complex formation and promoter escape. most works investigating promoter-proximal polii pausing have employed chromatin immunoprecipitation followed by sequencing to determine polii localization or in vitro transcriptional assays using nuclear extracts analyzed with radio-active gel electrophoresis. in order to gain greater mechanistic insight into the regulation of promoter-proximal polii pausing, we have been developing a diffusion-based single-molecule method using alternating laser excitation on the micro-second timescale (msalex). the method detects rna transcripts generated by a reconstituted human polii system in vitro using complementary doubly dye-labeled single-stranded dna (ssdna) probes. the human gene hspa b for heat shock protein (hsp ) is used as a model system due to its extensive characterization in drosophila. the method would provide a rapid, sensitive and robust avenue to screen for protein factors regulating promoter-proximal polii pausing. controlling of the pic composition using the reconstituted system allows for dissection of the functional roles of different pic components in facilitating regulation of polii pausing. we have demonstrated the hybridization of double dye-labeled ssdna probe to complementary ssdna mimicking rna transcripts and to transcripts generated with bacterial rna polymerase. also, a functional reconstituted human polii system has been verified using radioactive polyacrylamide gel electrophoresis of transcripts from in vitro transcription assays. malaria is a major global health problem. in , there were an estimated million case of malaria and deaths, most of them children under years old [ ] . among the malaria species that affect humans, plasmodium falciparum is the most deadly form. since no efficient vaccine is available yet, the fight against malaria includes vector control, protection from mosquito bites and artemisinin combined therapy. however, resistances to all known treatments have been observed. therefore, new antimalarial strategies involving novel targets and new mechanisms of action are needed. during its life cycle, in erythrocytic stage, which causes all the malaria symptoms, plasmodium falciparum relies on phospholipids to build the membranes necessary for daughter cell development. approximately % of parasite phospholipids consist of phosphatidylcholine (pc) and phosphatidylethanolamine (pe) synthesized by the parasite through the de novo kennedy pathways. in the pathway of phosphatidylcholine biosynthesis, the second step catalyzed by ctp:phosphocholine cytidylyltransferase [ec . . . ] is rate limiting and appears essential for the parasite survival at its blood stage [ ] [ ] . we are focused on the structural characterization of this enzyme, the identification of effectors by fragment-based drug design approach (fbdd) and then their optimization to eventually design a lead. the first reported crystal structure of the catalytic domain of the enzyme target (pfcct) has been solved at resolution . Å, enzyme-substrates complexes (cmp-, phosphocholine-and choline-bound forms) at resolutions . - Å and an enzyme-product (cdp-choline) complex structure at resolution . Å that give detailed images of binding pocket, demonstrate conformational changes between apo-and holo-protein forms and provide the information about the mechanism of the catalytic reaction at atomic level. the fbdd method uses a library of small molecules (fragments) with molecular weight that does not exceed da to explore target binding sites. although fragments often have too low affinities to evoke a biological response, their probability of binding is high because they are small enough to prevent unfavorable interactions with target protein-binding sites. moreover, they represent more attractive and synthetically tractable starting points for medicinal chemistry compared to more complex compounds. as the affinity is low, fragment screening usually depends on detecting binding rather than inhibition. screenings of a fragment library ( molecules) has been performed by fluorescence-based thermal shift assay and nuclear magnetic resonance saturation transfer difference (nmr std) [ ] . this combination of techniques identified so far fragment hits that are currently evaluated for their binding modes and affinities. co-crystallization of the protein-fragments complexes is carrying out to provide accurate information on the molecular interactions. topology of interactions will be used to rationally monitor every iterative round of the optimization process allowing subsequent rational design. [ ] world health organization, world malaria report (who press, geneva, switzerland), http://www. who.int/malaria/publications/world_malaria_report_ /wmr- -no-profiles.pdf?ua protein scaffolds play a crucial role in signaling pathways by generating signal specificity and increasing signal efficiency and amplitude. engineered protein scaffolds can be used as key regulators for signal transduction in artificial signal transduction cascades where they can regulate in-and output of the network. in this research a - - protein scaffold is developed which induces dimerization of proteins mediated by the small molecule stabilizer fusicoccin. as proof of principle caspase is used to constitute proximity induced dimerization. dimerization of caspase leads to its activation and consecutively initiates the caspase cascade involved in the programmed cell death pathway. caspase does not naturally bind to - - proteins, therefore the caspase monomer is conjugated to a - - binding motif which is known to bind into the binding grooves of a - - dimer. this interaction can be stabilized by the small molecule fusicoccin. we showed that upon addition of small molecule fusiccocin caspase dimerization is induced, resulting in caspase activity which is measured using a synthetic caspase substrate. moreover the biphasic effect of the - - scaffold could be proven. additionally, the activated caspase is also able to cleave its natural substrate caspase , downstream in the caspase cascade. these results indicate that the - - platform is a versatile small molecule induced dimerization platform which can be used as tool for engineering of a synthetic signaling network. the g e variant of the apoptosis inducing factor, responsible of a rare encephalopathy, is hampered in nad /h binding luca sorrentino , laura rigamonti , mirvan krasniqi , alessandra calogero , vittorio pandini , maria antonietta vanoni , alessandro aliverti the apoptosis inducing factor (aif) is a highly conserved mitochondrial flavoprotein known to play two opposite roles in eukaryotic cells: in mitochondria it is required for efficient oxidative phosphorylation (oxphos), while, when released into the cytoplasm, it triggers caspase-independent apoptosis ( ) . the mechanism of aif-induced apoptosis was extensively investigated, whereas its mitochondrial role is poorly understood. there are many evidences of aif importance for mitochondrial correct morphology and functions and recently the discovery of its direct interaction with chchd , a key regulator of respiratory complexes subunits import and folding in mitochondria, was reported ( ) . a unique feature of aif, probably pivotal for its vital function, is the ability to form a tight, air-stable charge-transfer (ct) complex with nad and undergo dimerization. although some aspects of aif interaction with nad / h have been analyzed, its precise mechanism is not fully understood. we investigated the effect of the pathogenic g e replacement, associated with oxphos defect and neurodegeneration ( ) , to understand how it could alter aif properties at the molecular level. to do so, we analysed how the wild type and the g e forms of murine aif interact with nad /h and nicotinamide mononucleotide (nmn / h), finding that the pathogenic replacement resulted in a dramatic and specific decrease of the rate for ct complex formation and consequent protein dimerization only in the case of the physiological ligand. our results demonstrate that the adenylate moiety of nad /h is crucial for the ligand binding process and that the g e replacement causes an alteration of the adenylate-binding site of aif that drastically decreases the affinity for and the association rate of the ligand. in addition, we shed new light on the mechanism of the dimerization process, demonstrating that fad reduction rather than nad /h binding initiates the conformational rearrangement of aif that leads to quaternary structure transitions. taken together, our results contribute to define how aif works at the molecular level in binding nad /h and undergoing dimerization and also point out that the g e replacement, responsible of a rare neurodegenerative disease, has the selective effect of slowing down the formation of aif dimeric ct complex. dipartimento di bioscienze, universit a degli studi di milano, dipartimento di scienze veterinarie e sanit a pubblica, universit a degli studi di mical, from the molecule interacting with casl, indicates a family of conserved cytoplasmic multidomain proteins that catalyze a nadph-dependent f-actin depolymerization activity through their essential n-terminal fad-containing monooxygenase-like domain (mo) in response to semaphorin signaling [ ] . this domain is followed by calponin homology (ch) and lim domains, proline-and glutamate-rich regions and a c-terminal coiled-coil motif that mediate the interaction with various proteins (e.g: crmp, casl, plexin, g proteins, ndr) [ ] . to contribute to establish the catalytic properties of mical mo and their modulation by the additional domains and by the interacting proteins, we have produced and are characterizing the human mical (mical-fl) and forms containing the mo [ ] , mo-ch and mo-ch-lim domains. all mical forms contain stoichiometric amounts of fad in the mo domain and zn ions in the lim domain. mical-mo catalyzes a nadph oxidase (h o -producing) activity. the ch, lim and c-terminal domains lower its catalytic efficiency (kcat/km, nadph) mainly due to an increase of km for nadph. the kcat is similar for all forms excepted for mical-fl where a -fold drop is observed, in agreement with the proposed autoinhibitory function of the c-terminal domain [ ] . the ph dependence of the kinetic parameters of mo, moch and mochlim is complex suggesting that it does not reflect the ionization state of individual groups, but rather the overall protein charge. mical-mo, -moch and -mochlim catalyze a nadph-dependent f-actin depolymerization with a similar apparent km for actin. f-actin (but not g-actin) stimulates the rate of nadph oxidation by increasing kcat and lowering knadph. the extent of nadph oxidation exceeds total f-actin which is in contrast with the proposal of specific modification of actin met or met reported for drosophila and mouse moch [ ] [ ] , but it suggests that f-actin stimulates the nadph oxidase activity or a case of substrate recycling. accordingly, with hmical mo and moch several actin residues are oxidized beside met and met . thus, the ch and lim domains do not seem to be important for the mical-actin interaction and actin modification may be mediated by in situ h o production. in hek t and cos- cells mouse collapsin response mediator protein- (mcrmp ) interacts with mical inhibiting h o production [ ] , suggesting that crmp could be a hydroxylatable substrate of mical-mo. we have produced the same mcrmp form ( - aa) and we have shown that under conditions that limit non specific interactions a mild stimulation (up to %) of nadph oxidation is observed. f-actin reversed the effect of mcrmp suggesting their competition for mical. these results suggest that crmp , a major microtubules regulator, is not the substrate of the mo domain, but actin and microtubules cytoskeleton components may be linked through the formation of crmp-mical complex in response to semaphorin-plexin signaling. experiments are in progress to complete the characterization of mochlim and full length mical forms. green fluorescent protein (gfp), owing to its genetically encoded strong fluorescence, has become one of the most important tools in modern biology [ ] . enhanced gfp (egfp, f l/s t-gfp), frequently used variants of this protein, is thermodynamically more stable and -times brighter than gfp [ ] . due to the improved fluorescent properties, egfp is commonly used as a fluorescent intracellular marker in bio-imaging in vitro and in vivo. despite sustained interest of the scientific community and numerous practical applications, the actual biological role of gfp remains elusive. recent reports put forward a hypothesis of antioxidant and photo-protective functions of gfp [ ] . in this study, we focused on the photo-protective role of egfp against reactive oxygen species (ros) photo-generated by visible light in water suspensions of nano-particular nitrogen-doped titanium oxide (n-doped nano-tio ), that is in the system: 'n-doped nano-tio )/visible light'. n-doped nano-tio (sumitomo tp-s ) was chosen as a photo-catalyst, since it is widely accepted that nitrogen doping enhances visible light photoactivity of tio . -hydroxy- , , , -tetramethylpiperidine-n-oxyl (tempol), a paramagnetic water-soluble compound, belonging to the nitroxide class o superoxide dismutase (sod) mimetics, was used as a target for photo-generated ros. a solar simulator, with the flux output intensity of kw/m , was used as a visible light source. electron spin resonance (esr) was employed to monitor the changes in the paramagnetic signal of tempol exposed to the action of ros in the absence and presence of egfp. in the absence of egfp and after min of illumination, due to a combined action of superoxide (o •-) and hydroxyl (oh•) radicals generated by the system 'n-doped nano-tio )/visible light, the esr signal of um tempol decayed by %. moreover, the growth of a new signal, interpreted as -oxo- , , , -tetramethyl- -piperidinyloxy (tempone), resulting from the attack of oh•radicals on tempol, was also observed. in contrast, in the presence of egfp ( . um) , the ros-induced decay of the esr signal of tempol was markedly smaller, not exceeding %. concomitantly, the growth of the esr signal of tempone was also partially inhibited ( % smaller amplitude), as compared to the process performed in the absence of egfp. in summary, our results point to a significant inhibition of the photodecomposition of tempol in the presence of egfp and support the hypothesis of the protective role of this fluorescent protein against ros generated by the system 'n-doped nano-tio )/visible light'. school of chemistry, national university of ireland galway, school of biochemistry and immunology, trinity college dublin by studying a variety of anionic ligands and their interactions with cationic cytochrome c, we are building knowledge of protein recognition geared towards regulating activity. in previous work it was shown that psulfonatocalix [ ] arene selectively binds to, and encapsulates, three lysine side chains on cytochrome c . here, the binding of two small molecule ligands to cytochrome c was investigated. nmr spectroscopy was used and in one case, a crystal structure of the complex was obtained (fig ) . the calixarene bound to cytochrome c, reveals a crystal packing assembly that suggests it is a key mediator of crystal formation. nmr data analysis indicates the calixarene's binding site on cytochrome c. the pillararene, a relatively new class of compound, is a symmetrical arrangement with a p-rich cavity , related structurally to calixarenes. this suggests good host-guest complexation properties. previously, the carboxylatopillararene showed selective binding to arginine, lysine and histidine . with this ligand, an interaction with cytochrome c was observed and a complex formed. additionally, biphasic binding behaviour was observed through analysis of the chemical shift perturbations. this may indicate more than one binding event taking place. the data from these studies indicate that recognition is occurring and again that lysine side chains play an essential role. the enzyme dihydrofolate reductase (dhfr) is necessary for the growth and development of all organisms. the structure and function of escherichia coli dhfr have been characterized in buffer. however, dhfr exists in living cells, where the protein concentration can exceed g/l. we know that weak, non-specific chemical interactions with cytosolic proteins alter protein conformation and dynamics, , both of which are expected to influence dhfr catalysis. investigators have examined steady-state enzyme kinetics under crowded conditions, but conclusions can be conflicting. , here, the effects of crowding on e. coli dhfr catalysis are assessed through specific activity measurements in solutions of synthetic polymers. these kinetics studies are complemented by in-cell and in vitro f nmr data from fluorinated tryptophan residues. preliminary results suggest that the effects of polymeric crowders on dhfr activity are non-monotonic, which may arise from the polymer's transition from the dilute to semi-dilute regime. the data suggest that synthetic polymers are not a valid representation of the cellular interior. biotechnology department, university of verona calcium (ca ) is one of the most important second messengers in eukaryotes. ca binding proteins can be subdivided into two categories: "ca buffers" that modulate ca ion concentrations in cells, and "ca sensors" that decode ca signals in a wide array of physiological processes in response to external stimuli. calmodulin (cam) is the prototypical example of ca sensor proteins in both animals and plants. in addition to conserved cam, plants possess a unique family of cam-like proteins (cmls). many of these cmls still remain uncharacterized and the investigation of their biochemical and biophysical properties will provide insight into ca signalling in plants. herein, a detailed characterization of arabidopsis thaliana cml is reported. cml is a protein of amino acids with a theoretical molecular weight of , da and % amino acid sequence identity with atcam . cml is predicted to have one functional ca binding site despite the presence of three ef-hand motifs (prosite). we overexpressed cml in e. coli and analyzed its biochemical and biophysical characteristics, i.e. calcium affinity and stoichiometry and eventual changes in conformation, thermal stability and proteolytic susceptibility upon ca binding. isothermal titration calorimetry (itc) and nuclear magnetic resonance (nmr) spectroscopy identified one ca binding site in cml and showed that ca and mg compete for the same binding site. the kd values determined by itc established that cml has higher affinity for ca than for mg . our data were consistent with the sequence based prediction of one functional calcium binding site. differential scanning calorimetry (dsc) showed that ca and mg have the same stabilizing effects on protein folding. apo-cml undergoes two thermal unfolding transitions, but in the presence of ca or mg only one unfolding event at an intermediate temperature occurs. limited proteolysis experiments showed that ca binding affords protection against cml digestion by trypsin. surprisingly, cml exhibits very few conformational changes upon calcium binding, which were evaluated by ans fluorescence and stokes radius measurements in the apo-and ca bound-forms. these results suggest that cml does not show the characteristics of a classical ca sensor protein. to better understand the physiological role of cml in plants, in vivo analysis will be performed. pb- fbp controls the hepatocyte morphology through rho signaling jun zhang , mingming ling , qianying zhang , yunhong wang , deqiang wang the department of cell biology and genetics, the formin-binding protein (fbp ) widely expressed in eukaryotic cells was previously identified to play a role in morphological maintenance in hepatocyte, but the molecular mechanism keeps still unclear so far. in the present investigation, it was found that rho family proteins cdc /rac signaling was involved in the morphological regulation controlled by fbp . knockdown of endogenous fbp expression with rnai technique or dominant negative mutant of fbp could trigger the cell morphological remodeling from the epithelioid to fibroid following the significant down-regulation of cdc / rac activities and dephosphorylation of paxillin. while the rho protein specific activator could restore the cdc /rac activities, and in turn abrogated the silence effect. overexpression of wild type fbp could not result in any of the morphological transition. furthermore, withdrawal of the silence could induce morphological recovery when the fbp expression, cdc /rac activities and paxillin phosphorylation were restored to the normal level. the experimental evidences strongly indicated that fbp was implicated in morphological control probably via rho signaling pathway in hepatocyte. key words: fbp ; rho signaling; paxillin; morphological control; hepatocyte this work was supported by a grant from national natural science foundation of china (nsfc, no. cytochrome c oxidase (cco) is the final enzyme in the respiratory chain of mitochondria but also an integral part of the metabolism of many types of bacteria. in a complex, stepwise redox-reaction, cco catalyzes the reduction of molecular oxygen to water and utilizes the resulting free energy to pump protons across the membrane thereby creating an electrochemical gradient [ , ] . to investigate proton pumping spectroscopically it is possible to label the entrance of the proton entrance channel with fluorescein, a ph sensitive dye, which allows determining time resolved local changes in proton concentration at the cytoplasmic cco surface and related properties. it has already been shown that the redox state of copper and heme centers affects such properties at the cytoplasmic surface. [ ] this study is a theoretical approach to investigate changes of pka values of the fluorescein label at the entrance of the k-channel for different protonation pattern in both oxidized and reduced cco by performing molecular dynamics (md) simulations. further work is based on calculations of pka values of the fluorescein using software karlsberg [ , ] . methods for genetically and synthetically manipulating protein structure enable a greater flexibility in the study of protein function. we have shown that using inteins as traceless, cleavable purification tags enables the separation of full length unnatural amino acid (uaa) containing proteins from their corresponding truncation products. this method has been used to incorporate uaas in previously unattainable positions in a variety of proteins using a myriad of uaas, inteins, and purification tags. in other applications, we have used e. coli aminoacyl transferase (aat) to selectively modify the n-termini of proteins with uaas in denaturing conditions and conditions that maintain folding. applications of particular interest include overcoming the need for an n-terminal cys residue in expressed protein ligation, transfer of reactive handles for "click" chemistry labeling of proteins, and transfer of fluorogenic molecules for photophysical experiments. we have found that aat can transfer protected cysteine, homocysteine, and selenocysteine to expressed proteins. after ligation, these residues can be converted to met or ala, making the ligation traceless. we continue to develop variants of aat to broaden the substrate scope of both its transferred substrate and n-terminal recognition element. in addition, expressed protein ligation is being used to incorporate backbone modifications, such as the thioamide, into various positions in the protein calmodulin to determine how these modifications can impact the structure and function of an ordered protein. in general, by working at the interface of several protein modification technologies, we have made beneficial discoveries that might be missed by more focused approaches. function and modularity of cw_ motives in the c-terminal region of the endolysin cpl- encoded by the cp pneumococcal bacteriophage manuel iglesias-bexiga , , noelia bernardo-garc ıa , rub en mart ınez-buey , noem ı bustamante , , guadalupe garc ıa , , marta bruix , juan hermoso , margarita men endez , dept. of biological physical-chemistry, iqfr-csic, ciber of respiratory diseases (ciberes), department of crystallography and structural biology, iqfr-csic, bacteriophage lytic murein-hydrolases have been proposed as enzybiotics, an efficient way to fight bacterial infections. however, the use of these enzymes is normally restricted to gram-positive bacteria since the outer membrane of the gram-negative bacteria hampers the access of the hydrolases to the peptidoglycan substrates. all the murein hydrolases reported in the pneumococcal system, both from host or phage origin, depend on the aminoalcohol choline to be fully active. there is only a unique exception to this rule, the cpl- lysozyme. this hydrolase is encoded by the lytic pneumococcal phage cp- and, instead of the common cell wall binding module (cwbm) that recognizes choline, cpl- harbors a completely different cell wall binding structure. recent studies have revealed that reducing the net charge of the cwbm, from . to . , leads to an improvement in the antibacterial activity of cpl- ( ) . the cwbm of cpl- is composed by three identical repeats of amino acids, the cw_ motives, and it folds both in the presence and in the absence of the n-terminal catalytic module ( ) . this module shows the capacity of recognize the glcnac-murnac-l-ala-d-isogln muropeptide (gmdp), structurally related with the peptidoglycan basic unit ( ) . here, we report the high resolution structure of the cell wall binding module of the cpl- endolysin. each cw_ repeat is composed of a bundle of three a-helices with a highly negative electrostatic charge at the surface. the strong inter-repeat interactions and the high ionic strength used in the crystallization conditions allow them overcoming the electrostatic repulsions inducing a closed-packed structure with a three-fold symmetry. the module dimensions ( x x Å) and the repeat arrangement in the crystal structure are inconsistent with the gmdp binding characterization, the activity displayed by cpl- truncated variants with one or two cw_ repeats, or the experimental determined hydrodynamic properties. using the small angle x-ray scattering (saxs) technique and the atsas computational platform ( ), a different arrangement of the cw_ repeats is envisaged in solution (fig. ) , whose rather opened structure ( x x Å) is consistent with the experimental data. additionally, employing the saxs-based structure and the honeycomb structure proposed for the peptidoglycan, a model, where each cw_ repeat of the cell wall binding module fit in adjacent glycan chains, has been derived. in , the protein structure initiative (psi) was started as to determine three-dimensional structures of proteins within every family. once solved, structures are deposited into the protein data bank (pdb) and termed structural genomics (sg) proteins. as of june , there are over , sg proteins deposited in the pdb and most of them are of unknown or uncertain biochemical function. in addition, many of these sg proteins have a putative functional assignment based on their sequence and structural similarities with proteins of known function; such comparisons can be made against large databases using programs such as blast or dali. however, these putative functional assignments are often incorrect. this project analyzes members of the crotonase superfamily (cs). the cs consists of five diverse functional subgroups that are well characterized structurally and functionally, representing different types of reactivity, including hydrolase, isomerase, hydratase, and dehalogenase activities. this superfamily also contains at least sg proteins, so it is ideal to test predictions of protein function. our approach is based on local structure matching at the computationally predicted active site. first, partial order optimum likelihood (pool) is used to predict the functionally important residues of each sg protein and of the proteins of known function in the superfamily. next, structurally aligned local sites of activity (salsa) is used to align the predicted catalytic residues of the well-characterized members in the superfamily. from this analysis we generate chemical signatures for each functional subgroup and compare them to the sets of catalytic residues predicted for the sg proteins. we demonstrate based on these computational methods that the majority of the putative annotations in the cs superfamily are likely incorrect. currently, biochemical assays are being used to test these predictions. preliminary biochemical results show that one sg protein, thermus thermophilus q sls _thet , classified as a probable enoyl-coa hydratase, possesses hydrolase activity as predicted by our methods. the outcomes of this project will be to successfully classify the biochemical functions of sg proteins based on their local structure at the predicted active sites and to provide a conceptual framework for the functional classification of the remaining sg proteins within the pdb. this work is supported by nsf-che- . directly observing the synergistic dynamics in f-actin and microtubule assembly jun zhang , deqiang wang the department of cell biology and genetics, key laboratory of molecular biology on infectious disease although important in cellular activities, little attention was paid to the synergistic effects of actin and microtubule cytoskeleton assembly. with the time-lapse atomic force microscope (tl-afm), we directly observed the large-scale dynamic structure of actin filaments formed in the presence or absence of microtubulin in solution. in absence of microtubulin, the g-actin could be polymerized into ordered filamentous structures with different diameter from the slimmest filament of single f-actin to giant filament in tree-like branched aggregates. the polymerized actin filaments, to which our most intense attention was attracted, was discretely arranged and showed obvious polymorphism in structures completely distinct from those in the presence of microtubulin. the supra-molecular complex structures of the latter were mainly composed of single f-actin and/or multifilaments clearly consisting of several single f-actin and regularly cross-linked with the assembled microtubular bundles. the experimental results demonstrated that the f-actin dynamics could be coordinated by microtubule assembly. further analyses implied that the interactions between f-actin and microtubule could prevent the emergence of structural polymorphism of f-actin alone, and give rise to organization of specific complex structures instead. it was suggested that dynamic synergy between the f-actin and microtubule would be implicated in living cells. the adaptor protein - - is found in a diverse range of pathologically relevant protein-protein interactions (ppis). as - - is a hub protein with very diverse interactions, it is able to influence the intracellular localization of their binding partners and they are key regulators of signal transduction processes as well as regulators of cell cycle functions.nevertheless, there are only few examples of - - acting extracellularly. one of the extracellular targets for - - is aminopeptidase n (apn). apn is an extracellular trans-membrane enzyme that acts as a receptor for - - . binding to apn, - - excreted by keratinocytes can upregulate the excretion of matrix metalloproteinase- (mmp ) in fibroblasts. mmp , by breaking down collagens, is key in the remodeling of the extracellular matrix. modulation of the - - /apn interaction thereby may play a crucial role in the fundamental understanding and ultimately treatment of wound healing, respiratory diseases and tumor growth. in the eukaryotic cell, the - - dimer operates as an adapter platform for binding partners. a wide range of classes of (small) molecules, natural products and peptides has been used to modulate the ppis, providing either stabilization or inhibition of the interactions of - - with its binding partner. binding partner fragments or peptides are known to bind to the - - binding groove via arecognition motif containing a phosphorylated serine or threonine. making use of the dimeric structure of - - , novel small-molecule inhibitors may be tethered to exploit the bivalent effect. from a large virtual screening and experimental validation, a scaffold containing a phenyl phosphonic moiety was identified, showing inhibitory properties for - - ppis. potent derivatives of this scaffold were bridged by polyethylene glycol (peg) linkers of varying lengths, thereby facilitating the compound to reach both binding sites of the - - dimer and concurrently increasing the compound's solubility in aqueous solution. similar bivalent inhibitors have been proven to synergistically increase their efficacy. biophysical evaluation by means of fluorescence polarization (fp) inhibition competition assays, revealed an increase of the half maximal inhibitory concentration (ic ) from approximately lm for the monomeric phenyl phosphonate to approximately . lm for the bivalent inhibitor with a Å linker. this demonstrates a -fold increase of inhibitory effect towards - - and its binding partner peptide mimic. extensive thermodynamic, kinetic and structural analysis of the interaction is in progress.phosphonic moieties have been shown to pass the cell membrane poorly, due to their highly charged character. by being able to specifically inhibit the extracellular interaction between - - and apn, these inhibitors are prevented from interfering with the extensive intracellular - - interactome. hence, these bivalent phenyl phosphonate inhibitors provide a promising strategy towards extracellular application. the mre complex is an oligomeric assembly comprising of dimmers of mre and rad proteins in archea and additionally nbs subunit present in eukaryote. it is the central player in the dna damage response -a functional network comprising dna damage sensing, signal transduction, cell cycle regulation and dna double strand breaks (dsbs) repair [ ] . recent structural studies revealed that rad hinge domain is rather a short kink in the coiled-coil region and adopts unusual dimerization mode by intermolecular coordination of zn(ii) and formation of so-called zinc hook domain [ ] . to date, very limited structural data on the zinc hook domain have been reported, the only known structure was resolved for rad homologue from hyperthermophilic archaeon -p. furiosus. unusual zn(ii) coordination mode in zinc hook domain raises question of how zinc hook domain assembles to form interprotein zinc binding site with sufficient stability to function at low intracellular free zn(ii) concentrations [ ] . our study on minimal zinc hook domain fragment ( aa) indicated low femtomolar affinity towards zn(ii) [ ] . extended zinc hook domain fragment ( aa) reveals even zeptomolar affinity. therefore, our main goal was to probe the thermodynamic and structural effects that are hidden in the small interprotein interface and are responsible for the dimerization of the large and critical protein machinery. probing of those effects was achieved by detailed biophysical characterizations (including potentiometry, nmr, hdx ms and cd spectroscopy) of protein fragments of zinc hook domains with a number of point mutations. we showed that extremely high stability of zinc hook domain from p. furiosus is achieved by the formation of hydrogen bond network in b-hairpins and interprotein hydrophobic core. eindhoven university of technology dna-based molecular circuits have become a very attractive tool in molecular imaging, synthetic biology, molecular diagnostics and biomolecular computing. the highly modular and predictable nature of watson-crick base pairing allows the construction of complex circuits using a limited set of logic gates and building blocks. however, the lack of generic approaches to interface dna-based molecular circuits with protein activity limits their application in biomedicine and molecular diagnostics. here we present a new, highly modular approach to control the activity of a reporter enzyme based on the dna-directed assembly and disassembly of a complex between tem -b-lactamase and its inhibitor protein blip. both proteins are conjugated to a unique oligonucleotide, allowing the assembly of the enzyme-inhibitor pair and inhibition of enzyme activity by the addition of a complementary template strand. addition of an oligonucleotide that is complementary to a loop sequence in the template results in the formation of a rigid dsdna spacer that disrupts the enzyme-inhibitor complex, restoring enzyme activity. using this noncovalent approach allowed easy tuning of the template and target sequences with only a single set of oligonucleotide-functionalized enzyme and inhibitor. to show the modularity of the system, a panel of different template sequences were selected. only in the presence of their complementary viral dna sequences restoration of enzyme activity was observed. in addition to this excellent specificity the system showed to by higly sensitive towards its target, since the presence of as little as fmol of target resulted in an observable increase in enzyme activity. the use of a stable and well-characterized enzyme-inhibitor pair, complemented by the modular design of our reversible dna-directed protein switch make it an attractive system to implement in dna-based molecular circuits. several studies demonstrated important roles of human carbonic anhydrases (hcas) in a variety of physiological and pathological processes. consequently, in recent years the catalytically active hca isoforms have become an interesting target for the design of inhibitors with biomedical applications [ ] . derivatized sulfonamides of type r-so nh represent the class of ca inhibitors (cais) mostly used and best characterized. the large number of crystallographic studies so far available on these molecules clarified the main factors responsible for the binding of the sulfonamide moiety to the ca active site. in particular, it has been highlighted that even though these molecules generally behave as very potent cais, they do not show selectivity for the different isoforms. indeed, the sulfonamide moiety plays a predominant role in the interaction with the enzyme, while any change in the nature of the r substituent has generally a rather marginal effect on the enzyme-inhibitor affinity. these characteristics make difficult the design of sulfonamide derivatives selective for the different ca isoforms. consequently, much efforts were dedicated in last years to the development of new inhibitors that, although presenting lower affinity for the ca active site, would be able to be more selective toward the different isoforms. carboxylic acids have been recently investigated as cais, showing that these molecules can adopt different binding modes to the enzyme active site. in particular, they can coordinate directly to the zinc ion or be anchored to the zinc-bound water molecule. however, the structural reasons responsible of this peculiar behavior have not been clarified yet. in a general research project aimed at providing insights into the binding mode of these molecules to cas, we have undertaken the characterization of two carboxylic acids, namely an ortho-substituted benzoic acid [ ] and a saccharine derivative, by means of kinetic, crystallographic and theoretical studies. exploring the mechanism of fibril formation using fluorescently labelled human lysozyme variants ana bernardo gancedo 'exploring the mechanism of fibril formation using fluorescently labelled human lysozyme variants' human lysozyme is a widely characterised protein whose mutational variants misfold into fibrils that are associated with systemic amyloidosis ( ) . although the process of aggregation for human lysozyme has been well studied, the details of early events within this process are not fully characterised. single molecule fluorescence microscopy has been used to determine the oligomeric distributions present in the aggregation process of a number of disease-related intrinsic disordered proteins (idps) ( ) . recent advances in site-specific labelling of human lysozyme ( ) have made this protein amenable to these single molecule fluorescence studies. we have introduced alexa-fluorophores into the i t variant of human lysozyme and have demonstrated that the process of in vitro fibril formation is not significantly altered. using these fluorophore-labelled proteins we can apply single molecule fluorescence to study the early aggregation events within this system, allowing us to compare protein aggregation in a globular protein and with the aggregation process of idp's. abstract protein structure, folding and function, while specific interactions with lipid molecules can also contribute towards the biological activity of some membrane proteins. improving understanding of the interactions has resulted in the development of artificial lipid systems that allow the bilayer properties to be rationally manipulated in vitro to control protein behaviour. the bacterial transporter lacy is a well known integral membrane protein from the major facilitor superfamily, responsible for the protondriven uptake of d-lactose in e. coli. with a high resolution structure available and considerable understanding of mechanistic detail, and with observed changes to both structure and function in different bilayer environments, lacy is a good model system for examining the behaviour of a major class of membrane proteins in these lipid systems. purified lacy has been reconstituted into liposomes and droplet interface bilayer systems of varying lipid composition and the effect on protein function and bilayer properties examined. targeting abeta oligomers by trehalose-conjugated peptides: a molecular dynamics study alzheimer's disease (ad) is currently one of the most common and devastating forms of dementia correlated with beta-amyloid peptide (abeta) accumulation in human brain tissue [ , ] . inhibiting abeta selfoligomerization in brain tissue remains one of the main strategies to prevent or treat this disorder. as a consequence, in recent years much efforts have been spent in the understanding of the amyloid fibril growth process and its modulation by putative drug molecules. an interesting class of compounds able to prevent abeta fibrillogenesis, is represented by beta-sheet-breaker (bsb) peptides [ ] . although these molecules are thought to recognize in a self-complementary manner the abeta hydrophobic core region, however their precise mechanism of interaction is still unclear. in this context, we have studied the structural basis underlying the inhibitory effect of abeta( - ) fibrillogenesis explicated by two promising trehaloseconjugated bsb peptides (ac-lpffd-th (thct) and th-succinyl-lpffd-nh (thnt)) [ ] using an all-atom molecular dynamics (md) approach [ , ] . the pentameric nmr structure [ ] of abeta has been used to model amyloid protofibril, and the two protofibril ends have been investigated as putative binding sites. our simulations suggest that the interaction with the two protofibril ends occurs through different binding modes. in particular, binding on the odd edge (chain a) is guided by a well defined hydrophobic cleft, which is common to both ligands (thct and thnt). moreover, targeting chain a entails a significant structure destabilization leading to a partial loss of b structure and is an energetically favoured process, as assessed by mm/pbsa calculations. a significant contribution of the trehalose moiety to complexes stabilities emerged from our results. the basic structural unit of chromatin is the nucleosome, which is composed of histone proteins forming a scaffold with about base pairs of dna wrapped around. chromatin compacts eukaryotic genomes and regulates gene activity, which is mediated in part by posttranslational modifications (ptms) on the n-terminal tails of the histones. uncovering the detailed relationship between histone tail modifications and gene activity is a major topic of biomedical sciences and general techniques for generating nucleosomes with defined modification patterns in large numbers would greatly facilitate such investigations. to this end we are establishing a chemical toolbox for designer chromatin with defined histone ptm patterns. a protein semysinthesis approach is used that bases on "ligation-ready nucleosomes" with truncated histone h that can be ligated with the corresponding synthetic histone tail. we resorted to sortase-mediated ligation as chemoselective ligation method. here we report our recent developments in establishing the envisioned chemical toolbox for designer chromatin. evaluating cation-pi and pi-pi interaction in proteins using various biophysical methods in proteins the aromatic residues phenylalanine (phe), tyrosine (tyr), and tryptophan (trp) can be involved in aromatic interactions known as cation-pi and pi-pi interactions (dougherty ). compared to other non covalent interactions in proteins, like h-bonds, dipole-dipole, or van der waals interactions, relatively little is known about the pi-pi and the cation-pi interactions. the strength of both aromatic interactions is dependent on the pi-electron density in the aromatic residues. a lowering of electron density can be created by introducing strong electron-withdrawing substituents like fluorine atoms in the aromatic ring (dougherty ). in this way a nearly isosteric change in the aromatic system results in a marked change in electron density. substitution with methyl groups is known to slightly increase the electron density. the response to low cellular oxygen levels in humans and other animals is induced by the hypoxia inducible transcription factors (hifs). these transcription factors are regulated by hypoxia inducible factor prolyl hydroxylases (phds), which act as 'oxygen sensors' by hydroxylating hifs, thus leading to the proteomic degradation of the transcription factors. over the last years, there have been multiple reports that describe additional phd substrates other than hifs. among them are the large subunit of rna pol ii, several transcription factors, and components of signalling pathways. validating these reports is of major medicinal relevance given that phd inhibitors are now in the late stage phase clinical trials. in order to investigate the selectivity of phds, the reported proteins have been tested as substrates for hydroxylation by mass spectrometry, and as binders or competitors of the phds. initial work on peptides that contain the putative hydroxylation sites has indicated that the phds are much more selective for their well-established substrate hif. however, in ongoing work these initial results are going to be validated on protein level by co-expressing phds with the reported substrates. additionally, peptides of reported substrates were screened for their ability to alter the kinetics of hif-hydroxylation by phd . an inhibitory effect of at least two different peptides on phd was observed, suggesting that there is an interaction between the prolyl hydroxylase and these peptides. in order to investigate the mode of binding and inhibition, nmr studies have been carried out and binding of the two inhibitory peptides on phd has been shown. altogether, these results indicate that, although phds might be more selective for hif as a substrate as it was initially thought, the enzymatic activity of the prolyl hydroxylases is possibly influenced by a number of other proteins that can directly bind to phds. non-natural aminoacids via the mio-enzyme toolkit alina filip , judith h bartha-v ari , gergely b an oczy , l aszl o poppe , csaba paizs , florin-dan irimie biocatalysis and biotransformation research group, department of chemistry, ubb, department of organic chemistry and technology an attractive enzymatic route to enantiomerically pure to the highly valuable a-or b-aromatic amino acids involves the use of aromatic ammonia lyases (als) and aminomutases (ams). all these enzymes have in common an auto-catalically formed -methylene- , -dihydroimidazole- -one (mio) electrophilic prosthetic group, and show high structural and sequence similarities. the recent advances in improving the functional properties of these enzymes increased both their biocatalytic and therapeutic applications. we aimed to create a library of recombinant mio-enzymes consisting of the pals and pams with large substrate promiscuity in order to provide access to various non-natural aminoacids through enzymatic ammonia addition and/or ammonia elimination reactions of the substrate library already available in our researchgroup. the developed complementary substrate and enzyme library would provide the mio-enzyme toolkit useful for the synthesis of nonnatural aminoacids. the synthetic gene of the enzymes (pcpal, rtpal, avpal, papam) were cloned into pet b_j expression vector using xhoi and bpu i cloning sites. the plasmid dna was transformed to several e.coli host strains (rosetta, bl , origami ) in order to optimize the expression yields. the enzymes containing an n-terminal his -tag were purified with affinity chromatography, followed by ion-exchange or/and size-exclusion chromatography, obtaining pure and homogenous proteins, in their tetrameric, presumably native fold. the enzyme activity and the kinetic parameters of the purified enzymes was determined towards the natural substrate l-phenylalanine, as well as towards novel bulkier aromatic substrates (heteroaryl alanines, styryl alanines, biphenylalanines). furthermore to enhance their biocatalytic applicability we covalently immobilized the enzymes to carboxylated single-walled carbon nanotubes (swcnt cooh) using linkers with different lengths, and tested the activity and recycling of the immobilized enzyme. antibodies that bind protein antigens are indispensable tools in biochemical research and modern medicine. utilizing a phage display selection strategy, we have obtained synthetic antigen binders (sabs), based on a fab fragment of igg, to a wide array of proteins as distinct as membrane proteins, structural proteins, scaffold proteins and nuclear targets. here we demonstrate the applicability of the sabs towards the native, full-length proteins in cells. we show that the generated sabs are able to pull-down endogenous proteins from mammalian cell extracts along with their natural binding partners. we developed a method of utilizing our high affinity and specificity binders as fluorescently labeled tools to visualize target proteins in their native environment in the cells without the need of secondary antibodies or blocking reagents. our system also includes a method of efficient delivery of generated antibodies to living cells, where they can perform their function. the sabs have been successfully used for altering biological processes in a controllable manner. in vitro evolution from pluripotent peptide libraries with natural neurotoxin scaffolds to target receptors, proteases and trophic factors tai kubo , mohammed naimuddin , seigo ono national institute of advanced industrial science and technology (aist) in vitro evolution from pluripotent peptide libraries with natural neurotoxin scaffolds to target receptors, proteases and trophic factors small molecule natural products are precious resources for drug discovery. during millions of years of evolution, natural products must have been exposed to various selection pressures and have been refined in structure and function to obtain the present features. in some peptide neurotoxins, however, the basic molecular scaffold mainly configured by disulfide (s-s) bridges and/or alpha/beta structures, is strictly conserved within each family even under the evolution pressure. on the other hand the loop regions, which are not heavily involved in scaffold formation, are highly diverged. this mode of molecular evolution named 'accelerated evolution', is reasonable to quickly adapt to the vigorous change of the environment. the evolutionally selected scaffold is compact harboring both rigidity and flexibility in nature, and it may support a topology appropriate for target recognition and selective interaction. inspired by the system, we designed random peptide libraries from the peptide neurotoxins of the accelerated evolution. a three-finger ( f) shaped snake neurotoxin consists of huge family evolved by accelerated gene evolution. we prepared a f-peptide library by introducing random sequences in each fingertip. another random peptide library with an ick (inhibitor cystine knot) motif was prepared based on a neurotoxin gtx - from spider; originally identified as a t-type ca channel modulator. each library was subjected to in-vitro evolution directed to specific target molecules. for the f-peptide library cdna display method was applied to select binders. when interleukin- (il- ) receptors were targeted, the selected f peptides showed binding affinities (kd nm) comparable to the native ligand il- . when trypsin was targeted, peptides with serine protease inhibitor activities similar to sti and bpti (ki nm) were isolated. specific binders to a trophic factor vegf were also generated from the f library. to target membrane proteins, we developed a unique in-vitro evolution system, and named it as the periss (intra periplasm secretion and selection) method. in the system, target membrane proteins are expressed in inner membrane of e. coli and peptides are secreted to the periplasmic space, in between the inner and outer membranes; and the space is served for interaction and selection. the periss method enabled us to identify a peptide specific to muscarinic receptor m subtype from the ick peptide library. in conclusion, it was proved that the library designed from the scaffold of peptide toxin, which evolved in the mode of accelerated gene evolution, has pluripotency in target recognition, interaction and even bioactivity. phenylalanine ammonia lyase from petroselinum cripsum (pcpal) belongs to the class of enzymes containing -methylideneimidazole- -one (mio) as a prostetic group and it is responsible for the conversion of l-phenylalanine into trans-cinnamic acid. this reaction is reversibile under high ammonia concentration. we analyzed several factors that can influence the enantioselective synthesis of nitrophenylalanine mediated by whole cells as well as purified mio-containing and mio-less pcpals. first we investigated the behaviour of the enzymes depending on the ammonia concentration. we also inspected the influence of the ph on the pcpal catalyzed biotransformations. based on our results, we concluded that variation of ammonia concentration and the ph leads to decrease of enantioselectivity, suggesting that pcpal is able to catalyze the formation of both l-and d-enantiomers of electron-deficient structures. all microbial cellulase appears to have a conserved 'sg' amino acid sequence at an identical position in the n-terminal domain. the properties of the n-terminal amino acid sequence were also predicted computationally. this analysis showed that n-terminal sequence of the enzyme is unstable. the nterminal sequence also showed potential cleavage sites by different proteases which may contribute to its instability. the secondary structure analysis showed that the n-terminal sequence has % of the a.a. sequence in extended strand and % in random coil conformation. the n-terminal sequence was also analyzed for potential phosphorylation sites. while no potential serine and threonine sites were predicted, two tyrosine phosphorylation sites were predicted in the n-terminal sequence. the n-terminal sequence was also examined for the presence of kinase specific phosphorylation sites. the results showed the presence of one potential site which may be phosphorylated by pkc at position of the n-terminal sequence. the analysis for the prediction of the presence of oglcnac sites revealed that two such sites may potentially be present in the sequence. we have also predicted the ligand binding site in the n-terminal sequence of the protein. protein arginine methylation catalyzed by protein arginine methyltransferases (prmts), is a pivotal protein post-translational modification involved in a growing number of physiological and pathological processes including signal transduction, proliferation, differentiation and malignancy. prmt accounts for the majority of protein arginine methyltransferase activity in mammalian cells and, in consistence, a large amount of cellular substrates have been identified. several studies have reported that the activity of prmt changes upon stimulation in various cellular processes. in mammalian cells, prmt exists in a high molecular weight complex. the interacting partners of prmt , such as antiproliferative proteins btg and btg , protein phosphatase a, the orphan receptor tr , and ccr -associated factor (hcaf ) are shown to play a role in modulating the methyltransferase activity and the substrate selectivity of prmt . due to the pivotal roles of prmt in physiological and pathological conditions, intensive efforts have been put on the search of small synthetic chemical molecules which can efficiently modulate the activity of prmt for the potential development of therapeutics. in light of this, the intracellular small molecules that either transmit extracellular stimulation or act as cofactor to dictate the activity of prmts in cells are still poorly understood. our study focused on examining how cellular ions might affect the activity of prmt and found that divalent and monovalent ions differentially modulated the catalytic activity of prmt toward different substrates. oligomerisation properties of light-dependent protochlorophyllide oxidoreductase prothoracicotropic hormone (ptth) is one of the most important neuropeptide regulators for insect molting and metamorphosis. however, preparation of its recombinant protein has hardly been successful, because it is a homodimer protein with very complicated disulfide-bond structure. for example, silkworm ptth has three intramolecular disulfide bonds in its -residue polypeptide chain, and the two chains are further linked by an additional intermolecular disulfide bond to form the homomeric dimer. although the recombinant silkworm ptth was previously expressed in escherichia coli, the product was obtained only in precipitation fractions, and refolding of the precipitated protein provided the active dimer ptth in very poor yield. under such reductive conditions as in cytosol of the e. coli cells, formation of the correct disulfide-bond arrangement must be difficult. alternatively, for the heterologous expression of the silkworm ptth, we employed brevibacillus choshinensis (formally referred to as bacillus brevis), which has achieved good results in expression of various disulfide-bond-containing proteins. in this study, the silkworm ptth was expressed in the brevibacillus cells with an additional his -tag sequence at the c-terminus, for easier detection and purification. first of all, since the brevibacillus bacteria are equipped with a secretory system of the expressed proteins, a secretory signal sequence to be attached before the silkworm ptth was carefully selected. among four candidates in a commerciallyavailable kit, a signal sequence derived from an intrinsic cell-wall protein mwp gave better results in expression levels of the protein. second, incubation time of the cells was optimized, because an oligomerization state of the secreted ptth in the cell culture medium changed with the time. in the medium, various ptth oligomers including a monomer and a dimer were initially observed, but higher oligomers became a major portion of the secreted product after longer incubation than h. incubation for - h may be suitable for obtaining the native dimer form of the silkworm ptth. to remove the undesired monomer and higher oligomers, which mostly retained free sulfhydryl groups, the secreted proteins were treated with maleimide-peg -biotin. in the purification using a ni -nta column, the dimer of the his -tagged silkworm ptth was eluted with an imidazole gradient, separately ahead of other biotinylated proteins, probably due to interaction of the peg spacer with the ni -nta groups of the resin. after the reversed-phase hplc purification, the final product showed a single band on the nonreductive sds-page, and it had adequate ecdysone-releasing activity from isolated silkworm prothoracic glands. the brevibacillus bacteria are most promising host cells for the heterologous production of the insect ptth. role of the disulfide bridges in the transmembrane region of the insect prothoracicotropichormone receptor, torso torso is an insect cellular-membrane protein, which was recently identified as a receptor for prothoracicotropic hormone (ptth). although ptth is one of the important regulatory molecules in insect molting and metamorphosis, activation mechanism of torso by the ligand has not been elucidated yet. in this study, an oligomerization manner of the silkworm torso was examined, using heterologous expression in drosophila s cultured cells, because torso is a single-polypeptide receptor tyrosine kinase (rtk), and activation of such rtks is often triggered by the ligand-induced receptor dimerization on the cellular membrane. when activated with silkworm ptth, dimerization of the silkworm torso in the s cells was observed, using a cross-linking reagent bs , and the subsequent receptor autophosphorylation and downstream erk phosphorylation were also detected. surprisingly, however, the torso dimerization was revealed to occur even without the ligand stimulation, while the autophosphorylation and the erk phosphorylation were held in response to the stimulation. when fractionated by non-reductive sds-page, the silkworm torso showed an obvious dimer band, in addition to a faint monomer band, both with and without the ptth simulation, even though the receptor was not treated with the cross-linking reagent. this indicates that the torso protein is expressed originally as a disulfide-bond-linked dimer. in addition, by examining oligomerization states of several truncation and substitution mutants, cysteine residues in the transmembrane region were found to participate in the intermolecular disulfide bridges, linking the two receptor molecules in the dimer. when all of the three cysteines in the transmembrane region were replaced by phenylalanines, the disulfide-bond-linked torso dimerization was not observed, but spontaneous, ligand-independent association of the torso molecules was detected using the crosslinker bs . this spontaneous dimerization caused the apparent torso autophosphorylation, but it could not induce the downstream erk phosphorylation. consequently, without the intermolecular disulfide bridges, torso loses its responsiveness to the ptth stimulation. in conclusion, the disulfide bridges in the transmembrane region may play a role to preserve suitable relative position between the two torso molecules, which could induce ligand-dependent autophosphorylation leading to activation of the downstream signaling pathways in the cells. the yeast enzyme neutral trehalase (nth , ec . . . ) from saccharomyces cerevisiae hydrolyses the non-reducing disaccharide trehalose which serves as an energy source and a universal stress protectant in many different organisms. enzymatic activity of nth is enhanced by the yeast - - protein (bmh and bmh ) binding in a phosphorylation-dependent manner. nth activity is also regulated by ca binding to the ef-hand-like motif containing domain of nth [ ] .the native tbe page and analytical ultracentrifugation show that nth forms very stable complexes with bmh and bmh [ ] . to study the structure of nth alone and its complex with the - - protein we used circular dichroism, h/d exchange coupled to mass spectrometry, chemical cross-linking [ ] and small angle x-ray scattering (saxs) [ ] . at the same time protein crystallography of nth alone and its complex with bmh is performed.the low resolution structure of pnth :bmh protein complex revealed that binding of bmh induces a rearrangement of the whole nth molecule and that the region containing the ef-hand motif forms a separate domain which interacts with both bmh and catalytic domain of nth . we proved that integrity of the ef-hand motif is crucial for the bmh mediated activation of nth and ca binding. our data suggest that the ef hand-like motif functions as the intermediary through which bmh modulates the function of the catalytic domain of nth . these structural changes probably enable the substrate entry into the enzyme active site [ ] . our study of - - protein complex with the fully active enzyme nth offers a unique structural view of nth activation enabling us to better understand the role of the - - proteins in regulation of other enzymes. the assembly of self-regulating synthetic biochemical pathways in vitro has great potential as alternative catalysts for the high-yield production of low value/high volume commodity chemicals from biomass. high yields of low-value/high volume compounds that are required for economic viability is particularly difficult via traditional in vivo metabolic engineering of microbes due to competing biochemical pathways and toxicity. we have developed an alternative approach, called synthetic biochemistry, where the glycolysis pathway of central metabolism is reconstituted in vitro with an anabolic pathway that can produce useful compounds at high yield. in the specific synthetic biochemistry system described, reducing equivalents, atp, and carbon from glycolysis are funneled through the anabolic mevalonate pathway to produce the monoterpene limonene from glucose. the successful implementation of the in vitro pathway required development of a molecular purge-valve consisting of an nad and nadp specific reductase (ie wild-type and mutant pyruvate dehydrogenase), and nadh oxidase, noxe, to maintain proper nadp /nadph cofactor balance while allowing continuous carbon flux. we find that the purge-valve concept is readily transportable to other nad(p)h generating steps in central metabolism and can be used to convert glucose to limonene at high yield. chitinases (ec . . . ) are enzymes that randomly hydrolyze b- , glycosidic bonds of chitin and produce n-acetylchitooligosaccharide ((glcnac)n) that has various physiological functions such as immunostimulatory activity. most of fish takes crustacean such as shrimp and crab as food. therefore, the fish has chitinase in the stomach to chemically disrupt the chitinous envelope of crustacean. four chitinase isozymes ( - kda), pachia [ ] and pachib [ ] , and ptchia and ptchib, [ ] were purified from the stomach of silver croaker pennahia argentatus and threeline grunt parapristipoma trilineatum, by ammonium sulfate fractionation and column chromatographies, respectively. all the chitinases were stable and showed activity in the acidic ph range (ph - ). pachia and ptchia preferentially degraded the second glycosidic bond from the non-reducing end of (glcnac)n and pachib and ptchib had a preference for the third glycosidic bond of those. all the chitinases showed different substrate specificity toward insoluble long substrates. moreover, chitinase cdnas (pachi- and pachi- ) encoding pachia and pachib, and cdnas (ptchi- and ptchi- ) encoding ptchia and ptchib were obtained by cdna cloning using the rt-pcr and race method. the deduced amino acid sequences of all the chitinase cdnas contained n-terminal signal peptide, gh family catalytic domain, linker region, and chitin-binding domain. phylogenetic tree analysis of vertebrate chitinase revealed that fish stomach chitinases form unique chitinase isozyme groups, acidic fish chitinase- (afcase- ) including pachia and ptchia, and acidic fish chitinase- (afcase- ) including pachib and ptchib, which was different from an acidic mammalian chitinase (amcase) group. [ , ] the previously reported purified fish stomach chitinases [ ] can also be classified into two chitinase isozyme groups, afcase- and afcase- , by the n-terminal amino acid sequence. this study suggested that fish have excellent chitin degrading enzymatic system in which two different chitinases isozyme groups, afcase- and afcase- , with different degradation patterns are expressed in the stomach. recently, the enzymes produced by psychrophilic organisms have gained huge interest especially in the studies of temperature adaptation of the protein. previously, a cold-adapted yeast, glaciozyma antarctica pi was isolated from a marine environment in antarctica and the yeast was known to produce lipolytic and proteolytic enzymes. a gene encoding a unique recombinant bifunctional enzyme (lippi ) with cold active lipase with protease activity was successfully expressed, purified and characterized. temperature profile of the bifunctional lippi enzyme showed that the lipase functions optimally at c whereas the protease was more active at c. ph profile showed that both lippi lipase and protease were active at near neutral condition. activity of lippi lipase and protease were also activated in the presence of cacl but its protease counterpart seemed to be more active in the presence of zncl . effect of surfactants showed lippi lipase was activated by tween and sls and in contrast, lippi protease was almost deactivated in all surfactants tested. the presence of organic solvents did not affect both the lipase and protease activities. the lipase was more stable at solvents with higher log p value whereas the protease was slightly activated at low log p value particularly with dimethylsulfonyl. inhibitor studies revealed that lippi lipase was partially inhibited with edta and pmsf whereby the lippi protease was inhibited by pepstatin, edta and pmsf. lippi enzyme was successfully crystallized via vapour diffusion method. crystal of lippi enzyme was diffracted via synchrotron radiation. the three-dimensional structure of cold-adapted pi provided insight into cold adaptation and better understanding of the structural properties of lippi enzyme. the bifunctional properties of the enzyme could be potential candidate for low temperature industrial application. conformation-specific antibodies as enhancers and inhibitors of phosphatase activity of dep malgorzata nocula-lugowska , mateusz lugowski , anthony a. kossiakoff the university of chicago dep- (cd /ptp-h) is a transmembrane receptor-like protein tyrosine phosphatase (ptp) that has been implicated in the density-dependent regulation of cell growth, differentiation and transformation. it counteracts protein kinases by dephosphorylating a number of their substrates as well as the kinases themselves, thus potentially controlling the specificity of signals. for example egfr, vegfr , met, pdgf b receptor have been shown to be dephosphorylated by this phosphatase. dep- has been shown to act as a tumor suppressor and it has been proposed as a molecular target in antiangiogenesis therapy. as a result, both enhancers and inhibitors of dep- activity have the potential of elucidating pathways responsible for abnormal cell behavior. we generated synthetic antibodies against intracellular catalytic domain of dep- that act as modulators of the enzyme's phosphatase activity. by applying a combination of selection pressures an array of antibodies has been raised from phage display libraries of fab fragments which are capable of either enhancing or inhibiting dep- activity. in phosphatase assays with catalytic domain of dep- the antibodies demonstrate non-competitive or mixed kinetics. the crystal structure of dep- -inhibitor complex shows that this antibody binds to the part of the protein that is distant from the active site and acts by locking the enzyme in the nonnatural catalytically inactive state by hindering the closure of the wpd loop which is crucial for the reaction to occur. by contrast, as judged from the crystal structure of a complex of dep- with the antibody that enhances its phosphatase activity, this antibody seems to act by stabilizing the naturally found active state of dep- with wpd loop in the closed conformation. the antibodies are also able to recognize dep- in cells, as they stain dep- in immunofluorescence experiments. to test the applicability of raised antibodies in cells the activator was additionally used to pull down full-length endogenous dep- after being delivered to live cells. inhibition and enhancement of dep- activity by locking the enzyme in conformations which are either natural or imposed by allosteric binding of antibodies seems to be a mechanism that can be utilized to modulate activity of other tyrosine phosphatases. investigating acinetobacter baumannii pathogenesis: crystal structure of wbjb epimerase from a polysaccharide biosynthesis cluster oxygen homeostasis is regulated by hypoxia inducible factor, a transcription factor. when the oxygen level becomes too low (hypoxia), hypoxia-inducible-factor (hif- a) activates the expression of over a hundred genes, associated with angiogenesis, erythropoiesis, vegf (vascular endothelial growth factor), cell migration, and energy metabolism etc. hif- a cellular level is highly dependent on oxygen concentration and regulated by oxygen sensor enzyme, hif prolyl hydroxylase (phd plant sulphite reductase (sir) forms an electron transfer complex with ferredoxin (fd) for the reductive conversion of sulphite to sulphide. although previous studies have highlighted electrostatic interactions between oppositely-charged residues of the two proteins, detailed thermoenergetics of the intermolecular interaction for the complexation remains unknown. we herein carried out isothermal calorimetry of fd:sir complex formation at various nacl concentrations. driving force plot constructed from calorimetry showed that the complex was thermodynamically stabilized by both enthalpy and entropy through favourable electrostatic and non-electrostatic interactions. increasing nacl concentrations weakened interprotein affinity and contribution of the negative enthalpy changes became decreased, while no such significant decrease was found in the contribution of positive entropy changes. furthermore, a negative heat capacity change obtained from the enthalpy changes at distinct temperature indicated a contribution of hydrophobic interactions. these findings suggested that both electrostatic and nonelectrostatic interprotein interactions were energetically important for the complex formation. fddependent sir activity assay revealed a bell shaped activity curve with a maximum under a certain nacl concentration, while the methyl viologen-dependent assay of sir exhibited a profile of saturating curve, suggesting that an optimized interprotein interaction is a crucial factor in control of fd-dependent-sir activity. a residue-based nmr measurement of n-labeled fd upon complex formation with sir revealed that charged and non-charged residues were differentially contributed in the complex formation depending on nacl concentrations. we proposed that non-electrostatic forces were also critical for forming the fd:sir complex, and an optimized complex conformation for maximum enzymatic activity was achievable by a delicate balance among non-covalent intermolecular forces. these results may be extended for understanding of complexation between redox proteins containing biased charge clusters. ornithine transcarbamylase has a spatially extended active site as computationally predicted lisa ngu , kevin ramos , nicholas delateur , penny beuning , mary jo ondrechen understanding how an enzyme catalyzes a reaction is a fundamental problem in protein science. biochemical experimentation has revealed catalytic mechanisms of many enzymes; however these studies have focused almost exclusively on amino acid residues in direct contact with the reacting substrate molecule(s). here we report on the computational prediction and experimental verification of the importance of distal residues in enzyme catalysis, using e. coli ornithine transcarbamylase as an example. partial order optimum likelihood (pool), developed at northeastern university, is a machine learning technique that only requires the tertiary structure of a protein to predict important catalytic residues, based on computed, residue-specific electrostatic and chemical properties. pool has been shown to predict accurately the catalytic residues and to discern between compact and spatially extended active sites. dynamic conformational changes during catalysis and strong electrostatic interactions give rise to significant coupling between remote residues and the canonical active site residues of an enzyme. this suggests that at least some enzyme active sites are spatially extended, with second-and third-shell residues playing significant roles in catalysis. in this project, we focus on ornithine transcarbamylase (otc), for which dynamic processes are believed to play a role in its catalytic mechanism. otc is reported to undergo induced-fit conformational changes upon binding carbamoyl phosphate, which affects the subsequent binding of ornithine. residues predicted by pool to be catalytically important include five in direct contact with the substrate, r , h , d , c and r . pool also predicted remote residues to form a spatially extended, triple-layer active site. guided by computational predictions and using site-directed mutagenesis and kinetics assays of asp , his , glu and arg variants, we show that these pool-predicted remote residues, located in the second and third layers, are important for catalysis. alternative energy is a major focus of current research efforts. biodiesel, a mixture of fatty acid alkyl esters, is one of the most versatile alternative fuels currently in use. this is due to the fact that it is similar to gasoline and compatible with diesel engines found throughout the existing global infrastructure. biodiesel precursor lipids are abundant in cultivated feedstock organisms such as algae and bacteria. however, the standard process for converting oil to biodiesel is heat-intensive and requires complete removal of water, reducing the overall net energy gained in its production. our work constitutes an attempt to explore enzymatic synthesis of biodiesel from lipids such as those derived from emerging fuel crops. previous literature describes fatty acid alkyl ester formation in human patients with mrsa staphylococcus aureus wound lesions. these esters are formed by partially characterized esterase activity from an unidentified source. we have identified two mrsa enzymes responsible for this activity by using a combination of size exclusion chromatography, gas chromatography-mass spectrometry, and mass spectrometric protein sequencing. these two highly similar enzymes in the glycerol ester hydrolase (geh) family of proteins catalyze the synthesis of fatty acid alkyl esters in aqueous conditions at or near room temperature. we have demonstrated that other non-staphylococcal lipases do not exhibit this behavior. we have expressed these staphylococcal esterases in e. coli, and shown via gas chromatography that the expressed proteins catalyze the formation of fatty acid alkyl esters. based on sequence similarity to homologous proteins that have already been crystallized, we have predicted a structure for these enzymes and have engineered mutant fusions with higher rates of catalysis. our design hypothesis is that increased avidity for substrate molecules will yield a higher substrate concentration in the vicinity to the enzyme. to increase substrate concentration we have designed and expressed one of the enzymes as a chimeric fusion with the drosophila melanogaster alcohol-binding protein lush. gc-ms determination of biodiesel production rate indicates that the chimeric fusion has a lower-order rate constant with respect to ethanol. in other words, the fusion enzyme is less dependent on substrate concentration and is a superior catalyst at low ethanol concentrations. this result indicates that the rationally designed modification of binding avidity constitutes a potential avenue for improving the ability of enzymes to catalyze reactions with low-concentration or low-solubility substrates. functional elements of a human antizyme essential for binding and inhibiting human ornithine decarboxylase proteases are ubiquitous enzymes that catalyze the hydrolysis of peptide bonds within protein substrates; they have served as key model enzymes for studying the molecular basis for catalytic power and specificity. protease substrate specificity is most often defined in terms of linear sequence motifs that flank the cleavage site; however, the natural substrates of proteases are proteins with -dimensional shapes and complex conformational dynamics that are not well represented by -dimensional sequence alone. these structural and dynamical properties can impact recognition and binding of substrates by proteases, as well as the efficiency of catalysis itself. in this study, we explore the importance of substrate structure and dynamics for proteolysis using as our model the cleavage of the kunitz-bpti family of canonical serine protease inhibitors by mesotrypsin. bovine pancreatic trypsin inhibitor (bpti), an archetypal serine protease inhibitor of the kunitz family, has a high affinity interaction with trypsin, yet its peptide bond hydrolysis is many orders of magnitude slower than other peptide substrates. mesotrypsin, a trypsin variant, has been shown to hydrolyze kunitz family inhibitors at accelerated rates; this is especially true of human kunitz domain inhibitors. amyloid precursor protein inhibitor (appi) and amyloid precursor like protein- (aplp ), two human kunitz domain family members, are hydrolyzed by mesotrypsin several hundred times faster than bpti. here, we present a new, unpublished crystal structure of a cleavage intermediate aplp bound to mesotrypsin, refined to . Å resolution, revealing a dramatic substrate conformational change we hypothesize to be required during cleavage of a kunitz domain. using this structure along with published structures of appi and bpti complexes, we have modeled acyl-enzyme intermediates of mesotrypsin, and we have carried out molecular dynamic simulations that explore the transition of the initially formed native-like acyl-enzyme through the conformational transformation that allows the progression of the hydrolysis reaction. we further identify a specific hydrogen bond, present in bpti but not appi, which forms a stabilizing feature of the bpti scaffold. using site directed mutagenesis, we probe the contribution of this bond to the proteolytic stability of bpti. collectively our data for these highly structured substrates show that proteolysis rates are limited by a necessary conformational change in the substrate as the reaction progresses. rigid substrates possessing stabilizing features that render them highly resistant to this conformational change are proteolyzed more slowly than more flexible substrates of similar structure. lpmos are copper metalloenzymes that carry out the oxidative cleavage of the b- , -glycosidic bond, generating new chain ends that can subsequently be processed by cellulases, boosting the cellulose degradation. lpmos have a b-sandwich conformation with a flat binding surface, allowing for the enzyme to bind to crystalline cellulose. the cu ion, required for activity, is located in a so-called "histidine brace", in which the n-terminal histidine is highly conserved. regioselectivity according to the carbon atom being oxidized, lpmo types are identified: type and type oxidizing at the c and the c respectively, type lpmos oxidizing both the c and the c adjacent to the glycosidic linkage. we were able to express a type- lpmo (phanerochaete chrysosporium gh d) and a type- lpmo (trichoderma reesei cel a) in p. pastoris. this has proven to be very challenging, as lpmo activity requires a perfect cleavage of the signal sequence. after activity assays on pasc, characteristic hpaec-pad traces were obtained which will serve as a reference for engineering experiments. enzyme engineering using the dm database, a structure based multiple sequence alignment tool, it is possible to identify residues specifically conserved in subsets of protein sequences. by defining a subset for each lpmo type, we were able to identify residues contributing to regioselectivity. these positions are now being rationally engineered in subsequent rounds of mutagenesis, using trcel a as a template. the effect of the mutations will be determined by analyzing the hpaec-pad trace released from pasc. the main goal is to investigate the possibility of deleting the c specificity in a type lpmo. folding topology determines substrate binding order in the ribokinase superfamily alejandra herrera-morand e , victor castro-fern andez , madrid, españa ribokinase superfamily comprises three enzyme families: the adp-dependent sugar kinases family, the atpdependent coenzyme kinases family and the atp-dependent sugar kinases family. in all these families there is a large domain composed by a rossmann motif but only the atp-dependent enzymes have a b-meander motif in the c-terminal end. interestingly, these enzymes display an ordered kinetic mechanism where the substrate that will be phosphorylated binds first to the enzyme. the adp-dependent enzymes present a topological re-ordering of the secondary structural elements which produces an equivalent tertiary structure, which can be thought as a non-circular permutation (ncp) of the bmeander region. these enzymes also display an ordered kinetic mechanism but with an inversed order being the nucleotide the first substrate to bind to the enzyme. as this b-meander region of the proteins constitutes almost entirely the nucleotide binding site, and given that the permutation is the major structural difference between adp and atp-dependent kinases, it could the responsible for the nucleotide specificity. to test this hypothesis we introduce, by permutation, an atp-dependent topology in the homologous adp-dependent glucokinase from t. litoralis (pergk). size exclusion chromatography and circular dichroism spectra show that both the wild type and the permutated enzyme eluted as monomers with similar hydrodynamic behavior, and have the same secondary structure content. kinetic assays employing atp or adp as substrate demonstrate that even in the presence of mm atp, the pergk enzyme is not able to carry out the phosphoryl transfer. to test if the ncp has an impact in the kinetic constants and substrate binding order we determine the kinetic mechanism through classical protocols, involving initial velocity studies, product inhibition and dead end inhibitors. the results demonstrate that the pergk enzyme presents an altered substrate binding order compared to the wild type enzyme, where glucose was the first substrate to bind to the enzyme and glucose- -p the last product to be released. also, ligand-induced conformational changes were determined in the crystal structures. the apo, the enzyme-glucose and enzyme-glucose-adpbs structures were determined at . Å, . Å and . Å resolutions, respectively. structure analysis reveals that glucose binding provokes major conformational changes in the pergk enzyme, whereas adp binding does not cause further changes in the conformation of the protein. the results show that although the permutation has no effect on the nucleotide preference it provokes a change in the substrate binding order that correlates well with that those observed in the crystal structures. also, they demonstrate that during the evolutionary history of the ribokinase superfamily folding topology dictates the substrate binding order (fondecyt ). background: human ceruloplasmin (cp) is a circulating copper-containing glycoprotein produced in the liver and first described as a component of alpha -globulin fraction of human plasma. cp belongs to the multicopper oxidase family and it is nowadays regarded as a "moonlighting" protein, because it changes its function according to substrate, localization and expression. cp plays a key role in copper transport and iron metabolism and it is also a potent inhibitor of leukocyte myeloperoxidase (mpo) (kd nm), a major source of oxidants in vivo. the protein is extremely susceptible to proteolysis. in fact, cp is a structural homolog of coagulation factors v and viii, that are physiological substrates of thrombin (fiia). interestingly, thrombin participates in both haemostatic and inflammatory responses: in some focus of inflammation, such as rheumatoid arthritis (ra), the high activity of fiia has been documented. it was demonstrated that fiia can promote the chemotaxis of neutrophils and monocytes and their adhesion to endothelial cells, to increase vascular permeability. all these effect are mediated by par- interaction, that are abundantly expressed in inflamed rheumatoid synovial tissues. aims: in this study the interaction of cp with thrombin was investigated to confirm the participation of fiia in "spontaneous" proteolytic degradation of cp. in fact, in vivo the integrity of cp is essential for its role in the transport or metabolism of copper. results: our results indicated that thrombin cleaves cp in vitro at arg-ser and lys-val bonds, generating a nicked species that retains the native-like fold and the ferroxidase activity of the intact protein, whereas the mpo inhibitory function of cp is abrogated. analysis of the synovial fluid of ra patients reveals that cp is proteolytically degraded to a variable extent, with a fragmentation pattern similar to that observed with fiia in vitro, and that proteolysis is blocked by hirudin, a highly potent and specific thrombin inhibitor. we demonstrate that fiia has intrinsic affinity for cp (kd - nm), independently of proteolysis, and inhibits cp ferroxidase activity (ki nm). mapping of thrombin binding sites with specific exosite-directed ligands (i.e. hirugen, fibrinogen gamma-peptide) and thrombin analogues having the exosites variably compromised (i.e. prothrombin, prethrombin- , alpha-thrombin), reveals that the positively charged exosite-ii of thrombin binds to the negative upper region of cp, while the protease active site and exosite-i remain accessible. these results suggest that thrombin can exacerbate inflammation in ra by impairing via proteolysis the mpo inhibitory function of cp and by competitively inhibiting cp ferroxidase activity. an artificial pathway for isobutene production by direct fermentation: combining metabolic engineering and protein engineering benoit villiers , franc¸ois stricher the purpose of global bioenergies is to develop innovative metabolic pathways for the production of light olefins from renewable resources, by direct fermentation. light olefins (ethylene, propylene, linear butylene, isobutene and butadiene) are the core of the petrochemical industry. however, microorganisms do not naturally produce light olefins and no bioprocess to convert renewable resources to these molecules has been industrialized so far. global bioenergies has developed an artificial metabolic pathway including all the necessary enzymatic reactions from feedstock to isobutene. the metabolic route leading to isobutene can be divided in three parts, the first one being the use of natural reactions occurring in the host microorganism. second, heterologous natural reactions were introduced into the same host microorganism. finally, in contrast with most former approaches, non-naturally occurring reactions as enzymatic key steps were used, for example the decarboxylation of hydroxyisovaleric acid into isobutene. such non-natural critical steps were made possible by taking advantages of the natural catalytic and substrate promiscuity of exogenous enzymes. candidate enzymes are then evolved using systematic, random and semi-rational approaches in successive rounds in order to reach the desired catalytic efficiency. since all these reactions are enzymatic, isobutene can be obtained by direct fermentation, e.g. a process wherein all the chemical transformations are carried on by the host microorganism. the scale-up of this process began in november in a pilot plant installed in pomacle-bazancourt, france, with an annual capacity of tons of oxidation-grade isobutene. importantly, production of a volatile compound such as isobutene (and other light olefins) by direct fermentation presents two major advantages: first, the product is spontaneously removed from the culture broth, which alleviates the limitations linked with titer issues. second, the purification process is considerably easier and cheaper since no energy consuming methods such as distillation or phase separation are necessary to purify the end product. for the first time, batches of industrially produced isobutene from renewable resources have been obtained in the first half of . this isobutene has been in turn converted into isooctane, an additive currently used to improve gasoline quality, which could also be used as a standalone fuel. a demonstration plant is planned in leuna, germany, with an annual capacity of tons of polymer-grade isobutene and ibn-one, a joint venture with cristal union ( th european beet processor), has been formed to build and operate the first plant in france converting renewable resources into isobutene. finally, while the isobutene process is progressing towards industrial scale, global bioenergies is also developing new artificial metabolic pathways enabling direct bio-production of butadiene and propylene. the development of a coupled enzyme assay to detect isochorismate pyruvate lyase activity protein folding is typically defined in terms of the spatial arrangement of structural elements, i.e. helices, sheets and loops. we have, however, been developing an alternative and complementary paradigm based on conserved hydropathic interaction networks within proteins. these networks can be viewed as environments comprised of a mixture of polar and hydrophobic interaction fields, and may be the most important factor driving protein folding. this concept applies even to the lowest structural level within a protein: the sidechain conformations (or rotamers). exhaustive statistical analysis of existing crystallographic structures of proteins showed rotameric preferences and led to the creation of rotamer libraries frequently used in multiple aspects of structural biology, e.g., crystallography of relatively low-resolution structures, homology modeling and biomolecular nmr. however, little is actually known about the forces and factors driving the preference or suitability of one rotamer over another. in our study, tyrosine was analyzed since its sidechain has a comprehensive set of hydropathic properties that made it ideal as a proof of concept residue. construction of d hydropathic interaction maps of tyrosine residues in our dataset, reveals the environment around each, in terms of hydrophobic (p-p stacking, etc.) and polar (hydrogen bonding, etc.) interactions. after partitioning the tyrosines into backbonedependent bins, a map similarity metric based on the correlation coefficient was applied to each mapmap pair to build matrices suitable for clustering. notably, the first bin representing tyrosines, reduced to unique hydropathic environments with most diversity arising from favorable hydrophobic interactions with many different residue partner types. polar interactions for tyrosine include ubiquitous hydrogen bonding with the phenolic oh and somewhat surprisingly a handful of unique environments for the tyrosine backbone. all but one of the environments are dominated by a single rotamer, the exception being an environment defined by a paucity of interactions with the tyrosine ring and as a consequence its rotamer is indeterminate. this is consistent with it being composed of mostly surface residues. each tyrosine residue attempts to fulfill its hydropathic valences and thus, structural water molecules are seen in a variety of roles throughout these environments. alanine was analyzed using the same protocol as well. having the smallest sidechain (and small hydropathic interaction maps), alanine allowed us to investigate a significantly larger database, permitting us to examine the correlation between hydropathic maps and various structural features. in conclusion, the analysis of hydropathic environments strongly suggests that the orientation of a residue in a three-dimensional structure is a direct consequence of its hydropathic environment, which leads us to propose a new paradigm, interaction homology, as a key factor in protein structure. it is not the surrounding residues that direct sidechain conformations, but rather the hydropathic "field" of the surrounding atoms. folding studies of independent domains of lysine, arginine, ornithine binding protein (lao) protein folding problem has been addressed from the past years until nowadays, however, we still can not explain how proteins acquire their native structure from their amino acid sequence. different approaches has been taken in order to study protein folding, for example, the comparative study of folding mechanism between homologues proteins with high identity of sequence and structure, and the study of independent regions within a single protein. previously in our laboratory, thermodynamic and kinetic folding properties of lysine, ornithine, arginine binding protein (lao), a amino acid periplasmic binding protein (pbp), composed by two rossmann fold domains (one continuous and the other discontinuous) attached by a hinge region, has been studied. even there is a functional research about binding characteristics of histidine binding protei ns (his j) domains of when expressed independently (chu, b. ); there are no folding studies in these conditions for this or another pbps. it should be noted that his j shares % of sequence identity and tertiary structure (rmsd Å) with lao. in order to know the folding effect of encoding different domains in the same poly peptidic chain, as well as its influence in function, we are studying the thermodynamic and kinetic characteristics of folding of independently expressed lobes of lao, and comparing with those of native protein. by now, we expressed and purified the discontinuous domain. circular dichroism (cd) and fluorescence intensity spectra show that this independent domain has primary and tertiary structure. thermal denaturation has a single cooperative transition, which indicates this domain is folded. thermodynamic analysis of temperature and urea-induced experiments suggest that lao's folding characteristics are not just the addition of those from independent domains. furthermore, folding and refolding kinetics suggest the presence of a burst phase intermediate. a hypothesis to reconcile the physical and chemical unfolding of proteins a comprehensive view of protein folding is crucial for understanding how misfolding can cause neurodegenerative diseases and cancer. when using physical or chemical perturbations, nmr spectroscopy is a powerful tool to reveal a shift in the native conformation toward local intermediates that act as seeds for misfolding. high pressure (hp) or urea is commonly used to disturb folding species. pressure favors the reversible unfolding of proteins by causing changes in the volumetric properties of the proteinsolvent system. however, no mechanistic model has fully elucidated the effects of urea on structure unfolding, even though protein-urea interactions are considered to be crucial. here, we provide nmr spectroscopy and d reconstructions from x-ray scattering to develop the "push-and-pull" hypothesis, which helps to explain the initial mechanism of chemical unfolding in light of the physical events triggered by hp. in studying mpnep from moniliophthora perniciosa, we tracked two cooperative units using hp-nmr as mpnep moved uphill in the energy landscape; this process contrasts with the overall structural unfolding that occurs upon reaching a threshold concentration of urea. at subdenaturing concentrations of urea, we were able to trap a state in which urea is preferentially bound to the protein (as determined by nmr intensities and chemical shifts); this state is still folded and not additionally exposed to solvent [fluorescence and small-angle x-ray scattering (saxs)]. this state has a higher susceptibility to pressure denaturation (lower p / and larger dvu); thus, urea and hp share concomitant effects of urea binding and pulling and water-inducing pushing, respectively. these observations explain the differences between the molecular mechanisms that control the physical and chemical unfolding of proteins, thus opening up new possibilities for the study of protein folding and providing an interpretation of the nature of cooperativity in the folding and unfolding processes. zinc: a promoter or inhibitor for iapp aggregation? feng ding , praveen nedumpully-govindan zinc ions have been found to play an important and yet complex role in human islet amyloid polypeptide (hiapp) aggregation, which is associated with b-cell death in type-ii diabetes (t d). both concentration-dependent promotion and inhibition of iapp aggregation by zinc ions have been observed in vitro. similarly, at the population level, both positive and negative correlations were reported between the activity of a b-cell specific zinc transporter and t d risk. zinc ions are able to bind a single histidine in hiapp and coordinate the formation of zinc-bound hiapp oligomers. we hypothesize that the relative zinc/hiapp concentration determines the population of zinc-bound hiapp oligomers with different molecular weights. we have applied molecular dynamics (md) simulations to systematically study the structure and dynamics of a range of zinc-coordinated hiapp oligomers, including monomers, dimers, trimers, tetramers, and hexamers. our computational results suggest that different zinc-bound oligomers have distinct aggregation propensities. high-molecular weight oligomers ( peptides) have higher aggregation propensity than zinc-free and zinc-bound hiapp monomers at mm concentration in silico. therefore, our results provide a molecular insight into the complex role of direct zinc binding on hiapp aggregation. at low zinc/hiapp stoichiometry, zinc binding promotes aggregation. as the stoichiometry increases and zinc ions bind to single hiapp peptides, the aggregation of hiapp is inhibited due to electrostatic repulsion between the charged zinc ions. our computational study sheds light on the complex role of zinc on hiapp aggregation and t d development. biomolecules function in the densely crowded and highly heterogeneous cell, which is filled up to a volume of % with macromolecules [ ] . often, artificial macromolecular crowding agents are used to mimic these conditions in vitro and the excluded volume theory is applied to explain the observed effects [ ] . however, recent studies emphasize the role of further contributions aside from a pure volume effect including enthalpic and solvent effects [ , ] . we study cosolute effects at high molecular and macromolecular concentrations via a thermodynamic analysis of the thermal unfolding of ubiquitin in the presence of different concentrations of cosolutes (glucose, dextran, polyethylene glycol, potassium chloride) [ ] . in contrast to the excluded volume theory, we observed enthalpic stabilization and entropic destabilization forces for all tested cosolutes. the enthalpic stabilization mechanism of ubiquitin in macromolecular polysaccharide solutions of dextran was thereby similar to the effects observed in monomeric glucose. further, it remains unclear how such cosolutes reflect the physicochemical properties of the complex cell environment as a characterization of the in-cell crowding effect is lacking. thus, we developed a fret-based macromolecular crowding sensor to study the crowding effect in living cells [ ] . the averaged conformation of the sensor is similar to dilute aqueous buffer and cell lysate. we find that the in-cell crowding effect is distributed heterogeneously and can change significantly upon osmotic stress. the presented method allows to systematically study in-cell crowding effects and understand them as a modulator of biomolecular function. the stability of biomolecules under co-solvent conditions is dependent on the nature of the co-solvent [ ] . this can alter a protein's properties and structural features through biomolecular interactions between its functional groups and the co-solvent molecules. ionic liquids (ils) represent a rather diverse class of co-solvents. the design flexibility of these molten salts is an attractive feature, allowing the properties of the il to be tuned to meet the requirements of different applications [ ] . particularly, the modulation of reaction pathways between folding states, offering possibilities to control irreversibility in non-native protein aggregation [ ] . this has led us to investigate the impact of ils as co-solvents with the well-known protein denaturant urea. urea is considered to be a non-ionic chaotrope disturbing considerable the grid of hydrogen bonds with the protein backbone. urea interacts preferentially with the protein surface, mainly apolar residues and that dispersion, rather than electrostatic interactions, is the main energetic contribution to explain the stabilization of the unfolded state of the protein and the irreversibility of the unfolding process in the presence of urea [ ] . a large body of multidomain protein folding work has been devoted to study monomeric proteins. how do multidomain multimeric protein fold, avoiding accumulation of stable intermediate is yet to be studied in detail. our present study is focussed on understanding the folding and assembly of the domains of a homodimeric l-aspraginase from a hyperthermophile pyrococcus furiosus (pfa). each monomer of pfa consists of distinct n-and c-terminal domains (npfa and cpfa, respectively), connected by a linker. the folding mechanism of each domain with respect to full length protein was studied by mutating one out of two tryptophans, one in each domain. domains were purified and studied individually to obtain parallel account of the folding of each domain in isolation. subunit assembly was studied by analytical size exclusion chromatography (sec), multiangle light scattering and functional activity. through far uv cd, intrinsic trp fluorescence and sec, we demonstrated that domain folding and subunit association were intimately linked in full length pfa. interestingly, en route to its folding there was complete absence of hydrophobic intermediates as probed by ans fluorescence. folding of npfa was highly cooperative and, it provides interacting surfaces for cpfa to fold and also facilitates subunit assembly. the folding cooperativity of isolated domains was very less compared to the folding cooperativity of their full length counterparts, as indicated by equilibrium m values. to our surprise, during ph induced denaturation, at ph and , the dimer dissociates into highly hydrophobic folded monomers which readily underwent amyloidogenesis. we showed that at such extreme conditions, cooperativity in folding process in multidomain multimeric protein is not solely governed by the folding of individual domains, rather by concomitant folding and association of domains directly into a quaternary structure. in other case, where subunit folding occurred prior to association, protein readily underwent extensive aggregation. groel assisted folding of multiple recombinant proteins simultaneously over-expressed in e.coli megha goyal , tapan kumar chaudhuri aggregation prone recombinant proteins very often form inclusion bodies and also exhibits poor yield of functional protein during in vitro refolding process from chemically denatured form. bacterial chaperonin groel provides folding assistance to several proteins, when over-expressed with one of the recombinant proteins. there are instances that groel in presence of few other co-expressed chaperones like dnaj, dnak etc provides better yield of folded protein during homologous and heterologous expression. considering the ongoing events in the cells, it is known that molecular chaperone groel assists in the folding of various proteins in the cytoplasm. hence attempt to fold multiple recombinant proteins over-expressing simultaneously with the co-expression of chaperones can be worth trying. this approach may cut down various complexities in the functional recombinant protein preparation, including time and effective cost. keeping this view in mind, folding of two simultaneously expressed aggregation prone proteins, kda e.coli maltodextrin glucosidase (malz) and kda yeast mitochondrial aconitase have been investigated with the co-expression of groel and groes in e.coli cytosol. it has been previously reported that both the chosen proteins undergo co-expressed groel-groes assisted folding in e.coli cytosol, when they over-express alone. in this study we have optimized the overexpression of malz and aconitase simultaneously in e.coli. further optimisation was carried out to coexpress groel along with malz and aconitase. based on the basic philosophy that soluble protein mainly contains folded fraction, the event of groel/es assisted folding of simultaneously overexpressed proteins, malz and aconitase was monitored through the attainment of soluble proteins under various sets of conditions such as temperature. the major outcome of the present study is that, with the groel-groes assistance, the yield of soluble proteins (malz and aconitase) together constitutes higher percentage of folded protein in contrast to the percent yield when a single protein was overexpressed. significance of this type of study relies on the fact that the cells can over-produce higher amount of recombinant proteins, when multiple over-expression takes place. not only pushing up cell's capability of over-expression, co-expression of groel and groes efficiently assists in the folding of multiple proteins simultaneously over-expressed in e.coli. amyloid fibrils associated with serious diseases including alzheimer's, parkinson's, and prion diseases promoted the challenge of studying protein misfolding, leading to the development of amyloid structural biology. amyloid fibrils form in supersaturated solutions via a nucleation and growth mechanism. although the structural features of amyloid fibrils have become increasingly clearer, knowledge on the thermodynamics of fibrillation is limited. furthermore, protein aggregation is not a target of calorimetry, one of the most powerful approaches used to study proteins. here, with b -microglobulin, a protein responsible for dialysis-related amyloidosis, we show direct heat measurements of the formation of amyloid fibrils using isothermal titration calorimetry (itc). the spontaneous fibrillation after a lag phase was accompanied by exothermic heat. the thermodynamic parameters of fibrillation obtained under various protein concentrations and temperatures were consistent with the main-chain dominated structural model of fibrils, in which overall packing was less than that of the native structures. we also characterized the thermodynamics of amorphous aggregation, enabling the comparison of protein folding, amyloid fibrillation, and amorphous aggregation. in order to obtain general thermodynamic properties of protein aggregations, we further investigated aggregation of glucagon and insulin, two of the most famous amyloidogenic peptide hormones, using itc. we also observed characteristic heat of spontaneous amyloid fibrillation of both proteins after a lag time. taken all together, we showed that thermodynamic studies on amyloid fibrillation and amorphous aggregation were indeed possible by means of itc-based qualitative and quantitative calorimetric analyses. itc will become a promising approach for clarifying the thermodynamic properties of protein aggregates. the more case studies are required toward the establishment of thermodynamics of protein misfolding and aggregation when hydrophobic proteins are, for any reason, exposed to the cytosol they are rapidly captured by protective complexes which shield them from the aqueous surroundings and decide their fate (by either targeting them to their correct membrane homes or marking them for degradation by the ubiquitin/proteasome system). the bag holdase is a heterotrimeric protein complex, comprising bag , ubl a and trc , which works closely with the cochaperone sgta to triage hydrophobic proteins and pass them along the appropriate pathway. sgta also interacts with viral proteins and hormone receptors and is upregulated in numerous cancer types. these functions require further investigation to determine the scope of sgta as a therapeutic target. our lab has solved the solution structure of the n-terminal dimerization domain of sgta and characterised its interaction with two different ubiquitin-like (ubl) domains in the bag holdase (one from ubl a and the other from bag itself) using nmr chemical shift perturbation data and other biophysical techniques including isothermal titration calorimetry and microscale thermophoresis. at this meeting i will report on the progress we have made in structurally characterising further key players that participate in this quality control, with the aim of clarifying the intricate network of molecular interactions that governs these processes in health and disease. ensemble, ribbon and electrostatics spacefill views of the sgta dimerization domain structure. the final panel shows the structure overlaid with its yeast homologue. alpha synuclein is a small protein ( kda) expressed at high levels in dopaminergic neurons. fibrillar aggregates of a-synuclein inside the dopaminergic neuron are the major components of lewy bodies and lewy neuritis inclusion, which are considered as potential hallmark of parkinson's disease (pd). both in vitro as well as in vivo studies suggest that the soluble, oligomeric forms of a-syn are the more potent neurotoxic species, responsible for neuronal injury and death in pd. therefore, molecules that inhibit the toxicity of oligomers either by reducing their formation or by converting their more toxic oligomeric state to less-toxic fibrillar state would be effective agents for the drug development against pd. curcumin is one of the asian food ingredients which has shown a potential role as therapeutic agent against many neurological disorders including pd. however, the instability and low solubility makes it less attractive for use as potential therapeutic agent. the present work focuses on screening of the compounds similar to curcumin but having better effects on the morphology and toxicity of oligomeric and fibrillar assemblies of a-syn, which could be used as therapeutic agent preferentially over the naturally occurring curcumin. we synthesized and analyzed the effects of nine compounds, which are structurally similar to curcumin, on different stages of a-syn amyloid aggregation. here, we showed that curcumin and its analogs accelerate a-syn aggregation to produce morphologically different amyloid fibrils in vitro. however, there is no significant effect of curcumin and its analogs on the secondary structure of preformed a-syn fibrils. furthermore, these curcumin analogs showed differential binding affinities with the preformed a-syn aggregates, possibly due to difference in their chemical structures. the present data suggest the promising role of curcumin analogs in the treatment of a-synucleinopathy disorders. in vitro folding mechanisms determine the forces applied during co-translational folding there is currently much debate as to whether experiments conducted in vitro describe the folding of proteins in vivo. in particular, it is often suggested that the co-translational folding of nascent protein chains is dominated by the presence of the ribosome and associated chaperones, and that folding mechanisms will be affected by the vectorial nature of translation. here we use an arrest peptide assay to investigate the co-translational folding of a number of all-a spectrin domains that exhibit a range of thermodynamic stabilities and in vitro folding rates. our unexpected finding is that that the force exerted on the ribosome by these domains is not related to either the thermodynamic stability of the domain, or to the folding (loading) rate, but rather to the in vitro folding mechanism. we infer that the in vitro folding mechanisms of these domains are unaffected by the presence of the ribosome -even when part of the nascent chain is retained within the ribosome exit tunnel. there has been much work to date investigating the intermediates present in stalled translation complexes -but now, for the first time, we can begin to directly explore the rate limiting transition state in the co-translational folding of homologous proteins. can the structure of a protein (h . ) depend on the treatment of a solvent medium (explicit vs effective) in a coarse-grained computer simulation? ras pandey , barry farmer university of southern mississippi, air force research laboratory solvent medium plays a critical role in orchestrating the structure and dynamics of a protein. in computer simulation modeling of protein structure in a solvent medium, explicit, implicit, effectivemedium, approaches are often adopted to incorporate the effects of solvation. because of the complexity in incorporating all atomic and molecular details, the multiple components, reaching the large-scale, etc. implicit solvent or effective medium approach is generally more viable than the explicit solvent methods. some of the pertinent characteristics such as excluded volume of the solvent constituents, its concentration, and the underlying fluctuations which may be important in probing some issues are generally ignored in effective medium or implicit solvent approaches. using a coarse-grained approach, we investigate the structure and dynamics of a protein (a histone, h . ) in the presence of both effective as well as explicit solvent media over a range of temperatures with the monte carlo simulations. the protein is represented by a coarse-grained chain of residues whose interactions are described by knowledge-based residue-residue and hydropathy-index-based residuesolvent interactions. in effective medium approach, each empty lattice site around the protein structure acts as a solvent. only a fraction of lattice sites are occupied by mobile solvent constituents along with the protein chain in explicit solvent medium. large scale simulations are performed to analyze the structure of the protein for a range of residue-solvent interactions and temperature in both explicit and effective solvent media. we study a number of local (e.g. solvation and mobility profiles) and global (radius of gyration and structure factor) physical quantities as a function of temperature. we find that the response of the radius of gyration of the protein in explicit solvent is different from that in effective medium solvent. thus, the presence of fluctuations in explicit solvent approach have considerable effects on the structure and dynamics of protein h . . differences due to type of solvent on the response of some of these quantities as a function of temperature as well as general similarities will be presented. single-molecule vectorial folding and unfolding through membrane pores david protein folding and unfolding in vivo is frequently vectorial. for example, proteins are synthesized at the ribosome and emerge n-terminal first. as the polypeptide chain emerges from a nm wide pore is free to fold, interact with partners or misfold . in another example, proteins are unfolded at the proteasome by pulling from either the n or c terminus against a - nm wide pore, applying a tension on the residues surrounding the terminus of the protein . under this conditions, proteins may behave differently than when unfolded/refolded with temperature or urea. this may have important implications, as protein folding and unfolding in vivo is related to both function and disease. we noticed that vectorial folding is inherently linked to nanometer size pores. making use of nanopore technology we developed a method to monitor protein unfolding during membrane translocation at the single-molecule level . briefly, an oligonucleotide attached at either end of a protein threads a single protein nanopore inserted in a lipid membrane. in response to an applied membrane potential, the oligonucleotide pulls the protein through the pore and as it is forced to translocate it unfolds. analysing the ionic current we obtain the unfolding pathway and information on the polypeptide sequence. this methodology has shown that proteins unfold with different kinetics when pulled from one terminus or the other . remarkably, it is also possible to say whether the protein has been phosphorylated or not, and where . we have recently advanced our model system to study protein folding after translocation at the singlemolecule level . a single-protein molecule was translocated through a pore and forced to translocate back at predetermined times. we measured the stability of the refolded state at different times and we obtained the vectorial folding pathway of the protein. further, we observed that the protein was capable of co-translocational folding and that this premature folding contributed to the complete translocation of the protein. our results show that nanopore technology applied to proteins can be used to describe the vectorial folding and unfolding of proteins, providing insight to how these processes may work in vivo. further, single-molecule protein sequencing is a possibility that could revolutionise our knowledge on biological processes. thermodynamics studies of oligomeric proteins, which are the dominant protein natural form, have been often hampered because irreversible aggregation and/or slow reactions are common. there is not a single report on the reversible equilibrium thermal unfolding of proteins composed by (b/a) barrel subunits, albeit this "tim barrel" topology is one of the most abundant and versatile in nature. the eponymous tim barrel, triosephosphate isomerase (tim) is a ubiquitous glycolytic enzyme that catalyzes the isomerization of glyceraldehyde- -phosphate and dihydroxyacetone phosphate. the unfolding of several tims, mainly of eukaryotic organisms, has been extensively studied. regarding thermal unfolding, eighteen tims, mainly from eukaryotes, as diverse as amoebozoa, euglenozoa, ascomycota and chordata, have been studied. even though a full thermodynamic characterization has been hampered by irreversible aggregation and/or the presence of hysteresis in all of them, the activation parameters that describe the kinetic control of five eukaryotic tims have been reported. we characterized the structure, catalytic properties, association state and temperature-induced unfolding of the eponymous tim barrel, triosephosphate isomerase (tim), belonging to five species representative of different bacterial taxa: deinococcus radiodurans (drtim), nostoc punctiforme (nptim), gemmata obscuriglobus (gotim), clostridium perfringens (cptim) and streptomyces coelicolor (sctim). irreversibility and kinetic control were observed in the thermal unfolding of nptim and gotim, while for drtim, sctim and cptim, the thermal unfolding was found to follow a two-state equilibrium reversible process, a behavior not observed previously for others tims. shifts in the global stability curves of these three proteins are related to organismal temperature range of optimal growth and modulated by variations in maximum stability temperature and in the enthalpy change at that temperature. reversibility appears to correlate with low isoelectric point, the absence of residual structure in the unfolded state, small cavity volume in the native state structure, low conformational stability and a low melting temperature. furthermore, the strong coupling between dimer dissociation and monomer unfolding may reduce the possibility of aggregation and favor reversibility. it appears that there is a delicate balance between several contributions whose concerted interplay is necessary to achieve thermal reversibility in oligomeric enzymes. furthermore, the finding that the three reversible proteins come from organisms from different phyla suggests that unfolding reversibility may be more common than what is currently known supported by a critical step in the late phase of human immunodeficiency virus type (hiv- ) infection is targeting of the virally encoded gag proteins to the plasma membrane (pm) for assembly. prior to assembly, the hiv- gag polyprotein adopts a compact "folded over" conformation and exists in the monomeric or low-order oligomeric states. whereas it is established that the nucleocapsid domain of gag specifically recognizes motifs in the viral rna genome for packaging, there is compelling evidence that the myristoylated matrix (ma) domain also binds to cellular rna to prevent premature gag targeting to intracellular membranes. upon transport of gag to the pm, the interaction of ma with rna is exchanged for an interaction of ma with pm components. this molecular switch induces an extended conformation of gag, leading to formation of high-order gag oligomers on the pm. because gag is anchored and therefore captured by its interaction with the available phospholipids, the intracellular targeting of gag is likely to be determined by the relative strength of its interaction with the dominant lipids composing each membrane subcompartment. the key to understanding this essential molecular switch is elucidating at the molecular level the interaction of ma with specific pm components. for over two decades, biochemical, in vivo, in vitro and genetic studies have focused on factors that modulate binding of retroviral gag proteins to membranes but only recently the structural and molecular determinants of gag assembly have begun to emerge. in addition to the electrostatic interactions between a highly conserved basic region of ma and acidic phospholipids, it is now believed that the hydrophobicity of the membrane interior represented by the acyl chains and cholesterol also play important roles. we employ nmr methods to elucidate the molecular determinants of gag binding to the membrane. our structural studies revealed that phosphatidylinositol- , -bisphosphate (pi ( the production of functionally antibodies depends on the transition of immature b cells to mature plasma cells and is tightly linked to several "quality control" check points. during b cell development, the pre-b cell receptor (pre-bcr) is the first checkpoint which determines the viability and proliferation of the pre-b cell. the pre-bcr is composed of an immunoglobulin (ig) heavy chain molecule associated with an ig light chain-like molecule called the surrogate light chain (slc). the slc is composed by two proteins k and vpreb which possess a unique region at the n-or c-terminus, respectively. vpreb lacks a b-strand which is provided by the k protein allowing the non-covalent interaction essential for formation of the slc heterodimer. our understandings of the molecular mechanism of slc function and assembly are still at an early stage. in particular, we do not know how the slc associates and forms the pre-bcr for the selection of all heavy chains (hcs). our study focuses on dissecting the "fab fragment" of the pre-bcr to study the effect of the unexpected structural features of the slc to gain insight in hc selection. the analysis of the assembly of the slc revealed a significant difference between the single domains and the complexes in terms of stability and assembly. the folding behavior of the ch domain in the presence of the slc is key for the first quality control mechanism in the endoplasmic reticulum (er) prior to surface expression. our results show that the slc interacts with ch domain in a similar manner to the cl domain. thus, the folding of the naturally disordered ch domain upon interaction with the slc releases the hc retention in the er by bip. taken together, our study provides new insights into the folding and assembly of the "fab fragment" of the pre-bcr and paves the way for a detailed mechanistic understanding of hcs selection by the unique slc. though the - (sfgailss) region of human islet amyloid polypeptide (hiapp) has long been known to be crucial for amyloid fiber formation, lack of b-ordering of this region in structures of the final fiber as determined by both nma and x-ray has been puzzling. new evidence now suggests that the fgail region forms ordered b structures only in early intermediates. we present new dir studies on the fgail region of hiapp, with uniformly c o labeled amides, along with spectral and kinetic modelling. evolution of the peak frequency and d lineshape of the labeled region clearly present a transition from random coil to a stable b sheet, a conclusion which is substantiated by simulation of the d ir spectra. as determined from kinetic modeling, the fgail b-sheet creates a free energy barrier that is the cause of the lag phase during aggregation. these findings help to rationalize a broad range of previous fragment and mutation studies as well as provide a mechanism for fiber formation that has self-consistent kinetics and structures. the temperature dependence of protein stability in living cells studies addressing the consequence of crowding that exist in the interior of cells have reached an interesting stage. experimental data so far, predominantly from, small to medium sized proteins are indicating that, in general, natively folded proteins including, intrinsically disordered, gain structure and stability under conditions mimicking cell interior. however, on the other hand, a few studies on small proteins indicate destabilization of the native state. in very few instances, crowding resulted in compaction and aggregation of the unfolded and partially folded states. experimental data on the consequences of cell-like crowding situation on relatively large proteins with complex folding free energy landscape are absent. alpha subunit of tryptophan synthase, a kda tim barrel protein, provides a unique opportunity to address the consequence of crowding on the structure and stability of the native state and also on a partially folded state stable equilibrium intermediate populated in its (un)folding reactions. in the presence of increasing amounts the most commonly used crowding agent, ficoll- , a non-monotonous increase in the far uv-cd is observed for the native state. a steady increase up to mg/ml ficoll followed by a decrease in far-uv cd region is observed, indicating loss of structure at increased concentrations of the crowding agent. h- n hsqc nmr and fluorescence (fl) spectra confirm the of loss of structure at higher concentrations of ficoll- . loss of native base line in the urea induced unfolding reaction monitored by cd and fl clearly confirms the destabilization of the native state. similar to the structural changes observed for the native state, for the equilibrium intermediate state maximally populated at m urea also, non-monotonous changes in the far uv cd and fluorescence spectra are observed. the highly populated equilibrium intermediate shows an initial steady increase in the far uv cd signal followed by a sudden decrease. our results suggest that the structure of both native and partially folded states may be affected under crowding conditions. alpha- antitrypsin (aat) is a -kda serine protein inhibitor (serpin), which acts as an inhibitor of neutrophil elastase within the lungs. during inhibition, the protein undergoes a dramatic conformational change in which its exposed reactive centre loop (rcl) is cleaved and inserts into the central a-sheet as an extra beta-strand. this highly dynamic protein is also susceptible to mutations, resulting in misfolding and the accumulation of ordered polymers as intracellular inclusions within the endoplasmic reticulum of hepatocytes, where aat is synthesized. despite much knowledge of the folding and misfolding properties of aat as an isolated protein, very little is understood of how aat acquires its structure during biosynthesis. like all proteins, the biosynthesis of aat takes place on the ribosome, and protein folding occurs in a co-translational manner as the nascent polypeptide chain emerges from the ribosome's exit tunnel. this study aims to develop the biochemical and nmr structural strategies to characterize the co-translational folding characteristics of aat as it is being synthesized on the ribosome. for these studies, we have designed a series of secm-stalled ribosome nascent chain complexes (rnc) of aat of different lengths, which mimics the "snapshots" of the protein synthesis, capturing the folding process of the nascent chain during its emergence from the ribosome. using this library, we have recently developed a strategy to produce large quantities of the rncs both in vitro and in vivo within e. coli, a prerequisite for detailed biochemical and structural studies. using the aat-rncs, we are developing a suite of biochemical strategies to probe the capacity for aat nascent chains to adopt native structure on the ribosome. we have combined protease inhibition assays, western blot and native-page analysis to demonstrate that aat can fold while bound to the ribosome. in addition, we have employed a cysteine-based modification "pegylation" assay to probe lowresolution structural information of aat-rnc and this will guide our structural studies by nmr spectroscopy to provide a detailed understanding of aat folding on the ribosome at high resolution. thermodynamic properties of proteins vary with the environmental solvent condition (temperature, ions, ph, denaturants, etc.). although the effect of each environmental factor on proteins has been well studied, the complex effect of more than two environmental factors was not studied thoroughly. in this study, we investigate the simultaneous effect of urea denaturation (disruption of non-covalent bonds in proteins) and acid denaturation (titration of protein residues) on the nature of the folding transition for cyu protein. we performed the molecular dynamics simulations of bbl (pdb code: cyu) protein in various urea concentration at k. we calculated ph-dependent free energy landscape using the extended munoz-eaton model and described the phase diagram for the folding transition of bbl at various ph value and urea concentration. we mapped out the phase diagram of the folding transition of cyu, which clarifies the condition with which it undergoes the cooperative folding transition or the barrierless folding transition. biophysical analysis of partially folded states of myoglobin in presence of , , -trifluoroethanol paurnima talele , nand kishore the protein folding process involves one or more distinct populated intermediates. one such partially folded structure of particular importance observed during protein folding pathway is molten globule state. the properties of a molten globule state are intermediate between those of native and unfolded protein molecules. the importance of studying equilibrium molten globule is in its greater stability and flexible structure which has been shown to bind a variety of substrates and play a definite role in certain human diseases via aggregation, misfolding or some other mechanism. a protein must assume a stable and precisely ordered conformation to perform its biological function properly. the stability of a protein under specific conditions depends on its interactions with the solvent environment. therefore it is essential to understand protein folding intermediates, protein solvent interactions and protein stabilization. we have made attempts to thoroughly investigate the formation of stable molten globule state of the protein induced by alcohol using combination of calorimetric and spectroscopic techniques. the presentation will cover the topic on biophysical studies on partially folded states of myoglobin in presence of , , -trifluoroethanol. the thermal denaturation of myoglobin was studied in the presence of , , -trifluoroethanol (tfe) at various ph values using differential scanning calorimetry and uv-visible spectroscopy. the most obvious effect of tfe was lowering of the transition temperature with increasing concentration of tfe up to . mol•dm- , beyond which no thermal transitions were observed. the conformation of the protein was analyzed by a combination of fluorescence and circular dichroism measurements. at ph . and . , partially folded states of myoglobin were confirmed by cd spectroscopy. quantitative binding of ans to the tfe induced molten globule state of myoglobin was studied by using isothermal titration calorimetry (itc). the results enable quantitative estimation of the binding strength of ans with the molten globule state of myoglobin along with the enthalpic and entropic contributions to the binding process. the results also suggest occurrence of common structural features of the molten globule states of proteins offering two types of binding sites to ans molecules which has been widely used as a fluorescence probe to characterize partially folded states of proteins. modules. each cbr comprises a b-hairpin core followed by a short linker sequence. choline molecules are bound between two consecutive repeats through hydrophobic and cation-p interactions with aromatic side chains. apart from its biotechnological applications as an affinity tag for protein immobilization and purification, clyta is useful as a model for understanding the folding and stability of repeat proteins. in this sense, we proposed to get minimal peptides encompassing the sequence of a single cbr or even only its b-hairpin core able to maintain the native fold and the ability to bind choline. to that end, we first proceeded to analyze the peptide comprising the third b-hairpin core, denoted as clyt . based on cd and nmr data we demonstrate that the peptide clyt conserves its native bhairpin structure in aqueous solution, but forms a stable, amphipathic a-helix in detergent micelles and as well as in small lipid vesicles [ ] . considering the great differences in the distribution of hydrophobic and polar side chains shown by clyt b-hairpin and a-helix, we propose that amphipathic structures are stabilized in micelles or lipid vesicles. this "dual" behavior is the only up-to-now reported case of a micelle-induced conformational transition between two ordered peptide structures. to check whether other cbr repeats also undertake b-hairpin to a-helix transition in the presence of micelles, so that it represents a general tendency ascribed to all pneumococcal choline-binding modules, we will show new experimental evidences based on cd and nmr structural studies on peptides derived from the bhairpin cores of other clyta repeats, as well as in modified clyt peptides. continuing our studies of the effect of like-charged residues on protein-folding mechanisms, in this work, we investigated, by means of nmr spectroscopy and molecular-dynamics simulations, two short fragments of the human pin ww domain [hpin ( - ); hpin ( - )] and one single point mutation system derived from hpin ( - ) in which the original charged residues were replaced with non-polar alanine residues. results, for both original peptide fragments of hpin demonstrate the presence of ensembles of structures with a tendency to form a b-chain reversal. understanding the biology of huntington's disease via the pathogenic huntingtin monomer huntington's disease (hd) is caused by an abnormal extension of the polyglutamine (polyq) region within exon of the protein huntingtin from typically glutamines to over . disease onset correlates with the huntingtin misfolding and causing the formation of aggregates, however recent studies have postulated that pathogenic huntingtin monomer may form compact structures that are responsible for neuronal toxicity in hd. we sought to examine the conformation of huntingtin monomers, how polyq sequence length affects monomer structure and which protein-binding partners in the cell may exert a gain-of-toxic mechanism in pathology. hydrogen-deuterium exchange mass spectrometry was used to measure the degree of structure in both non-pathogenic ( q) and pathogenic ( q) huntingtin, with results showing that both forms exchanged % of potential nh hydrogen bond donors within seconds (n ), with little to no further exchange over the following ten minutes. this result suggested that the pathogenic conformations are not stabilized by slow exchanging hydrogen bonds. binding partners to the monomer were assessed in neuro a cell culture by immunoprecipitation and quantitative ms/ ms proteomics approaches after depletion of aggregates by pelleting. proteins that more prevalently co-precipitated with pathogenic huntingtin included fused in sarcoma (fus), glycine-trna ligase (gars), peroxiredoxin (prdx ), phosphatidylethanolamine-binding protein (pebp /rkip), and histone subunit hist h a, all of which were significantly enriched by two-fold or greater. rna-seq analysis indicated that none of these proteins had altered expression levels, suggesting that the binding interactions are not due to changes in background abundance. overall we found that the conformational differences are subtle, yet are sufficient to generate several specific proteome interactions that offer clues to a toxic gain-of-function mechanism in pathology. work is ongoing to probe the more subtle changes in conformation and the importance of these interactors to mediating mechanisms of dysfunction. hereditary tyrosinemia type i is an autosomal recesive disorder caused by deficiency of fumarylacetoacetate hydrolase (fah) enzyme. deficiency of fah leads to cellular accumulation of toxic metabolites which include mainly, succinylacetone (sa), maleylacetoacetate (maa) and fumarylacetoacetate (faa) in many body tissues. fah is mainly expressed in hepatocytes and renal proximal tubular epithelium. therefore, liver and kidney are the two primary organs affected by this disorder, and development of hepatocellular carcinoma is the major symptom. missense mutations leads to a loss of enzymatic efficiency which, in a high number of mutations, correlates with loss of kinetic and thermodynamic stability of the enzyme. in our ongoing project, we are trying to elucidate the molecular basis of tyrosinemia by means of biophisical and structural characterization of fah wild type along with its mutations. this knowledge should help us design new therapies based on the identification of pharmacological chaperones that could restore the altered enzymatic stability of the enzyme. human fah wild type and selected mutants were synthesized and inserted in an expression vector for e. coli. the proteins were purified in a fplc and, their thermodynamic and kinetic stability investigated using circular dichroism. our preliminary results confirm the loss of termodinamic stability of different mutants and its variability compared to wild type protein. repulsion between net charges of subunits during ferritin assembly daisuke sato , hideaki ohtomo , atsushi kurobe , satsuki takebe , yoshiteru yamada , kazuo fujiwara , masamichi ikeguchi department of bioinformatics, graduate school of engineering, soka university, jasri/spring- the organisms have a lot of spherical shell-shaped supermolecules consisting of identical or distinct subunits (e.g., ferritin, virus capsid, lumazine synthase and encapsulin). such multimeric proteins spontaneously assemble into their native structures from the subunits to acquire the specific functions. however, the assembly mechanism of such supermolecules has not been understood in detail. hence, to investigate the assembly mechanism is biologically important. escherichia coli non-heme ferritin (ftn) consists of identical subunits, which are assembled into a spherical shell-shape with / / symmetry. ftn is able to store iron inside cavity. the subunit includes a-d helices forming -helix bundle, a long bc-loop between b and c-helices and a short e-helix at the c-terminal. ftn dissociates into dimers at acidic ph. the dimer was shown to maintain the native-like secondary and tertiary structures by circular dichroism spectra and small angle x-ray scattering (saxs). the acid-dissociated ftn is able to reassemble into the native structure when ph increases. to clarify ftn assembly mechanism, we performed the stopped-flow time-resolved saxs (tr-saxs) experiments. the saxs profiles could be acquired every ms after the initiation of reassembly. the initial velocity calculated from the forward scattering intensity increment was proportional to the square of the protein concentration, implying that the reaction is second-order. we propose the sequential bimolecular reaction, in which two dimers bind to form tetramer, then another dimer attaches to the tetramer to form a hexamer, and so on. the assembly rate depended on ph and ion strength, indicating that the electrostatic interaction plays an important role in the assembly reaction. the assembly rate decreased with increasing ph in the range from . to . and increased with increasing nacl concentration. this indicates that there are repulsive electrostatic interactions between assembly units and that they increases with increasing ph from . to . . a possible interaction is the repulsion between net charges of dimers since pi of ftn is expected to be . . to test this possibility, we made several mutants with different net charges. as mutational sites, we selected charged residues that are far from the subunit interface. selected sites were glu , glu , glu , glu and glu . we constructed the mutants with one, two, three or four glu -> gln substitutions of selected sites. the structures of those mutants were similar to that of wild-type ftn. if aforementioned hypothesis is correct, the assembly rate is expected to increase with increasing the number of substitution. the result agreed well with this expectation and strongly suggested that the electrostatic repulsion between dimers is an important factor determining the assembly rate of ftn. improved modeling of protein unfolding rates and pathways through solvation and modeling of beta-barrels benjamin walcott , , lu ıs garreta , christopher bystroff , , department of biology, rensselaer polytechnic institute, center for biotechnology and interdisciplinary studies, department of computer science, universdad del valle, department of computer science, rensselaer polytechnic intitute an understanding of the folding and unfolding pathways of proteins is integral to improving our ability to associate the structural impact of point mutations and disease etiology. information gained here can also be used for protein structure prediction and design. to model unfolding pathways in proteins we utilize a computational method called geofold. this approach uses recursive hierarchical partitioning of protein structure and finite elements simulation. geofold considers three types of partitioning operations: translational motion (break), single point revolute joints (pivot), and rotation around two points (hinge). from these operations, a directed acyclic graph (dag) is constructed where nodes correspond to the substructures created by these operations and the edges represent the operations. for each operation in the dag, its dissociation and reassociation rates are determined as a function of solventaccessible surface area, hydrogen bonds, voids, and conformational entropy. finite element simulations are carried out to simulate the kinetics of unfolding. this model accurately predicts changes in unfolding pathways due to disulfides in a four-protein case-study, but it fails to produce a realistic pathway for b-barrel proteins such as green fluorescent protein (gfp). to better model these barrel proteins, a new partitioning operation is introduced involving the breaking of all contacts between an adjacent set of b-strands, called a seam. in addition, to improve the accuracy of kinetic modeling, several updates have been made to the energy function, including an improved solvation model and a contact-orderbased estimation of the reassociation rates. the predicted unfolding rates and pathways using this improved geofold are compared with experimentally measured values in kineticdb for proteins with multi-state unfolding kinetics, point mutations, circular permutations, and engineered disulfides. the presence of multiple domains in a protein can result in the formation of partially folded intermediates, leading to increased aggregation propensity. this can be reduced by cooperative, all-or-nothing folding of the multi-domain protein. in good agreement with ensemble folding experiments, a coarsegrained structure-based model of e. coli adenylate kinase (ake) folds cooperatively. ake has three domains, nmp, lid and core. we examine the role of the interfaces between these domains in facilitating folding cooperativity in ake. mutants in which these interfaces are deleted exhibit similar folding cooperativities as wild-type ake. on closer inspection, we observe that unlike a typical multi-domain protein in which one domain is singly-linked to its adjacent domain, nmp and lid are inserted into core, i.e. they are both connected to core by two linkers each. we create circular permutants of ake in which the inserted domains are converted to singly-linked domains, and find that they fold less cooperatively than wild-type ake. domain insertion in wild-type ake facilitates folding cooperativity even when the inserted domains have lower stabilities. the n-and c-termini of nmp and lid are constrained upon the folding of core and this facilitates their folding. thus, nmp and lid which undergo large conformational changes during catalysis can be smaller with fewer stabilizing interactions. in addition, inter-domain interactions need not be optimized for folding, and can be tuned for substrate binding, conformational transition and catalysis. analysis of protein domains using structural bioinformatics suggests several examples of multi-domain proteins in which domain insertion is likely to facilitate folding cooperativity. tuning cooperativity on the free energy landscape of protein folding pooja malhotra , jayant udgaonkar national centre for biological sciences, tata institute of fundamental research the mechanism by which a protein explores the free energy landscape during a folding or unfolding reaction is poorly understood. determining whether these reactions are slowed down by a continuum of small ( kbt) free energy barriers or by a few large (> kbt) free energy barriers is a major challenge. in this study the free energy landscape accessible to a small protein monellin is characterized under native-like conditions using hydrogen exchange in conjunction with mass spectrometry. cooperative and noncooperative opening processes could be directly distinguished from the mass distributions obtained in the ex limit. under native conditions, where the native state is maximally stable, the unfolded state is transiently sampled in an entirely non-cooperative and gradual manner. under conditions which stabilize the unfolded state or destabilize the native state of the protein, the slowest structure opening event becomes cooperative. the present study provides an understanding of the relationship between stability and folding cooperativity. it suggests that the cooperative transitions observed in unfolding reactions maybe a consequence of the changes in the stabilities of the unfolded state and the transition state. it also provides rare experimental evidence for a gradual unfolding transition on a very slow timescale. role of electrostatic repulsion between unique arginine residues on the assembly of a trimeric autotransporter translocator domain eriko aoki , kazuo fujiwara , masamichi ikeguchi haemophilus influenzae adhesin (hia) belongs to the trimeric autotransporter family. the autotransporter consists of an n-terminal signal peptide, an internal passenger domain and a c-terminal translocator domain. the signal peptide directs to export across the inner membrane via the sec system and is cleaved, the passenger domain is a virulence factor, and the translocator domain (hiat) is embedded in the outer membrane. the crystal structure of hia translocator domain (hiat) has shown that hiat forms a transmembrane b-barrel of b-strands, four of which are provided from each subunit. the b-barrel has a pore that is traversed by three a-helices, one of which is provided from each subunit. the protein has a unique arginine residue at . arg side chains from three subunits protrude from the b-strand toward the center of the barrel and are close to each other. these residues seem to have an unfavorable electrostatic effect on the assembly and decrease the trimer stability. to investigate the role of this residue on the trimer assembly and stability of hiat, we replaced this arginine with the neutral amino acid, methionine (r m) or the positively charged residue, lysine (r k), and properties of these mutants were investigated. hiat and two mutants were dissociated by formic-acid treatment, and they were able to reassemble in the presence of the detergent. to measure the time course of trimer reassembly, amounts of reassembled trimer and monomer were quantified by sds-page at different assembly times. although the neutralized mutation increased the rate of reassembly, the final amount of reassembled trimer decreased, especially at higher protein concentration. these suggest that the neutralized mutation cause the incorrect oligomer formation. the far-uv cd spectrum of reassembled wt hiat was nearly identical with that of the native wt hiat. however, the spectrum of the reassembled r m mutant was more intense that of the native r m mutant, although the proportion of trimer was much lower than that of the wt hiat. this suggests that the incorrect oligomer has a secondary structure different from the wt hiat. r k mutant showed assembly properties similar to those of the wt hiat. therefore, the repulsion between positively charged residues seems to be important for preventing hiat from misassembly. similar proximity of arginine residues is observed for hiv capsid protein, carboxysome shell protein, lumazine synthase and so on. the electrostatic repulsion between arginine residues may be a general mechanism for protein assembly. department of veterinary pathobiology, kagoshima university, institute for food sciences, hirosaki university, faculty of fisheries, kagoshima university, department of veterinary histopathology, kagoshima university, veterinary clinical training center, kagoshima university, department of veterinary anatomy, kagoshima university, sakamoto kurozu inc., the united graduate school of agricultural sciences, kagoshima university kurozu is a traditional japanese rice vinegar. during fermentation and aging of the kurozu liquid in an earthenware jar over year, solid residue called kurozu moromi is produced. in the present study, we evaluated whether concentrated kurozu or kurozu moromi could ameliorate cognitive dysfunction in the senescence accelerated p mouse. senescence accelerated p mice were fed . % (w/w) concentrated kurozu or . % (w/w) kurozu moromi for or weeks. kurozu suppressed cognitive dysfunction and amyloid accumulation in the brain, while kurozu moromi showed a tendency to ameliorate cognitive dysfunction, but the effect was not significant. we hypothesize the effect is caused by the antioxidant effect of concentrated kurozu, however, the level of lipid peroxidation in the brain did not differ in senescence accelerated p mice. dna microarray analysis indicated that concentrated kurozu increased hspa a mrna expression, a protein that prevents protein misfolding and aggregation. the increase in hspa a expression by kurozu was confirmed using quantitative real-time pcr and immunoblotting methods. therefore, the suppression of amyloid accumulation by concentrated kurozu may be associated with hspa a induction. however, concentrated kurozu could not increase hspa a expression in mouse primary neurons, suggesting it may not directly affect neurons. young-ho lee although amyloid fibrils are associated with a number of pathologies, their conformational stability remains largely unclear. we herein investigated the thermal stability of various amyloid fibrils. a-synuclein fibrils, freshly prepared at c at neutral ph, cold-denatured to monomers at - c and heat-denatured at - c. meanwhile, the fibrils of b -microglobulin, alzheimer's ab - /ab - peptides, and insulin exhibited only heat denaturation, although they showed a decrease in conformational stability at low temperature in the presence of chemical denaturants. a comparison of structural parameters with positive enthalpy and heat capacity changes which showed opposite signs to protein folding suggested that the burial of charged residues in the fibril cores contributed to the cold denaturation of a-synuclein fibrils. reinforced electrostatic repulsion at low temperatures may promote cold denaturation, leading to a unique thermodynamic property of amyloid fibrils. we propose that although cold-denaturation is common to both native proteins and misfolded fibrillar states, the main-chain dominated amyloid structures may explain amyloid-specific cold denaturation due to the unfavorable burial of charged side-chains in fibril cores. key structural differences between tbtim and tctim revealed by thermal unfolding molecular dynamics simulations angel piñeiro , miguel costas , andrea guti errez-quezada dept of applied physics, university of santiago de compostela, lab. of biophys. chem., dept of physical chemistry, fac. of chemistry, unam the thermal unfolding pattern obtained by differential scanning calorimetry for trypanosoma cruzi and trypanosoma brucei triosephosphate isomerase (tcim and tctim) are significantly different although the crystal structure of both proteins is almost indistinguishable and the sequences are highly homogolous. in order to explain these differences at molecular level a set of molecular dynamics simulations were performed at different temperatures between and k. the obtained trajectories were analyzed in detail and the residues that showed to be key in the unfolding pathway of each species were identified. a set of residues that behave significantly different between both proteins were selected and proposed for mutations. the general aim is to identify the minimum amount of residue mutations that allow providing tbtim with the behaviour of tctim and vice versa. experimental complementary work is also being performed on the same protein. repositioning som as a potent inhibitor of transthyretin amyloidogenesis and its associated cellular toxicity salvador ventura , ricardo sant'anna , maria ros ario almeida , nat alia reixach , raul insa , adrian velazquez-campoy , david reverter , n uria reig universitat aut onoma de barcelona, instituto de biologia molecular e celular, icbas, the scripps research institute, som-biotech, universidad de zaragoza transthyretin (ttr) is a plasma homotetrameric protein implicated in fatal amyloidosis. ttr tetramer dissociation precedes pathological ttr aggregation. despite ttr stabilizers are promising drugs to treat ttr amyloidoses, none of them is approved by the food and drug administration (fda). repositioning existing drugs for new indications is becoming increasingly important in drug development. here, we repurposed som , an fda-approved molecule for neurodegenerative diseases, as a very potent ttr aggregation inhibitor. som binds specifically to ttr in human plasma, stabilizes the tetramer in vivo and inhibits ttr cytotoxicity. in contrast to most ttr stabilizers, it exhibits high affinity for both ttr thyroxine -binding sites. the crystal structure of som -bound ttr explains why this molecule is a better amyloid inhibitor than tafamidis, so far the only drug in the market to treat the ttr amyloidoses. overall, som , already in clinical trials, is a strong candidate for therapeutic intervention in these diseases. neurometals as modulators of protein aggregation in neurodegenerative diseases s onia s. leal , joana s. crist ovão , cl audio m. gomes protein misfolding and aggregation is a hallmark across neurodegenerative diseases such as alzheimer's disease and amyotrophic lateral sclerosis (als). since these diseases are mostly sporadic, the formation of protein amyloids in the nervous system depends of chemical and biological triggers within the neuronal environment, such as metal ions [ ] . in this communication i will overview the metallobiology of neuronal calcium, zinc and copper, which are key players in brain function and have altered homeostasis in most neurodegenerative conditions. our recent work will illustrate how this allows establishing molecular mechanisms in neurodegenerative diseases [ ] [ ] [ ] [ ] [ ] . in the pursuit of this goal, in the last years we have been investigating superoxide dismutase (sod ), a cu/zn metalloenzyme that aggregates in the fatal neurodegenerative disorder als, as a model. in sod -als cases, this ubiquitous protein selectively aggregates in motor neurons, implicating a local biochemical factor in the process: interestingly, zn and ca levels are upregulated in the spinal and brain stem motor neurons of als patients, and increased ca triggers multiple pathophysiological processes which include direct effects on the sod aggregation cascade [ , ] . recently we established that calcium ions promote sod aggregation into non-fibrillar amyloid, suggesting a link to toxic effects of calcium overload in als [ ] . we showed that under physiological conditions, ca induces conformational changes on sod that increase sod b-sheet content and decrease sod critical concentration and nucleation time during aggregation kinetics. we also observed that calcium diverts sod aggregation from fibrils towards amorphous aggregates. interestingly, the same heterogeneity of conformations is found in als-derived protein inclusions. we thus hypothesized that transient variations and dysregulation of cellular ca and zn levels contribute to the formation of sod aggregates in als patients [ , ] . in a follow up study we combined experimental and computational approaches to show that the most frequent ligands for ca are negatively-charged gatekeeper residues located in boundary positions with respect to segments highly prone to edge-to-edge aggregation. calcium interactions thus diminish gatekeeping roles by shielding repulsive interactions via stacking between aggregating b-sheets, partly blocking fibril formation and promoting amyloidogenic oligomers such as those found in als inclusions. interestingly, many fals mutations occur at these positions, disclosing how ca interactions recreate effects similar to those of genetic defects, a finding with relevance to understand sporadic als pathomechanisms [ ] . the amino acid proline is well-known by its disorder promoting and helix breaking properties. prolines can be accommodated within transmembrane (tm) alpha-helices and participate in important biological tasks like signal transduction, ligand binding and helix-helix packing. x-ray crystallography and nmr indicate that proline residues in membrane proteins induce distortions of the helix geometry to different extents ranging from small bends to severe kinks. however, such studies provide essentially a static snapshot of membrane-embedded helices. therefore, the link between proline dynamics and function is not completely understood. in this work we have used singlemolecule f€ orster resonance energy transfer (smfret) and fluorescence correlation spectroscopy (fcs) to probe the structure and dynamics of the tm domain of human glycophorin a (gpa), a widely used model membrane protein for oligomerization studies. a fluorescent dye pair has been attached to both ends of the membrane-spanning region of gpa, which allowed monitoring the average distance and distance fluctuations between the attachment points. site-specifically double-labeled gpa has been reconstituted into two membrane-mimetic systems: sds micelles and phospholipid bilayers assembled into nanodiscs. using proline-scanning mutagenesis we have systematically evaluated the impact of proline residues in different positions along the membrane normal on transmembrane helix length and lateral packing. furthermore, we have investigated the distance distribution in tm helices containing native prolines, namely the insulin receptor and the nesprin protein. our results shed light into the relation between proline dynamics and the folding and function of tm helices. thermodynamic contributions of specific mutations of l e protein in the rna: protein interface region measured by analytical ultracentrifugation and gel shift assay bashkim kokona , , sara kim , margaret patchin , britt benner , susan white in saccharomyces cerevisiae, ribosomal protein l e acts as an autoregulator by inhibiting the splicing of its pre-mrna and translation of its mrna. the l e protein-rna binding site has been previously studied, revealing a rna kink-turn motif, which is characterized by a sharp bend in the phosphodiester backbone due to unpaired nucleotides and internal tertiary interactions. l e structural flexibility at the rna-binding interface makes such interaction an excellent model to explore the energetics of rna protein binding. we made l e k a, f a, and f w mutants to quantify the thermodynamic contributions of such interactions to the protein-rna complex. we used analytical ultracentrifugation sedimentation equilibrium (se) and sedimentation velocity (sv) to investigate conformational changes and protein-rna binding free energy changes due to mutations. our computed changes of binding free energy based on the sedimentation equilibrium experiments were consistent with the gel shift assay results. in addition, sedimentation velocity experiments on the l e wild type indicate that protein-rna interaction is highly dynamic and involves conformational changes of the kink-turn rna induced by l e protein. our results provide new insights on understanding the binding between ribosomal proteins and their rna molecules counterpart, which can be used to complement the x-ray structure. role of a non-native a-helix in the folding of equine b-lactoglobulin takahiro okabe , toshiaki miyajima , kanako nakagawa , seiichi tsukamoto , kazuo fujiwara , masamichi ikeguchi equine b-lactoglobulin is a small globular protein ( residues). although elg adopts a predominantly b-sheet structure consisting of nine anti-parallel b-strands (a-i) and one major a-helix in the native state, it has been shown that a non-native a-helical intermediate accumulates during the burstphase of folding reaction from the unfolded state in the concentrated denaturant. to ask whether the non-native helix formation is important for acquiring the native b-sheet structure, we determined first where the non-native a-helix is formed. a stable analogue of the burst-phase folding intermediate was observed at acid ph (a state). the amide hydrogen exchange experiment and proline-scanning mutagenesis experiment have shown that the non-native a-helix is formed at the region corresponding to the h strand in the a state. to investigate the role of this non-native a-helix on refolding reaction of elg, we constructed several mutant proteins, which were designed to destabilize the nonnative a-helix in the folding intermediate without perturbation on the native structure. a mutant, a t, fulfilled this requirement, that is, a t showed a native structure similar to that of the wildtype protein, and largely reduced cd intensity in the a state. then, the refolding kinetics were investigated by the cd and fluorescence stopped-flow method. a t mutation resulted in reduction of the burst-phase cd intensity, which confirmed that the non-native a-helix is formed around the h strand region. subsequent to the burst-phase, four kinetic phases were observed for a t and the wildtype protein. importantly, the folding rate constants of the four kinetic phases were similar between both proteins. furthermore, interrupted refolding experiments demonstrated that the native state was formed in the two parallel pathways in the two slower phases of the four kinetic phases. the relative amplitudes of the two pathways were similar between a t and the wild-type protein. these results clearly showed that the formation of the non-native helix has little effect on the folding rates and pathways, and suggested that the non-native helix formation may not be a severe kinetic trap for protein folding reaction. impact of the chaperonin cct in a-synuclein(a t) amyloid fibrils assembly ahudrey leal_quintero , javier martinez-sabando , jose mar ıa valpuesta , begoña sot centro nacional de biotecnolog ıa (cnb/csic)., centro nacional de biotecnolog ıa (cnb/csic)., centro nacional de biotecnolog ıa (cnb/csic)., centro nacional de biotecnolog ıa (cnb/csic) and fundaci on imdea-nanociencia cct is a eukaryotic chaperonin that uses atp hydrolysis to encapsulate and fold nascent protein chains. moreover, it has recently been shown that cct is able to inhibit amyloid fibers assembly and toxicity of the polyq extended mutant of huntingtin, the protein responsible of huntington disease. although this opens the possibility of cct being also able to modulate other amyloidopathies, this has not addressed yet. the work presented here intends to determine the effect of cct in the amyloid fibers assembly of a-synuclein(a t), one of the mutants responsible of parkinson disease. it is demonstrated that cct is able to inhibit a-synuclein(a t) fibrillation in a nucleotide independent way, suggesting that this effect is based on binding rather than on active folding. furthermore, using deletion mutants and assaying the interaction of cct with monomers, soluble oligomers and fibres, it has been possible to unravel the mechanism of this inhibition: cct interferes with fibers assembly by interacting with a-synuclein(a t) nac domain once soluble oligomers are formed, thus blocking the reaction before the fibers start to grow. amyloid-like aggregation of nucleophosmin regions associated with acute myeloid leukemia mutations daniela marasco , concetta di natale , valentina punzo , domenico riccardi , pasqualina scognamiglio , roberta cascella , cristina cecchi , fabrizio chiti , marilisa leone , luigi vitagliano department of pharmacy, cirpeb: centro interuniversitario di ricerca sui pepti, section of biochemistry, department of biomedical experimental and clinical scie, institute of biostructures and bioimaging nucleophosmin (npm ) is a multifunctional protein involved in a variety of biological processes and implicated in the pathogenesis of several human malignancies. npm has been identified as the most frequently mutated gene in acute myeloid leukemia (aml) patients, accounting for approximately % of cases ( ). the most frequent human npm mutations lead to variants with altered c-terminal sequences of the c-terminal domain (ctd) that, in its wild form, folds as a three helix bundle. aml modifications lead to (a) an unfolding of the ctd in the mutated protein and (b) its accumulation in the cytoplasm due to the loss of nuclear localization sequences with mutations of trp (mut e) and also of trp (mut a) ( ) . to gain insights into the role of isolated fragments in npm activities we dissected the ctd in its helical fragments. here we describe the unexpected structural behavior of the fragments corresponding to the helices h and h in both wild-type and aml-mutated variants. h region shows a remarkable tendency to form amyloid-like assemblies while only the muta sequence of h region is endowed with and b-sheet structure, under physiological conditions, as shown by circular dichroism, thioflavin t and dynamic light scattering. the aggregates of h , are also toxic to neuroblastoma cells, as determined by using the mtt reduction and ca influx assays ( ) . furthermore the effects of the local context on the different tendencies to aggregate of h and h were investigated and appeared to influence for the aggregation propensity of the entire ctd. since in aml mutants the ctd is not properly folded, we hypothesize that the aggregation propensity of npm regions may be implicated in aml etiology. these findings have implications to elucidate the pathogenesis of aml caused by npm mutants and aggregation phenomena should be seriously considered in studies aimed at unveiling the molecular mechanisms of this pathology. we report a resume of our study regarding the effects of microwaves in the range - mhz on a typical protein, myglobin. previous literature have concerned the effects on living and in vitro organic systems induced by high frequencies electromagnetic fields. we have focused our attention on a typical protein, myoglobin, because proteins are the simplest organic systems that are fundamentals in organic functions of livings. myoglobin is a protein found mainly in muscle tissue of vertebrates, consisting of a single protein chain with amino acids and one heme group that stores oxygen in the muscle cells. the physiological importance of myoglobin is mainly related to its ability to bind molecular oxygen. in particular, we focused our attention on the secondary structure of this protein in order to highlight whether exposure to microwaves unfold the protein producing transitions from a-helix component to b-sheet features. to this aim fourier transform infrared (ftir) spectroscopy have been used. the importance of this study is related to previous literature which indicated that transition from a-helix to b-sheet structure in a protein can be responsible for aggregation mechanisms that can lead to neurotoxicity and neurodegenerative disorders that can be considered as the first step to some pathologies [ ] [ ] [ ] . the aggregates consist of fibers containing unfolded proteins with a prevalent b-sheet structure termed amyloid [ ] . in our studies myoglobin in deuterium oxide (d o) solution was exposed for h to mobile phone microwaves at and mhz at a power density of w/m . ftir spectra were recorded by a spectrometer vertex v from bruker optics, following the protocol accurately described in [ ] [ ] [ ] . ftir spectroscopy analysis evidenced an increase in intensity of b-sheet structures and a significant shift to lower frequencies of about . cm- of the amide i vibration after exposure [ , ] . these results led to conclude that mobile phone microwaves induce proteins unfolding and formation of aggregates [ , ] . membrane proteins play a vital role in many biological processes, and yet remain poorly understood as they are frequently unstable in vitro. the goal of this project is to investigate the insertion and folding of membrane proteins into lipid bilayers, using a cell free expression system. we have used both e.colibased cell extracts (s ), and commercial translation systems (purexpress) in combination with synthetic liposomes of defined lipid composition. these studies will aid understanding of cooperative folding, folding intermediates, and the effects of the lipid bilayer on folding and insertion. model e.coli proteins have been investigated, as they can offer important insights into other proteins, and thus facilitate the further study of more biologically relevant proteins. it has been found that the rhomboid protease glpg spontaneously inserts into liposomes without the aid of an insertase such as secyeg. this spontaneously inserted glpg is functional, and is able to cleave bodipy-labeled casein, yielding a fluorescent product. the major facilitator superfamily (mfs) transport proteins lacy, galp and glpt have also been found to insert spontaneously into liposomes. it has been shown that the lipid composition of the liposomes has an effect on the amount of protein inserted into the bilayer, with all proteins tested to date preferring liposomes containing at least mol% dopg. ongoing and future work will involve the use of rare codons to alter the rate of translation, to investigate the effect this has on the final folded structure of the protein. preliminary work is also currently being done into whether the two domains of the mfs family transporters fold cooperatively or independently, thus aiding understanding into the folding and stability of membrane transport proteins. frederic greco , audrey toinon , nadege moreno , marie claire nicola€ ı rabies remains an important worldwide health problem that causes a fatal encephalomyelitis [ ] . currently, rabies in humans is under control in europe and north america following the use of efficient vaccines for dogs and wild animals. however, it still kills more than , people every year mainly in africa and asia [ ] . human vaccination prevents infection with very high efficacy. the vaccine contains an inactivated rabv produced on vero cells. rabv is an enveloped, negative single stranded rna virus which encodes five proteins, namely the nucleoprotein (n), the phosphoprotein (p), the matrix protein (m), the glycoprotein (g), and the viral rna polymerase (l) [ ] . the viral envelope is covered by trimer spikes of g-glycoprotein which is the most significant surface antigen for generating virus-neutralizing antibodies. here we illustrate the use of dsc (differential scanning calorimetry) to identify structural domains or proteins involved in thermal transitions. the dsc thermogram for intact beta-propiolactone inactivated rabv samples in pbs buffer reveals two major thermal transitions with a tm respectively at c and c. we have initially focused our investigations on one of the major proteins encode in rabv, glycoprotein g [ ] . glycoprotein g contains disulfide bridges on the ectodomain [ ] , is sensitive to bromelain cleavage [ ] and shows reversible conformation changes at low ph [ ] . considering these characteristics, our results provide evidence on the identity of one thermal transition observed by dsc. keywords: rabies virus, differential scanning calorimetry, protein unfolding domain swapping of the dna-binding domain of human foxp is facilitated by its low folding stability exequiel medina, sandro l. valenzuela, crist obal c ordova, c esar a. ram ırez-sarmiento and jorge babul departamento de biolog ıa, facultad de ciencias, universidad de chile, santiago, chile protein folding and dimerization (or oligomerization) are biologically relevant processes when reaching the quaternary structure is required for function. proteins that form dimers by exchanging segments or domains of their tertiary structure with another subunit, the so-called domain swapping phenomenon, are examples where folding and dimerization are tightly concerted processes. previous studies on domain swapping proteins, such as p suc and diphtheria toxin, have shown that, in general, a high kinetic barrier separates monomers and domain swapped dimers, and that this barrier can be lowered by promoting protein unfolding and refolding at high protein concentrations, thus favoring the swapped oligomer. recent crystal structures of the dna-binding domain of several human forkhead box (fox) proteins have shown that the p subfamily of these transcription factors (foxp) can form swapped dimers. the human foxp proteins are interesting models of domain swapping, because mutations of the dna-binding domain of these proteins are linked to diverse inherited disorders in humans, such as ipex and language deficits, and some of these mutations are located in the hinge region that connects the exchanged segment with the rest of the protein. moreover, foxp and foxp have been described to reach monomer-dimer equilibrium in solution after hours of incubation, suggesting that a low kinetic barrier separates both species. using foxp as a model of domain swapping, we analyzed the temperature and protein concentration effects on the dimer dissociation, obtaining the free energy change and enthalpy of the process by van't hoff analysis (dh of . kcal•mol- , ds of . kcal•mol- •k- and dg at c of . kcal•mol- ). these results indicate that the monomer-monomer association is an example of an enthalpy-driven process. to understand how foxp domains swap without protein unfolding, we performed equilibrium unfolding experiments using gndhcl as denaturant, showing that the wild-type protein has a low stability (dgu kcal•mol- , cm . m at c), in contrast to other domain swapping proteins with high kinetic barriers. we further explore the domain swapping mechanism of foxp through biased targeted molecular dynamics simulations, showing that the exchange process can occur by specific local destabilization and unfolding of the hinge region and helix h . to further corroborate that the low stability of wild-type foxp facilitates its domain swapping, we engineered a monomeric version of foxp through a single-point mutation in the hinge region, which has been previously described in the literature, and used this protein to visualize the effect of monomer stability in the dimer formation. comparison of the folding stability of the monomeric mutant a p and wild-type foxp shows that ddgu (mutant-wild-type) is . kcal/mol, concluding that the ability of foxp to domain swap rapidly can be explained through its low monomer stability and local unfolding of the exchange region. funding: fondecyt and . determining the coupled interactions that stabilize the structural framework of the ß-propeller fold loretta au , david green , , department of statistics, the university of chicago, department of applied mathematics and statistics, stony brook university, graduate program in biochemistry and structural biology, stony brook university, laufer center of physical and quantitative biology, stony brook university b-propeller proteins are a highly evolved family of repeat proteins that are involved in several biological pathways, such as signal transduction, cell-cycle modulation and transcription regulation, through interactions with diverse binding partners, despite having a similar fold. as for all repeat protein families, there is a consistent pattern in secondary structure for each repetitive region, in addition to the entire family. typically, four to ten propeller blades (each containing four anti-parallel b-sheets) are arranged in a toroidal shape, thus providing a large binding surface for ligands or other proteins. about % of known proteins adopt this distinctive fold, and although the requirements for tertiary structure and protein function are fundamentally encoded in primary structure, this relationship is not fully understood, and addressing it could provide insight on why the b-propeller fold is common. many techniques in comparative sequence analysis can successfully identify amino-acid conservation between closely related proteins, but molecular interactions between amino acids are often neglected, and further experimentation is still needed to determine the reasons underlying conservation. to explore how primary structure can dictate fold and function, we devised a computational approach to perform large-scale mutagenesis, by adapting the dead-end elimination and a* search algorithms (dee/a*), and also leveraged the structural conservation of each repeating region to understand how sequence variation influences protein fitness, defined here as a combination of stabilizing and binding interactions. dee/a* can evaluate low-energy protein sequences and their corresponding three-dimensional structures, and we used the bsubunit of a g-protein heterotrimer (pdb: gp , gia b g ) as a model system to demonstrate: ( ) how the multiple roles of individual amino acids in protein fitness can be deconvolved, and ( ) how epistatic interactions between them can contribute to structural stability. in doing so, we were able to identify important patterns in sequence complementarity between repeating regions that cannot be found using sequencebased methods alone. these results suggest that computational approaches can be used to determine important protein interactions, and help elucidate the prevalence of b-propeller proteins in biology. temperature induced conformational changes of the villin headpiece miniprotein stanislaw oldziej , wioletta _ zmudzi nska , anna hałabis the c-terminal subdomain of the actin-binding protein villin called hp (villin headpiece) has been used as a model protein in a number of studies of protein folding kinetics and protein folding mechanism [ , ] . the hp is a residue miniprotein with an alpha-helix bundle three-dimensional fold. the goal of our work was to determine conformational ensemble of polypeptide chain of the investigated miniprotein at a wide range of temperatures to get detailed information about how protein structure is influenced by temperature. d nmr spectra of the title miniprotein were registered at , and k. the three-dimensional structure of the hp based on restraints derived from nmr spectra registered at k is almost identical with structure deposited in the pdb database in the record f k [ ] . at higher temperatures ( and k) the general shape of the protein remains unchanged, with well packed hydrophobic core. however, with temperature increase alpha-helices start to melt. at k structure of the protein remains compact and in general shape similar to structure observed at k, but none of the alpha-helices could be observed. results obtained for hp protein are in agreement with previous observation for the trp-cage miniprotein [ ] , that with temperature increase regular secondary structure elements melt first before the break-up of the hydrophobic core of the protein. biological membranes provide a selective and chemically sealed barrier for cells. transport of ions and small molecules across the membrane is mediated by transporter proteins and the breakdown of a cell's ability to produce functionally folded membrane transport proteins can lead to dysfunction and has been implicated in many diseases . however little is known about the processes that govern the misfolding of a-helical integral membrane proteins, taking into account that these proteins fold and maintain functional structures within membranes of various organelles. the neurotransmitter sodium symporter (nss) protein family is an example of a-helical transporter proteins. the nss family encompasses a wide range of prokaryotic and eukaryotic ion-coupled transporters that regulate the transport of neurotransmitter molecules whose dysfunction has been implicated in multiple diseases and disor-ders . we have investigated the folding processes of prokaryotic homologue of the nss family leut responsible for the transport of neurotransmitters and amino acids to the sodium electrochemical gradient. previously folding processes of membrane transporters have mainly been characterised within detergent micelles. however, detergent micelles are not an accurate depiction of the environment of the membrane bilayer, with this in mind we have also attempted to investigate folding processes within a bilayer pd- nmr investigation of ph-induced unfolding of b domain of an escherichia coli mannitol transporter ii mannitol in the bacterial phosphotransferase system kim gowoon , yu taekyung , suh jeongyong the bacterial phosphotransferase system (pts) mediates sugar phosphorylation and translocation across the cytoplasmic membrane. cytoplasmic b domain (iib mtl) of the mannitol transporter enzyme ii mannitol, a pts family protein, delivers a phosphoryl group from a domain to an incoming mannitol that is translocated across the membrane. iib mtl is comprised of a four-stranded ß-sheet and three helices, representing a characteristic rossmann fold. we found that the iib mtl of escherichia coli unfolded at a mildly acidic condition. we made iib mtl mutants to investigate the mechanism of the ph-induced unfolding using nmr spectroscopy. we monitored backbone amide groups and side chain imidazole groups of histidine residues using d hsqc nmr, and pointed out a potential histidine residue that might be responsible for the unfolding. histidine residues may be generally important to the folding stability in response to environmental ph changes. can site-directed mutagenesis shed light on the refolding pattern of human glucose -phosphate dehydrogenase (g pd)? nurriza ab latif , , paul engel conway institute, univerversity college dublin, faculty of biosciences and medical engineering, universiti teknologi malaysia human glucose -phosphate dehydrogenase (g pd) is the first enzyme involved in the pentose phosphate pathway (ppp). this oligomeric enzyme catalyses the reaction of glucose -phosphate to form phosphogluconolactone with concomitant reduction of nadp to nadph. in erythrocytes nadph is important mainly for protection against oxidative stress. in connection with its role as the sole source of nadph, g pd deficiency commonly causes haemolytic disease and is known as the most common human enzyme deficiency globally. protein folding problems and instability are believed to be the major defects in the deficient enzymes. in this study, we employed site directed mutagenesis with hope to give more information on the role of -sh groups in the refolding of human g pd. two mutants were created: ) one in which all cys residues were replaced by ser and ) one in which only c and c were retained. the refolding of recombinant human g pd has been studied primarily by measuring the enzyme activity after refolding. we also used a combination of intrinsic protein fluorescence, ans ( -anilino- -naphthalenesulphonic acid) binding and limited proteolysis to look at the conformational change during the refolding. the results showed that gdnhcl-denatured recombinant human g pd wild type could be refolded and reactivated by rapid dilution technique. even though, as recombinants in e. coli, the mutants were well expressed and active, they remained inactive after attempts were made to refold them in vitro. the methods we applied may have provided some insights on the refolding pattern of this oligomeric protein, albeit qualitatively rather than quantitatively. a single aromatic core mutation converts a designed 'primitive' protein from halophile to mesophile folding connie tenorio , liam longo , ozan s. kumru , c. russell middaugh , michael blaber department of biomedical sciences, florida state university, department of pharmaceutical chemistry, university of kansas experiments in prebiotic protein design suggest that the origin of folded proteins may have favored halophile conditions. these results are consistent with salt induced peptide formation which shows that polymerization of amino acids is also promoted by high salt concentrations. as a result of various origin of life studies, a consensus on which amino acids likely populated early earth has emerged. these residues were synthesized by abiotic chemical and physical processes from molecules present in the surrounding environment. the properties of the consensus set of common prebiotic amino acids (a,d,e,g,i,l,p,s,t,v) are compatible with known features of halophile proteins, meaning these proteins are only stable in the presence of high salt concentrations. the halophile environment, thus, has a number of compelling aspects with regard to the origin of structured polypeptides. consequently, a proposed key step in evolution was, movement out of the halophile regime into a mesophile one commensurate with biosynthesis of "phase " amino acids -including the aromatic and basic amino acids. we tested the effects of aromatic residue addition to the core of a "primitive" designed protein enriched for the prebiotic amino acids (a, d, e, g, i, l, p, s, t, v) that required halophilic conditions for folding. the subsequent results show that the inclusion of just a single aromatic residue was sufficient for movement to a mesophile folding environment. thus, the inclusion of aromatic residues into the codon table could have conferred key stability to early proteins enabling adaptive radiation outside of a halophile environment. contact prediction methods that rely on sequence information alone, such as evfold, can be used for de novo d structure prediction and identification of functionally important residues in proteins. large multiple sequence alignments of protein families consisting of evolutionarily related and plausibly isostructural members reveal co-variation patterns that can be used to identify interactions between pairs of amino acids. we use a global probability model to disambiguate direct and indirect correlations. specifically, we use a maximum entropy approach called pseudo-likelihood maximization (plm) to distinguish causation (residue interactions) from correlation (correlated mutations) and compute evolutionary couplings (ecs). the inferred set of residue interactions can then be interpreted as physical contacts and used in de novo d structure prediction. furthermore, the interactions that are inferred can help guide experiments that measure the phenotypic consequences of protein substitutions, making the method useful for functional studies. the present work can be divided into three areas: (i) methodological improvements related to alignment, folding procedure, structure refinement and ranking; (ii) folding of proteins of known structure for benchmarking and prediction of proteins of unknown structure; and (iii) focused exploration of specific cases of interest. developing shuffle as a platform for expression and engineering of antibodies na ke , alana ali-reynolds , bryce causey , berkmen berkmen shuffle is a genetically engineered e.coli strain that allows disulfide bond formationin its cytoplasm with high fidelity. many proteins containing disulfide bonds have been successfully expressed in shuffle. in this study, we expressed, purified and characterized full-length monoclonal antibody igg in shuffle. for the first time, a fulllength igg can be functionally expressed in the cytoplasm compartment of an e.coli strain. in order to improve the folding and assembly of igg, we have investigated the expression of igg in various formats and vectors; we have co-expressed chaperones and other helper proteins with igg. several-fold increase in the yield of fulllength igg was observed. we characterized the shuffle produced igg and found it comparable to hybridoma produced igg. optimization of fermentation conditions for a large-scale production is in progress. we aim to develop shuffle as an easy, fast, robust platform for antibody engineering, screening and expression. experimental and computational studies of the effects of highly concentrated solutes on proteins: insights into the causes and consequences of quinary protein structure and cytoplasmic organization most studies of protein structure and function focus on pure, diluted samples; however, real-world biochemistry and typical biotechnological applications of proteins take place in complex media with very high concentrations of solutes ( - g/l) of varied size and chemical nature. on one side, this has recently fostered the study of proteins in vivo, in cell, or at least in media mimicking the native conditions. on the other hand, physical chemistry has for a long time studied the general effects of crowded and viscous conditions on proteins, looking mainly at coarse traits like diffusion and stability. but the general effects on traits relevant at atomic/residue resolutions have been less studied, and one fundamental issue remains unsolved: to what extent are proteins forced into interactions with highly concentrated solutes, and with what direct consequences? i will present here our ongoing efforts to dissect the fine effects of high solute concentrations and macromolecular crowding on proteins, based on nmr experiments and md simulations, two complementary techniques of high spatial and temporal resolutions. our results show that smaller solutes are prone to extensive interactions with proteins when at high concentrations while large solutes act chiefly through excluded-volume effects. overall, we observe location-specific perturbations of a protein's surface, its internal dynamics and internal dielectrics, and its hydration, all very dependently on the solute's size and chemical nature. our results support the growing notion that proteins should be studied in native-like media, adding that not only macromolecular crowders but also small molecules should be considered in these studies. last, the fact that high-concentration conditions affect far more than a protein's diffusion rate and stability suggests critical consequences of quinary protein structure and cytoplasmic organization on the regulation of proteins within cellular biochemistry. aldona jeli nska , anna lewandrowska , robert hołyst we developed an analytical technique for the study of interactions of ligands (e.g. cefaclor, etodolac, sulindac) with most abundant blood protein (e.g. bovine serum albumin) using the flow injection method. the experiments were conducted at high flow rates ( cm/s) in a long (> m), thin ( mm) and coiled capillaries. the compound of interest ( ml) was injected into carrier phase, which moved by the poisseule laminar flow. at the detection point we measure the concentration distribution of the analyte. the width of the final profile of the analyte concentration is inversely proportional to the effective diffusion coefficient of the analyte. from the differences between the widths of the concentration distribution of free and bound ligand we can determine value of the association constant. carbohydrate binding modules (cbms), which are defined as contiguous amino acid sequences within a carbohydrate-active enzyme, have been found in both hydrolytic and non-hydrolytic proteins and are classified into families, according to their primary structure similarity. the characterization of cbms by different methods has shown that these modules concentrate enzymes on the surface of polysaccharide substrates. it is thought that maintaining the enzyme in proximity with the substrate leads to more rapid degradation of the polysaccharide. therefore, the study of these kinds of modules or domains is relevant, since they are involved in multiple processes in organisms, like signaling, defense and metabolism; and some of them are involved in allergenic responses. in the present work we studied two different models: the first one is a cbm of the family from lactobacillus amylovorus (lacbm ) that binds starch these domains are present in a a-amylase like a repetitive tandem of five modules that are consecutive and do not present connectors. by means of itc and using a single recombinant lacbm domain we determined a ka . x m- for b-cyclodextrin and a ka . x m- for acyclodextrin. when the number of consecutive recombinant modules increased to three or five tandem modules, the ka values increased to m- ; however, these constants did not show an additive or a synergic effect. for these experiments we fitted the isotherms to different models and used different algorithms. additionally, we used circular dichroism in the uv-far region to determine if there existed conformational changes upon binding of the cyclodextrin molecules to the different tandem modules. we could only observe slight changes in a positive band centered around - nm, which has been explained in terms of p-p; interactions of the aromatic residues at the binding site. these cbms have been used as carriers for in vivo vaccine delivery and affinity tags. the second model is a hevein-like cbm of the family present in a chitinase-like protein from hevea brasiliensis (hbcbm ). hevein is a lectin from h. brasiliensis that shows a % identity with hbcbm . these cmbs are connected to the catalytic domain, in proteins such as chitinases, by a linker of approximately residues. in these experiments we used fluorescence techniques to determine the affinity constants for chitotriose. we previously reported a ka . x m- when using a hbcbm that has a met residue at the nterminal region. besides the aromatic residues at the binding site, the met residue also interacts with the ligand, as determined using crystallographic and docking techniques. the mutant hbcbm -r w that does not have the met residue showed a ka of . x m- with chitotriose, similar to the value reported for hevein using itc (ka . x m- ). interestingly, there exists an isoform of the hbcbm that has a connector between one cbm and a half cbm ( . xhbcbm ). this protein has a ka of . x m- with the same ligand. initiating vesicle formation at the golgi complex: auto-regulation and protein interactions govern the arf-gefs gea and gea margaret gustafson , j. chris fromme molecular decision-makers play critical roles in the effort to maintain efficient and accurate cellular functions. in the case of vesicular traffic at the golgi complex, the decision to initiate vesicle formation is made by a set of guanine nucleotide exchange factors (gefs) that activate the small gtpase arf , which is the master controller for the recruitment of cargos and coat proteins. saccharomyces cerevisiae possess three golgi arf-gefs, gea , gea , and sec , which work at distinct sub-compartments of the golgi to activate arf only when and where appropriate. in the case of sec at the trans-golgi network (tgn), this requires a positive feedback loop in which active arf relieves autoinhibition of sec , as well as recruitment to the golgi membrane and catalytic stimulation by signaling rab gtpases. we know far less about the decisionmaking process for gea and gea , which are responsible for retrograde traffic within the golgi and to the endoplasmic reticulum. i have found that both gea and gea can bind membranes weakly in vitro, an ability which is counteracted by their c-terminal hds domains. in addition, i have discovered membrane recruitment in vitro is aided by the rab gtpase ypt . however, these interactions cannot fully explain the distinct localization patterns of gea , gea , and sec , as all three have been shown to be recruited by ypt , which is found throughout the golgi. my work has revealed that in addition to the well-established distinct localization from sec , gea also occupies different golgi compartments from gea , so specific signals must exist which help the gefs decide where to go. my current efforts focus on understanding the roles of the other domains of gea and gea , identifying the signals which send them to different parts of the golgi, and unraveling the different roles they play in vesicle trafficking pathways. sequence variation in archaea through diversity-generating retroelements sumit handa , blair g paul , kharissa l shaw , david l valentine , partho ghosh department of chemistry and biochemistry, university of california san diego, marine science institute, university of california protein diversification is an essential tool for the survival and evolution for various species. diversitygenerating retroelements (dgr) in bacteria is known to generate massive variation in dna through an error prone reverse transcriptase and retrohoming, which leads to variation in protein sequence. recent discovery of dgrs in intraterrestrial archaeal systems have opened an opportunity to study this massive sequence variation in third domain of life (paul bg, et al. nat. comm.) here, we present the first crystal structure of variable protein from archaea with ligand-binding pocket is surface exposed. also, it has conserved c-type lectin (clec) fold, as shown by previous work on variable proteins, major tropism determinant (mtd) and treponema variable protein a (tvpa) which bind ligands through the clec fold. despite weak sequence identities ( - %) among these variable proteins, clec fold was found to be conserved. this variable ligand-binding site for archaea variable proteins can potentially generate variants. protein synthesis is a dynamic process mediated by a variety of proteins and enzymes. recent studies have shown that hydroxylation is a key post-translational modification involved in translation termination. in particular, the fe(ii)-and -oxoglutarate-dependent oxygenase, jumonji domaincontaining (jmjd ), regulates translation termination via the carbon hydroxylation of an invariant lysine residue, k , of the eukaryotic release factor, erf . in eukaryotes, translation termination is mediated by a release factor complex that includes erf . erf is comprised of three domains, and it is responsible for recognizing stop codons in mrna transcripts before triggering polypeptide release from the ribosome. the lysine residue hydroxylated by jmjd falls within the n-terminal domain and more specifically within the highly conserved niks motif. this motif has been identified by cross-linking and mutagenesis studies to play an essential role in stop codon recognition. while hydroxylation of k by jmjd has been found to increase translational termination efficiency, the exact molecular mechanism by which hydroxylation influences termination remains unclear. this work aims to understand how hydroxylation of erf affects translation termination by exploring the effect of hydroxylation on the structure, dynamics, stability, and binding of the n-terminal domain of erf (erf -n) using mass spectrometry, protein nmr spectroscopy, circular dichroism and differential scanning fluorimetry. in our efforts to understand the effect of hydroxylation, an additional jmjd -catalyzed modification, characterized by a da mass shift on k , was identified in vitro. the effect of this modification on erf was similarly explored. our findings suggest that hydroxylation has no effect on the in-solution nmr structure of erf -n, which experiences chemical shift changes localized to the target lysine residue. correspondingly, there are no significant differences in secondary structure content between wild type and hydroxylated erf -n. hydroxylation was also found to have no effect on protein stability or dynamics. interestingly however, the da modification appears to cause more significant chemical shift changes dispersed beyond the niks motif. this suggests a more global effect on the in-solution nmr structure despite the little differences observed in protein dynamics and secondary structure content. the da modification was also found to have a destabilizing effect on erf -n. neither hydroxylated nor da modified erf -n exhibited differences in rrna binding. while hydroxylation of erf was found to have little effect on protein structure, dynamics, stability, or binding, the da modification has marked effects on protein structure and stability. such differences suggest that this modification has the potential to play an important role in translation. functional and structural analysis of a gh ß-n-acetylglucosaminidase from the marine bacterium vibrio harveyi piyanat meekrathok , arthur t. porfetye , marco b€ urger , ingrid r. vetter , wipa suginta biochemistry-electrochemistry research unit, suranaree university of technology, max planck institute of molecular physiology vibrio harveyi b-n-acetylglucosaminidase (so-called vhglcnacase) is a new member of the gh glycoside hydrolase family responsible for the complete degradation of chitin fragments, with nacetylglucosamine (glcnac) monomers as the final products. however, the d structure of glcnacase is still unknown. in this study, crystal structure and function of glcnacase were investigated based on protein crystallography. size-exclusion chromatography and the native-page were employed to verify the protein state of glcnacase in a native form and the acidic active-site residues were mutated using sitedirected mutagenesis method. the effects of mutations on the binding and hydrolytic activities were studied by enzyme kinetics. to provide a structural basis of glcnacase, the wild-type enzyme was crystalized at k using a solution containing . m sodium acetate ph . and . m sodium malonate and recorded x-ray data. the wild-type enzyme was crystallized within days in the monoclinic crystal form, belonging to space group p , with unit-cell parameters a . , b . , c . Å. the crystal structures of v. harveyi glcnacase were solved and refined to highest resolution of . Å. structural investigation revealed that glcnacase comprises three distinct domains, designated as the n-terminal carbohydrate-binding domain, the a b topology domain and the tim-barrel catalytic domain. the substrate binding groove of glcnacase is a small pocket, which is suitable to accommodate a shortchain chitooligosaccharide. kinetic analysis revealed that a group of the adjacent d -h -e showed a significantly decreased activity as compared with the wild-type enzyme, and these residues might be important for enzyme catalysis. silencing the molecular timekeeper in human cancer alicia michael , stacy harvey , patrick sammons , amanda anderson , hema kopalle , alison banham , carrie partch university of california -santa cruz, university of oxford the circadian clock coordinates temporal control of physiology by regulating the expression of at least % of the genome on a daily basis. disruption of circadian rhythms through environmental stimuli (e.g. light at night) or genetic means can lead to the onset of diseases such as: diabetes, cardiovascular disease, premature aging and cancer. - the circadian clock orchestrates global changes in transcriptional regulation via the bhlh-pas transcription factor clock:bmal . pathways driven by other bhlh-pas transcription factors have a homologous repressor that modulates activity on a tissue-specific basis, but none have been identified for clock:bmal . we discovered that the cancer/testis antigen pasd fulfills this role to suppress circadian rhythms. pasd is evolutionarily related to clock and interacts with the clock:bmal complex to repress transcriptional activation. furthermore, deletion of one region, highly conserved with clock exon , alleviates repression by pasd to suggest that it utilizes molecular mimicry to interfere with clock:bmal function. structural and biochemical studies of the direct interaction of pasd with the clock:bmal complex using recombinant protein expression and biophysical techniques are currently underway. as a cancer/testis antigen, expression of pasd is natively restricted to gametogenic tissues but can be upregulated in somatic tissues as a consequence of oncogenic transformation. reducing pasd in human cancer cells significantly increases the amplitude of transcriptional oscillations to generate more robust circadian rhythms. our work suggests that mechanisms to suppress circadian cycling can be hard-wired in a tissue-specific manner and our data show that they can be co-opted in cancer cells to attenuate clock function. the scaffolding protein iqgap participates in various cellular functions such as cell-cell adhesion, cell polarization and migration, neuronal motility, and tumor cell invasion by binding to target proteins, including rac and cdc , two members of the rho family. to better understand the molecular basis of these interactions, we utilized in this study a novel time-resolved fluorescence spectroscopy to determine individual rate constants for iqgap interaction with fourteen different rho proteins. the results indicated that iqgap binds among rho proteins selectively to rac-and cdc -like proteins only in a gtp-dependent manner. moreover, the interaction of rho proteins with the c-terminal half of iqgap (grd-c), shorter fragment contains grd-gbd, only the grd and also grd-gbd with single and double phosphomimetic mutations s e and s d was performed. obtained results showed that, when both grd and gbd are existing, fluorescence changes is detected but for grd alone or in the case of s d or s e/s d no change was observed, suggesting that gbd and specifically, cysteine is critical for this interaction. furthermore, fluorescence polarization results showed that the grd-c interact with cdc and rac but not with rhoa, and interestingly the grd domain showed similar behavior, but with to folds lower affinity as compared with the grd-c. consistent with this, a gdp-bound form of cdc showed interaction with both grd and the grd-c in quiet comparable affinities. at last, competition experiments utilizing interacting partners of rac , e.g. tiam , p rhogap, plexin-b , p phox, pak and rhogdia, along with structural analysis, revealed two negative charged areas on the surface of rho-and rnd-like proteins, which might explain their inaccessible interaction with iqgap . the overlapping binding site of cdc and rac on the surface of iqgap together with the kinetic details of the selective interaction of iqgap with rac-and cdc -like proteins suggests that these interactions are most likely mediated via the same mechanism. ing dimerizes through its n-terminal domain, with a symmetric antiparallel coiled-coil structure , making it a bivalent reader of the h k me mark. ing is highly homologous with ing , but forms part of a different histone acetyl transferase complex . here, we show that ing is also a dimer and thus a bivalent reader of the h k me mark. however, the crystal structure of the n-terminal domain of ing shows an asymmetric dimer, different from the homologous ing domain. our nmr data (backbone assignment and paramagnetic relaxation effects) and saxs data indicate that the structure of the n-terminal domain of ing in solution is similar to ing , suggesting that the crystal structure of ing is likely a crystallization artifact. three point mutations in the n-terminal domain of ing have been described in oral squamous cell carcinoma: q r, i v, and c r . we have found that the n-terminal domains of the three mutants are dimeric coiled-coils but with different stability, as measured by thermal denaturation. while the q r mutant is as stable as the wild type, the i v and c r mutants are strongly destabilized, suggesting a role in cancer development at least for these two mutants. efforts so far, to combat alzheimer's disease (ad) have focused predominantly on inhibiting the activity of enzyme(s) that are responsible for the production of the main causative beta amyloid forming peptide. however, the inherent complexity associated with the network of pathways leading to the progress of the disease may involve additional targets for designing effective therapies. recent experimental findings have identified abelson's tyrosine kinase (c-abl), a non-receptor kinase involved in a variety of cellular functions as a new target for ad. in the present study we employed energy optimized multiple pharmacophore modeling strategy from multiple c-abl structures bound with ligands in the inactive atp binding conformation. virtual screening followed by docking of molecules from chembridge_cns database, and maybridge databases resulted in the identification of best scoring molecules. based on docking score and selectivity assessment and druggability parameters, four out of the molecules are predicted to show increased specificity for c-abl in comparison to closely related kinases. given the implied role of c-abl not only in ad but in parkinson's disease, the identified compounds may serve as leads to be developed as effective neurotherapeutics. rafael palomino , glenn millhauser , pietro sanna university of california santa cruz, the scripps research institute the central melanocortin system is recognized as a key regulator of energy balance and appetite. the hypothalamic melanocortin receptor, mc r, is a g-protein coupled receptor that is antagonized by the peptide ligand, agouti-related peptide (agrp), leading to increased feeding and weight gain. while much research has gone into how this ligand exerts its effects at the receptor, less is known regarding nonmelanocortin components of the pathway. syndecan- , a heparan sulfate proteoglycan, has previously been implicated in potentiating agrp antagonism, however details of this interaction are unclear. this work aims to investigate the role of syndecans at both a molecular level and in vivo. we hypothesize that agrp binds the glycosaminoglycan (gag) components of syndecans, and that this interaction increases the local concentration of the peptide near mc r. furthermore, we have previously shown that designed positive charge mutations to agrp lead to increased in vivo efficacy that is independent of mc r activity, and we hypothesize that this is due to greater affinity for the negatively charged gags. using isothermal titration calorimetry we have shown tight binding between agrp and heparan sulfate, the major gag component of syndecan- , and this affinity is strengthened by additional peptide positive charge. through nmr, we see that both positively charged and polar residues are necessary for binding various heparan sulfate polymers. these data implicate a specific region of agrp that is not required for mc r binding as being necessary in its role as a heparan sulfate binding protein. expanding on these findings, we are now using a syndecan knockout mouse line to explore the mechanism of differential feeding in our designed mutants. preliminary results indicate a reduction in weight gain in knockouts compared to their wildtype littermates post peptide administration. collectively, these data show that the physiologically relevant form of agrp, previously considered unable to interact with syndecans, is indeed a heparan sulfate binding protein. furthermore, our designed mutants have differential affinities for gags, with increased affinity correlating to increased feeding potency. finally, as the mc r pathway is thought to be a viable target for wasting disorders such as cachexia, we are interested in leveraging this data to improve the potency and stability of our designed agrp mutants. taken together, this work aims to develop new insights and probe the therapeutic potential of a critical metabolic pathway. evidence of a proteolytic phenomenon in the starch binding domain of the a-amylase from lactobacillus amylovorus zaira esmeralda s anchez cuapio , alejandra hern andez santoyo , sergio s anchez esquivel , romina rodr ıguez sanoja instituto de investigaciones biom edicas, universidad nacional aut onoma de m exico, instituto de qu ımica, universidad nacional aut onoma de m exico a-amylases are glycoside-hydrolases that catalyze the hydrolysis of internal a- , glycosidic bonds in starch and glycogen generating smaller oligosaccharides ( ). these multidomain proteins contain a catalytic barrel (b/a) and, in some cases, one or more non-catalytic domains whose function is generally described as carbohydrate binding module (cbm) and particularly as starch-binding domains (sbd). the sbd can bind granular starch increasing the local concentration of substrate at the active site of the enzyme and may also disrupt the structure of the starch surface ( ) . the a-amylase from lactobacillus amylovorus has a structure that consists of a catalytic domain (cd) and an unusual carboxy-terminal starch-binding domain with identical cbms (belonging to family ) in tandem ( ). each repeat acts as an independent fixing module with an additive or synergic effect between the units ( ). when we stored pure sbd from l. amylovorus we found multiple forms of low molecular weight with a constant pattern, which does not correspond to random degradation. interestingly, when the protein is stored at ph close to and edta is added, such proteolysis appears to decrease. so far, there is little information about the proteolytic process of amylases and the nature of it. here we show that divalent ions induce a proteolytic cleavage of the sbd, raising the possibility of an autoproteolytic activity. acknowledgments: this work is supported by grants papiit in - and conacyt . s anchez cuapio z is supported by a personal grant from consejo nacional de ciencia y tecnolog ıa, m exico. unnatural amino acid and related methods provided a special mechanism to implement site-specific spectroscopy active probe incorporation in a specific membrane protein in cells. the site specific incorporation resulted in a single signal during acquisition, resulting in unambiguous signal assignment. the protein specific labeling makes it possible for in situ membrane protein analysis using nmr or fluorescence detection. the f containing unnatural amino acid incorporation has been applied for dynamic studies of transporters in native lipid membrane, and the phosphorylation quantification analysis for tyrosine kinase in native lipid membrane with the aid of lipodisc. the fluorescent unnatural amino acid incorporation enabled the site-specific channel responses analysis upon ligand binding in a single cell. heather wiebe , noham weinberg , department of chemistry, simon fraser university, department of chemistry, university of the fraser valley the mechanism by which conformational changes, particularly folding and unfolding, occur in proteins and other biopolymers has been widely discussed in the literature. molecular dynamics (md) simulations of protein folding present a formidable challenge since these conformational changes occur on a time scale much longer than what can be afforded at the current level of computational technology. transition state (ts) theory offers a more economic description of kinetic properties of a reaction system by relating them to the properties of the ts, or for flexible systems, the ts ensemble (tse). the application of ts theory to protein folding is limited by ambiguity in the definition of the tse, although the experimentally observed first-order kinetics for folding of small single-domain proteins lends itself to interpretation by this theory. the pressure dependences of the folding rate constant can be used to obtain activation energies and activation volumes, which are rationalized as the properties of the folding tse. the large amount of activation volume data in the literature has gone largely uninterpreted at the quantitative level. we propose to utilize this data in conjunction with md-calculated volumetric properties to identify the tse for protein folding. the effect of pressure on reaction rates is expressed in terms of logarithmic pressure derivatives, known as activation volumes. according to ts theory, activation volumes can be identified as the difference in volume between the ts and reactant species: activation volumes dv ‡ have been experimentally determined for the folding of several proteins. the concept of activation volume can be extended to that of a volume profile, dv(y), which describes how the volume of a system changes along reaction coordinate y. if the position y ‡ of the ts along the reaction coordinate is unknown, it can be found by locating dv ‡ on the volume profile: such volume profiles can be built using our recently developed md-based displacement volume method.* using this method, volumes of single molecules can be calculated by taking the difference between the volume of pure solvent and solvent containing the desired solute. this method takes into account the strength and type of solvent-solute interactions as well as the geometrical configuration of the solute. in this work, we present the successful application of this method to several conformationally flexible systems. structure of the p paf/pcna complex and implications for clamp sliding on the dna during replication and repair to the canonical pip-box binding groove on the pcna front face. in contrast to other pcna interacting proteins, however, p paf also contacts the inside of, and passes through, the pcna ring. the mostly disordered p paf chain termini thus emerge at opposite faces of the ring, but remain protected from degradation by the s core proteasome. we also unveil a novel dna binding activity of p paf, both free and bound to pcna, which is mainly mediated by its conserved histone-like n-terminal tail. molecular modeling shows that a ternary complex with a duplex dna inside the pcna ring is energetically feasible and our electron micrographs show increased density inside the ring. we propose that p paf acts as a flexible drag that regulates pcna sliding along the dna, and may facilitate the switch from replicative to translesion synthesis polymerase binding upon dna damage. acknowledgements: this work has been mainly sponsored by mineco grant ctq - and juan de la cierva- contract to alfredo de biasio. metabolic syndrome (mets) is one of the leading causes of the death worldwide; however, exact pathophysiological mechanisms of mets remain largely unknown. growing evidence suggests that the increased availability of glucocorticoids at the tissue level play an important in mets development. one of the major determinants of glucocorticoid local action seems to be the enzyme b-hydroxysteroid dehydrogenase ( b-hsd ). this enzyme is a well-known member of the short-chain dehydrogenase/reductase (sdr) superfamily. it is an important carbonyl reducing enzyme that, besides its role fine-tuning of glucocorticoids actions, is involved in the biotransformation of drugs and in the development of lung cancer through metabolism of the tobacco specific carcinogen nnk. the phylogenetically closest relative of b-hsd is dhrs enzyme from the same superfamily. unlike b-hsd , dhrs is poorly characterized however it can be supposed at least partially overlapping function to b-hsd . moreover its possible association with similar pathological conditions in human as b-hsd has already been indicated by several studies. the aim of this study is the basic biochemical characterization of dhrs . the enzyme is a member of cluster of "classical" sdr; such members are considered to be retinoid and steroid metabolizing enzymes, so characterization the enzyme was based on this assumption. dhrs was prepared in recombinant form in the sf cell line. it was proved that this enzymes is an integral membrane-bound enzyme localized in the endoplasmic reticulum with luminal orientation, similarly to b-hsd . known substrates of b-hsd and related enzymes were tested also as substrates of dhrs . it was proved that dhrs is nadph-dependent reductase with important substrates as steroid hormones cortisone and androstene- , -dione, all-transretinal and also xenobiotics as , -naphtoquinone or carcinogen nnk at least in vitro. for better understanding of the catalytic function of dhrs its structural model was prepared and it is used also for the identification of additional substrates by ligand virtual screening. dhrs enzyme is expressed in several human tissues as adrenals, liver, prostate, small intestine and kidney. these brand new initial results point to the possible involvement of dhrs in important cellular processes that deserve further investigation. these results will lay the foundation for an understanding of dhrs role in human physiology resp. pathophysiology. this project was supported by grant agency of charles university ( /c/ ) and unce / ). structure-based functional identification of helicobacter pylori hp as a nuclease with both dna nicking and rnase activities bong-jin lee , ki-young lee hp is a conserved, uncharacterized protein from helicobacter pylori. here, we determined the solution structure of hp using three-dimensional nuclear magnetic resonance (nmr) spectroscopy, revealing that this protein is structurally most similar to a small muts-related (smr) domain that exhibits nicking endonuclease activity. we also demonstrated for the first time that hp is a nicking endonuclease and a purine-specific ribonuclease through gel electrophoresis and fluorescence spectroscopy. the nuclease activities for dna and rna were maximally increased by mn( ) and mg( ) ions, respectively, and decreased by cu ( ) ions. using nmr chemical shift perturbations, the metal and nucleotide binding sites of hp were determined to be spatially divided but close to each other. the lysine residues (lys , lys and lys ) are clustered and form the nucleotide binding site. moreover, site-directed mutagenesis was used to define the catalytic active site of hp , revealing that this site contains two acidic residues, asp and glu , in the metal binding site. the nucleotide binding and active sites are not conserved in the structural homologues of hp . this study will contribute to improving our understanding of the structure and functionality of a wide spectrum of nucleases. high-fidelity recombinant protein production in a silkworm bioreactor sungjo park , in-wook hwang , tatsuya kato , enoch park , andre terzic center for regenerative medicine, mayo clinic, laboratory of biotechnology, shizuoka university the domesticated silkworm, bombyx mori, is an attractive host naturally equipped with a proficient posttranslational modification machinery adequate to fulfill stringent demands of authentic recombinant protein production. silkworm-based protein expression has originally relied on a prototype baculovirus vector system that employs silkworm as a bioreactor in place of more traditional cell lines. recent development of the silkworm trophic b. mori nucleopolyhedrovirus (bmnpv) bacmid launches a second generation of silkworm-based protein production technology. introducing the recombinant bacmid dna into silkworms expedites heterologous protein expression by eliminating prior virus construction and amplification steps. salient examples of heterologous eukaryotic proteins produced in silkworms are acetyl-coa carboxylase , malonyl-coa decarboxylase, spot /mig heterodimer and a , -sialyltransferase with consistent high levels of protein expression. thus, equipped with a fail-safe post-translational modification machinery, eukaryotic proteins are readily bioengineered using a silkworm-based protein expression platform. studies exploring potential applications of synthetic antifreeze proteins in the frozen food industry ho zee (charles) kong , conrad perera , ivanhoe leung , nazimah hamid , viji sarojini school of chemical sciences, the university of auckland., school of applied sciences, auckland university of technology in nature, certain species of plants, insects and fish produce a group of antifreeze glycoproteins and polypeptides which enable them to survive the freezing temperatures of their natural habitat. these naturally occurring antifreeze proteins (afps) were first discovered in polar fishes such as antarctic notothenioids and winter flounder. these afps have the ability to bind to ice crystals and restrict their size and morphology; decrease the freezing point of water and inhibit the ice recrystallization processes. ice crystal formation is of primary concern to the frozen food industry, as ice crystal formation during freezing can be disruptive to and cause damage to the cellular structures in food. the unique properties of afps can be developed into a potential solution to minimize freeze-thaw damage to frozen food. a number of tailor made synthetic analogues based on the naturally occurring afps were successfully designed and synthesized. antifreeze activity studies of the afps were carried out using the clifton nanoliter osmometer attached with a microscope. the afps exhibited thermal hysteresis as well as modification of ice crystal morphology, confirming their antifreeze activity in vitro. the ability of these synthetic afps in preserving the texture and structure of frozen food was evaluated using the techniques of scanning electron microscopy. the afps showed great potential to preserve the cellular structures of frozen food samples during freeze-thaw process. additionally, secondary structure analysis of the afps was carried out using circular dichroism. this presentation will summarize our current results on the design, synthesis and anti-freeze activity analysis of the synthetic afps. invasive fungal infections remain a leading cause of death in immunocompromised patients. current antifungal agents have a host of issues including limited efficacy, host toxicity and an alarming increase in resistance. current research in our laboratories is focused on targeting the calcineurin signaling pathway that has been shown to be required for fungal pathogenesis. calcineurin is a highly conserved serine-threonine-specific ca -calmodulin-activated phosphatase important in mediating fungal pathogenesis and stress responses. it is a key regulator of a signal transduction network required for survival of the most common pathogenic fungi in humans, making it an ideal target for fungal drug development. calcineurin is a heterodimer of a catalytic (a) and regulatory (b) subunit. phosphatase activity requires association of the two subunits. calcineurin is also the target of the immunosuppressant fk , which functions as an inhibitor by first complexing with the peptidyl-prolyl cis-trans isomerase immunophilin, fkbp . the fkbp -fk complex subsequently binds to calcineurin in a groove between the a and b subunits and inhibits its activity. although fungal calcineurins are targeted by fk , it also targets mammalian calcineurin and is thus immunosuppressive in the host. in order to improve therapeutic efficacy, we have undertaken a unique effort that utilizes both structural biology and molecular mycology in an effort to overcome the fungal versus human specificity barrier. the nmr studies to be presented here have been focused on determining the resonance assignments and solution structures for the fkbp proteins from the pathogenic fungi candida albicans, candida glabrata and aspergillus fumigatus. notably, the x-ray crystallography structures of the wild-type candida albicans and aspergillus fumigatus fkbp proteins revealed an intriguing intermolecular interaction involving four residues in the 's loop including pro (in c. albicans) and pro (in a. fumigatus) which are stabilized in the cis conformation. these data suggest that the protein might use itself as an enzyme substrate. in efforts to establish if this interaction remains in a solution environment, we have determined the nmr structure and measured the t relaxation rates for the wild-type a. fumigatus fkbp protein and for the p g mutant variant that adopts a dramatically different orientation of the 's loop and does not form an intermolecular interaction in the crystal structure. the nmr chemical shift data indicate that, while the remainder of the protein structure remains unchanged, the 's loops in the two variants are indeed different. in addition, the t relaxation rates of the residues in this region are dramatically dissimilar in the two variants, but remain identical throughout the rest of the protein. we have also begun inhibitor binding studies of all of the fkbp proteins from each of the pathogens by titrating the fk inhibitor into native and mutant fkbp proteins in order to examine conformational changes associated in the protein upon complex formation. using this approach we plan to determine the relative kd values for binding of each inhibitor to the fkbp protein from each pathogen for comparison of binding proclivities. lupin (lupinus angustifolius l.) b-conglutin proteins: structure functional features, catalytic mechanism modeling and cross-allergenicity identification using protein threading and molecular docking methods lupin is an important pulse, which displays a wide range of benefits in agriculture, particularly these involved in possible plant pathogen suppression. furthermore, lupin seed proteins promote different positive health aspects, preventing cardiovascular disease, and reduction of glucose and cholesterol blood levels. "sweet lupine" seeds seem to be promising as a source of innovative food ingredients due to averaged protein content similar to soybean and an adequate composition of essential amino acids. thus, lupin seeds may be important source of proteins for human and animal consumption. however, and as drawback feature, the number of allergic people to lupin seed proteins is rising, becoming a serious and a growing problem in the western world, because of the rapid introduction of lupin seeds as new ingredients in traditional and novel foods. the goals of this study are the characterization the structure-functional properties of lupinus angustifolius l or narrow leafed lupin (nll) b-conglutin proteins, with a focus in its catalytic mechanism, and its molecular cross-allergenicity with other legumes, i.e. peanut, by extensive analysis using different computer-aided molecular approaches covering (i) physicochemical properties and functional-regulatory motifs, (ii) sequence analysis, -d and d structural (threading) modeling comparative study and molecular docking, (iii) conservational and evolutionary analysis, (iv) catalytic mechanism modeling, and (v) sequence, structure-docking based b-cell epitopes prediction, while t-cell epitopes were predicted by inhibitory concentration and binding score methods. b-conglutins (vicilin-like or s proteins) are seed proteins typically found in reserve tissues (endosperm and cotyledon). they belong to the cupin superfamily of proteins, containing a globular domain constituted by a conserved b-barrel. two barrels were found in all b-conglutin protein isoforms and an additional mobile n-terminal arm constituted bye a-helices. molecular modeling analysis has shown that one of this barrel contain a semi-conserved metal binding motive (hyx. . .r), typically found in oxalate oxidase (oxox) enzymes. interestingly, our results revealed considerable structural differences between b-conglutin isoforms, particularly affecting -d elements (loops and coils), and numerous micro-heterogeneities are present in fundamental residues directly involved in epitopes variability, which might be a major contributor to the observed differences in cross-reactivity among legumes. we also identified multiple forms of b-conglutins polypeptides ranging from - kda, with ige-binding characteristics in atopic patients. thus, b-conglutins might be considered as major allergen in different species of lupin, including the "sweet lupin" group, since several of these polypeptides were recognized by human iges, having the potential to trigger an immune response leading to allergy symptoms. influenza virus is one of the most prevalent pathogens causing respiratory illness which often leads to serious post influenza complications such as pneumonia and myocarditis. some viruses, as the avian influenza h n , are especially dangerous and draw special attention of who. this highly pathogenic virus spreads quickly among domestic poultry and wild birds resulting in high mortality. what is more distressing, the h n virus may be transmitted to humans. because of antigenic drift it is impossible to deliver an effective vaccine against all subtypes of the h n virus. moreover, traditional egg-based production of influenza vaccines is time-and cost-consuming, what makes it inadequate in case of a pandemic. hence, we have developed an efficient production process of influenza vaccine based on a recombinant hemagglutinin antigen (rha). recombinant vaccines underlay strict regulations and quality requirements. the purpose of this work was to develop a battery of analytical methods that allow to evaluate key quality attributes of rha on each stage of production. at first, we have focused on rha structure as a crucial issue for its activity. the primary structure of rha was confirmed by peptide mapping and tof/tof fragmentation (hplc, maldi tof/tof). furthermore, ftir analysis was used to evaluate the secondary structure of the protein. the disulfide bonds, which stabilize the tertiary structure, were assigned by peptide mapping. additionally, free thiols were measured using ellman's reagent. moreover, we have employed rp-hplc, sec-mals and dls to explore oligomerization of rha. these techniques appeared to be useful not only to confirm existence of native oligomers, but also to find and discard misfolded fraction, aggregates and truncated forms. in addition, two analytical methods (rp-hplc and cge) were developed to assess the purity of rha as required by ich guidelines. we also have determined isoelectric point and heterogeneity of rha by cief. afterward, developed methods were applied in the stability studies that provide a valuable insight into a chemical degradation process and conformational changes of rha during storage. this work was supported by innovative economy operational program, grant no. wnd-poig. . . - - / - as a part of project "centre of medicinal product biotechnology. package of innovative biopharmaceuticals for human and animal therapy and prophylactics." muscle cell atrophy via hsp gene silencing was counteracted by celastrol-mediated hsp overexpression molecular chaperone heat shock proteins (hsp) are known to assist protein quality control under various stresses. although overexpression of hsp was found to promote muscle mass retention in an unloading state, it is unclear whether muscle atrophy is induced by suppression of hsp expression and is counteracted by active hsp overexpression. in this study, we pre-treated hsp sirna to rat l cells for the hsp gene-silencing, and determined myotube diameter, hsp expression and anabolic and catabolic signaling activities in the absence or presence of triterpene celastrol (cel), the hsp inducer. relative to a negative control (nc), muscle cell diameter was reduced by % in the sirna-treated group, increased . -fold in the cel-treated group and remained at the size of nc in the sirna cel group. hsp expression was decreased % by sirna whereas the level was increased -to -fold in the cel and sirna cel groups. expression of foxo and atrogin- was increased . -to . -fold by sirna, which was abolished by cel treatment. finally, phosphorylation of akt , s k and erk / was not affected by sirna, but was elevated -to -fold in the cel and sirna cel groups. these results suggest that hsp downregulation by hsp gene-silencing led to muscle cell atrophy principally via elevation of catabolic activities. such anti-atrophic effect was counteracted by cel-mediated hsp overexpression. the centers for disease control and prevention report that at least million people in the united states will become ill due to antibiotic resistant pathogens leading to , deaths each year. in order to circumvent these resistance mechanisms, it is essential to quantitatively understand how the function of the protein(s) involved relates directly to resistance. integral membrane efflux pumps are known determinants of single-drug and multi-drug resistance in a wide variety of pathogenic organisms. these transporters are proteins whose characterization typically requires reconstitution in an artificial membrane. subsequently, these important proteins are difficult to characterize by traditional in vitro studies. my project aims to determine the physicochemical parameters of the efflux pump tetb utilizing molecular biology and mathematical modeling. tetb is composed of transmembrane (tm) alpha-helices and is found within the inner membrane of gram-negative bacteria. this protein allows for the efflux of tetracycline (tet), doxycycline (dox), and minocycline (mcn) antibiotics from the cytoplasm into the periplasm. these tetracyclines are a bacteriostatic class of antibiotics that inhibit protein synthesis by binding to the s ribosomal, therefore, blocking the binding of aminoacyl-trna. for cells grown in tetracyclines, the efflux mechanism of tetb decreases the cytosolic antibiotic concentration allowing for the rate of protein translation to increase. i have inserted a tet(b) expression system into the chromosome of an escherichia coli lab strain and have determined its growth profile under various concentrations of tet, mcn, and dox using a high-throughput -well plate format. the growth rate profiles correlate with tetb pumping rates for each drug. tetb more readily pumps out tet compared with dox and mcn and we observe that cells expressing tetb can grow at higher tet concentrations compared with dox and mcn. the shapes of the growth rate profiles produced in the different drugs give insight into the physicochemical mechanism of tetb. we have built a preliminary mathematical model that can simulate these growth profiles and predict efflux pump physicochemical parameters. we are currently working on understanding how efflux expression effects bacterial growth by testing ribosome binding site (rbs) sequences of varying strengths in our tet(b) expression system. future work is geared toward modeling more complex efflux pumps such as the tripartite pumps which traverse both bacterial membranes and cause multi-drug resistance. collectively, this project aims to build an in vivo system which will allow for the characterization of a variety of efflux pumps without the arduous tasks of protein purification and subsequent reconstitution. ( ) identified a small transmembrane region of both kcne and kcne that are essential for their unique modulation of the kcnq channel. by swapping a triplet motif in the transmembrane region of kcne and kcne , we can flip the primary function of these two proteins. while the key for kcne and kcne 's unique modulating is believed to lie in this triplet motif, the mechanism and structural changes involved in this modulation is not fully understood. by using nmr spectroscopy, biochemical studies, and computational docking, we aim to look at the structural and conformational differences between kcne and the triple mutant kcne substituted with the three essential kcne residues. we have expressed and purified n-labled kcne triple-mutant in sufficient quantities for nmr studies in lmpg detergent micelles and other membrane mimetics, and we have collected d nmr spectra using a trosy-based pulse sequence. partial backbone assignments of kcne triple mutant have been determined by aligning and transfer assignments of the wt kcne previous determined in our lab. with the structure of kcne triple mutant determined, we aim to computationally dock the triple mutant into a model of the full-length kcnq channel in the open and closed state. lastly, we will compare the known structure of kcne docked to a model of kcnq to that of the kcne triple mutant to determine key interactions, significant structural and conformational changes, and how the triple motif region gives rise to its specific structural and functional differences. with this information, we can begin to understand the mechanism of the functional diversity of the kcne family on kcnq potassium channel. biochemical characterization of brassica napus diacylglycerol acyltransferase and its regulatory domain .a) expressed in saccharomyces cerevisiae. purified bnadgat in n-dodecyl-b-d-maltopyranoside (ddm) micelles behaves as dimers, which can associate further to form tetramers. the acyl donor preference of the major dimeric form with sn- , -diolein as acceptor follows the following order: a-linolenoyl-coa > oleoyl-coa palmitoyl-coa > linoleoyl-coa > stearoyl-coa. the first residues of bnac.dg-at .a corresponding to a soluble regulatory region was expressed in escherichia coli and purified. truncation of this soluble domain reveals that the dimeric interface is located within residues - , while the first residues allow formation of tetramers. this n-terminal region was implicated as an allosteric exosite for acyl-coas as revealed by previous lipidex- binding studies. in the current study, circular dichroism spectroscopy and isothermal titration calorimetry were used to probe the binding kinetics and thermodynamics. dgat appears to shift between two oligomerization states, a phenomenon that may be related to regulation of enzyme activity and mediated by the n-terminal domain. alteration of lysine and arginine content as a strategy to modify such an interaction was found to increase the activity of rdrp in vitro. further, deletion of c terminal amino acid residues also resulted in increase in the polymerase activity that was comparable to the full length rdrp-p complex. it was proposed that the conserved c terminal disordered domain of rdrp was responsible for interaction with p and modulation of the activity. in the present study, role of the c terminal disordered domain was further investigated by determining the oligomeric status of the complex and the c terminal deletion mutants of rdrp and also by quantitating the rdrp-p interaction using surface plasmon resonance. size exclusion chromatography revealed that rdrp eluted in the void volume of the column whereas a significant fraction of the rdrp-p complex eluted at a position corresponding to the size of the : complex of rdrp and p ( kda). activity measurements indicated that the heterodimeric complex was more active than the aggregate eluting in the void fraction. interestingly, the c terminal deletion mutants of rdrp (c del & c del rdrp) were also found to be less aggregated as compared to full length rdrp and some of the protein eluted at a position corresponding to the respective monomers. these monomers were also more active than the aggregate fractions. these results demonstrate that the increase in activity observed either upon interaction with p or deletion of the c terminal domain could be due to the change in the oligomeric state of rdrp. in order to further analyze the interaction of rdrp with p surface plasmon resonance was used. rdrp and its deletion mutants were immobilized on biacore sensor surface and p protein was used as an analyte. full length rdrp and c del rdrp were shown to interact with p with kd values of . and um respectively. however, c del and c del rdrp did not show any binding with p . these results suggest that the region - from the c terminus of rdrp is essential for the interaction with p . further, the c del rdrp was inactive although c del rdrp continued to be active suggesting that residues - from the c terminus are crucial for rdrp activity. further studies are in progress to identify the residues within these motifs that may be essential for the activity or interaction with p . aggregation of androgen receptor in spinal bulbar muscular atrophy is a multistep process spinal bulbar muscular atrophy (sbma) is a member of the polyglutamine (polyq) expansion diseases, like huntington disease, and it is caused by a genetic expansion of the polycag tract in exon of androgen receptor (ar) that codes for the polyq region. sbma is a late onset disease, which involves a progressive degeneration of the motor neurons and consequent muscular atrophy. there is still no treatment available for this disease. ar is a nuclear receptor that responds to testosterone and that regulates the expression of the masculine phenotype. it is composed of an intrinsically disordered nterminal domain (ntd) that bears the polyq tract, a dna binding domain and a ligand binding domain. aggregates of ar protein with an extended polyq are observed in the motor neurons of sbma patients. in vitro studies showed that aggregation of androgen receptor takes place only in presence of testosterone and that the cleavage of the protein by caspase is a crucial event for cytotoxicity. however, there is no clear knowledge of the mechanism of aggregation, for this protein. an increasing body of evidence supports the hypothesis that the aggregation of these proteins is controlled by regions flanking the polyq tract, by regulating the rate of aggregation depending on their secondary structure. we have applied nuclear magnetic resonance (nmr) and circular dichroism for generating information on the secondary structure of the n-terminal cleavage product of ar by caspase and we have studied its aggregation with a set of biophysical methods, like dynamic light scattering, an hplc sedimentation assay and transmission electron microscopy. we have found that the polyq tract of ar presents a high degree of helicity. we attribute this conformation to the n-terminal flanking region, characterized by high helicity and we have tested this hypothesis by performing mutations. we have also observed that the rate of the first step of oligomerization is not dependent on the number of glutamine repeats, but instead is due to self interactions of a region n-terminal to and far from the polyq. its progression to fibril is dependent to the number of glutamines in the tract. we have therefore identified two steps in the aggregation process of ar, where a motif far from the polyq at its n-terminal drives the early oligomerization, followed by the interaction of the polyq chains that stabilize it and determine the progression to fibrils. these findings shed a light for possible interventions on the ar oligomerization process, thus suggesting a different strategy to study the onset of the disease in sbma patients. destabilizing the transient helical conformation of islet amyloid polypeptide hastens peptide self-assembly and potentiates cytotoxicity carole anne de carufel , phuong trang nguyen , alexandre arnold , isabelle marcotte , steve bourgault amyloidogenic polypeptides can be divided into two different structural classes: those that are intrinsically disordered and those that show a well-defined structure in their monomeric soluble state. natively folded proteins, such as transthyretin, have to unfold (or misfold), at least partially, to form amyloids. in contrast, intrinsically disordered polypeptides, such as the islet amyloid polypeptides (iapp) and abeta peptide, need to undergo conformational rearrangements allowing the formation of locally ordered structure(s) to initiate the amyloidogenic process. studies have shown that iapp and abeta adopt an alphahelix conformation in the initial steps of amyloidogenesis. this intermediate is believed to be on-pathway to fibril formation, although this hypothesis is still the matter of debate. in this study, we designed human iapp (hiapp) derivatives in which alpha-helix destabilizing substitutions were incorporated into the putative helical segment of iapp to probe the initial structural event in amyloid formation. using trifluoroethanol titration, we observed by cd spectroscopy that strategic incorporation of d-amino acids at positions and leads to an iapp derivative (diapp) that cannot fold into a helix. in homogeneous solution, hiapp and diapp show similar kinetics of fibrillization, as measured by thioflavin t fluorescence. although their amyloid fibrils display different characteristics by afm, iapp and diapp are able to self-associate to form amyloids when mixed together and when seeded with one another. studies in heterogeneous environment, notably in presence of glycosaminoglycans and model membranes of dopc/dopg ( : ), showed a helical intermediate for hiapp while only a beta-sheet secondary structure was apparent for diapp. while the rate of amyloid fibril formation was increased for both peptides, diapp was drastically affected by these anionic biomolecules with an absence of lag phase. the incapacity of adopting a transient helical conformation accentuates cell toxicity, supported by the caspase / activation level and the increase in intracellular calcium level. overall, this study indicates that the helical intermediate is offpathway to iapp amyloid formation and offers novel mechanistic insights for the development of molecular identities modulating peptide self-assembly and iapp-induced cytotoxicity. for an organism to survive, its proteins must adopt complex conformations in a challenging environment where macromolecular crowding can derail even robust biological pathways. the situation is perilous: many diseases arise from improper folding of just a single protein. to cope, cells employ a repertoire of molecular chaperones and remodeling factors that usher unfolded proteins into active conformations, sequester them, or target them for degradation. yet, not all aggregated proteins are the result of mis-folding. yeast prions are self-templating protein-based mechanisms of inheritance that rely upon chaperones for their propagation. the best studied of these is the prion domain (nm) of sup , which forms an amyloid that can adopt several distinct conformations (strains) that produce distinct phenotypes. using genetic, biochemical, spectroscopic, and solid state nmr techniques, we investigated the structural and dynamic underpinnings of sup amyloids and found that prion strains differ in both their atomic structure as well as their dynamic motions. interestingly, these mobility differences correlate with differences in the interaction with molecular chaperones in vivo. limitations on the specificity and sensitivity of biophysical techniques typically restrict structural investigations to purified systems at concentrations that are orders of magnitude above endogenous levels. therefore, i developed an approach to apply a sensitivity-enhancement technique for nmr, dynamic nuclear polarization (dnp), to investigate interactions between sup and molecular chaperones at endogenous concentrations in their native environments. critically, i found that the cellular environment induced structural changes in a region of sup that is intrinsically disordered in purified samples but known genetically to influence prion propagation from one generation to the next. this approach enables structural and mechanistic investigation of proteins in biologically relevant contexts. genetic instability within regions encoding repetitive proteins as a driver of adaptation stephen fuchs more than ten percent of all eukaryotic proteins contain within them a region of repetitive amino acid sequence. these repetitive domains range from short stretches of a single amino acid to multiple copies of longer, heterogeneous amino acid sequences and generally show lack of defined structure. they play diverse roles in cells including acting as structural proteins, promoting cell-cell interactions, and mediating the assembly of molecular machines. tandem repeat proteins are known to be variable in length within cellular populations although the mechanisms dictating this variability have not been elucidated. here we describe work uncovering specific features within the coding sequences of repetitive proteins that contribute to tandem repeat instability in yeast. furthermore, we demonstrate that cells will expand and/or contract repetitive regions in order to adapt to environmental stresses and describe a role for dna repair proteins in this process. lastly, we demonstrate how these mechanisms are likely conserved in higher eukaryotes, including humans. this study uncovers the molecular basis for an important aspect of natural protein evolution and describes a novel mechanism for adaptation in response to environmental changes. a proline-tryptophan turn in the intrinsically disordered domain of ns a protein is essential for hepatitis hepatitis c virus (hcv) nonstructural protein a (ns a) and its interaction with the human chaperone cyclophilin a (cypa), a peptidyl-prolyl cis-trans isomerase (ppiase), are both targets for highly potent and promising antiviral drugs that are in late stage of clinical development [ , ] . despite its high interest in the development of drugs to counteract the worldwide hcv burden, ns a is still an enigmatic multifunctional protein poorly characterized at the molecular level. ns a is required for hcv rna replication and is involved in viral particles formation and regulation of host pathways. thus far, no enzymatic activity or precise molecular function has been ascribed to ns a that is composed of a highly structured domain (-d ), as well as two intrinsically disordered domains (-d ) and (-d ). ns a-d structure has been solved by x-ray crystallography and ns a-d and -d have been characterized by nmr spectroscopy. these two last domains do not adopt a stable d structure but rather exist as an ensemble of highly dynamic conformers. using nmr spectroscopy, hcv ns a-d has been shown to establish a direct interaction with the human cypa and to be a substrate for the enzymatic ppiase activity of cypa [ ] . the cypa interaction site in ns a-d is composed of nearly residues that correspond to the most conserved region of the domain, with proline residues being strictly conserved among all hcv genotypes. whereas ns a-d is mainly disordered, some of its nmr resonances, corresponding to residues in the cypa binding site, display unexpected h and n nmr chemical shifts for an intrinsically disordered domain. thus we have further characterized this region by nmr spectroscopy. a short structural motif in the disordered ns a-d has been identified and we solved its nmr structure. in a cellular assay, we showed that this structural motif, a minimal pro -trp turn, is essential for hcv rna replication. we demonstrated that this pro-trp (pw) turn is required for proper interaction with the host cypa and influenced its enzymatic ppiase activity on residue p of ns a-d . this work provides a molecular basis for further understanding of the function of the intrinsically disordered domain of hcv ns a protein. in addition, our work highlights how very small structural motifs present in intrinsically disordered proteins can exert a specific function. [ ] [ ] . this -residue peptide also shows toxicity towards mammalian cells but at higher concentrations, suggesting its possible usefulness as a treatment for trypanosomiasis. here we present the peptide's relative cytotoxicity for bloodstream and procyclic forms of t. brucei and for mammalian cells, the fate of the peptide in t. brucei using fluorescently-labelled bt- , and its three dimensional structure using nmr spectroscopy.minimum inhibitory assays confirmed the peptide's selective toxicity towards both bloodstream and procyclic forms of t. brucei, demonstrating its potential to serve as a starting point for a trypanocidal drug. fluorescence spectrophotometric experiments, carried out using fluorescein labelled bt- , show that the peptide is released from the external surface of the parasite into the suspending medium under de-energized conditions but retained in energized cells. heteronuclear and homonuclear biomolecular nmr experiments (tocsy, noesy, h- c-hsqc, h- n-hsqc, etc) folowed by structural calculations (chemical-shift based as well as simulated annealing techniques) in the free state indicate that this peptide is mostly unstructured in aqueous solution, suggesting that there is a major conformational change upon binding to t. brucei that is required for uptake. we suggest that the evolutionary pressure that selected for the intrinsically disordered structure of this peptide was the advantage it conferred upon the host to bind to many different surface structures throughout the microbiological world. physikalische biologie, heinrich heine university, structural biochemistry (ics- ), research centre j€ ulich, chemistry and biotechnology, swedish university of agricultural sciences (slu) the misfolding and amyloid formation of proteins featuring intrinsically disordered regions is a pathological hallmark of several neurodegenerative diseases, including alzheimer's disease and parkinson's disease. engineered binding proteins targeting amyloidogenic proteins aid in the elucidation of the aggregation mechanism and suggest therapeutic strategies. we have constructed phage display libraries enriched in binders to amyloidogenic intrinsically disordered proteins, using zab , a protein with high affinity for the amyloid-beta peptide, as a scaffold. binding proteins selected from these libraries are termed beta-wrapins (beta-wrap proteins). the beta-wrapins as and hi exhibit nanomolar affinity for monomeric alpha-synuclein or islet amyloid polypeptide, respectively. as and hi potently inhibit in vitro amyloid formation and toxicity at substoichiometric concentration ratios, indicating that they interfere with the nucleation and/or elongation of amyloid fibrils. the nmr structures of the betawrapin:target complexes reveal beta-hairpin motifs in alpha-synuclein and islet amyloid polypeptide which are stabilized by coupled folding and binding. in the case of alpha-synuclein, the beta-hairpin is formed in the sequence region - which contains the beta-strand segments b and b of amyloid fibril models and most disease-related mutations. we show by disulfide engineering, biophysical techniques, and cell viability assays that intramolecular tertiary interactions between the b and b segments of alpha-synuclein interfere with its aggregation, and moreover inhibit aggregation of amyloid-beta peptide and islet amyloid polypeptide. our results reveal a common preference of different amyloidogenic proteins for formation of beta-hairpin motifs and demonstrate a critical role of hairpin conformers in the control of amyloid formation. interaction profiling through proteomic peptide phage display cecilia blikstad , moon-hyeong seo , norman davey , roland arnold , sachdev s sidhu , philip m kim , ylva ivarsson department of chemistry -bmc, donnelly centre a considerable part of the human proteome is intrinsically disordered. the disordered regions are enriched in short motifs serving as docking sites for peptide binding domains. domain-motif interactions are crucial for the wiring of signaling pathways. these interactions are typically transient and difficult to capture through most conventional high-throughput methods. we therefore developed a novel approach for the large-scale profiling of domain-motifs interactions called proteomic peptide phage display (prop-pd) ( ). in prop-pd we combine bioinformatics, oligonucleotide arrays, peptide phage display and next-generation sequencing. this allows the interrogation of domain-motif interactions on a proteome-wide scale and the de novo motif discovery.in our pilot experiment we generated two distinct phage libraries, one displaying all human c-terminal sequences and one displaying c-termini of known virus proteins. we used the prop-pd libraries to identify interactions of human postsynaptic density /discs large/zonula occludens- (pdz) domains. we successfully identified novel pdz domain interactions of potential relevance to cellular signaling pathways and validated a subset of interactions with a high success rate. recently, we created a prop-pd library that displays peptides representing the disordered regions of the human proteome. we validate our disorderome library against a range of peptide binding domains, which provides novel insights into their binding preferences and suggest interactions of potential biological relevance as will be presented here. prop-pd can be used to uncover protein-protein interactions of potential biological relevance in high-throughput experiments and provides information that is complementary to other methods. prop-pd is scalable and can be developed to any target proteome of interest. phosducin is a kda phosphoprotein that regulates visual signal transduction by interacting with the gtbg; subunit of the retinal g-protein transducin. the function of pdc is regulated by phosphorylation at ser and ser in a process that involves the binding of phosphorylated pdc to the regulatory - - protein, but the molecular mechanism of the regulation by - - protein is still unknown. pdc was also suggested to be involved in transcriptional control, the regulation of transmission at the photoreceptorto-on-bipolar cell synapse, and the regulation of the sympathetic activity and blood pressure [ ] [ ] [ ] . here, the solution structure of pdc and its interaction with the - - protein were investigated using small angle x-ray scattering, circular dichroism, quenching of tryptophan fluorescence, analytical ultracentrifugation, hydrogen-deuterium exchange coupled to mass spectrometry and nuclear magnetic resonance. we show that the - - protein interacts with and sterically occludes both the n-and c-terminal gtbg binding interfaces of phosphorylated pdc, thus providing a mechanistic explanation for the - - depedent inhibition of pdc function. the - - protein dimer interacts with pdc using surfaces both inside and outside its central channel. the n-terminal domain of pdc, where both phosphorylation sites and the - - binding motifs are located, is intrinsically disordered protein which remains likely highly flexible when bound to - - indicating the fuzzy-like character of this complex. in addition, it has been speculated that the - - protein binding decreases the rate of pdc dephosphorylation after a light stimulus through its interaction with phosphorylated ser and ser , thus lengthening the time that pdc remains phosphorylated after a light exposure. pdc is dephosphorylated in vivo by protein phosphatases known to cause neurodegenerative disease in a polyglutamine-length dependent manner. despite intense study, the molecular basis of polyq toxicity in hd or any of the other diseases has only partially been elucidated and potential routes to therapeutic intervention are sparse. the use of genetically tractable model organisms to identify the cellular pathologies caused by mutant huntingtin expression is essential to our understanding of the disease pathology in humans. in eukaryotes, many of the protein folding homeostasis pathways are highly conserved and yeast cells expressing a glutamine-expanded fragment of huntingtin exon exhibit a polyq length-dependent toxicity that recapitulates many of the basic protein folding defects associated with polyq diseases in neurons. taking an unbiased approach, we screened an overexpression library of the entire yeast genome for suppressors and enhancers of polyq toxicity and identified seven proteins with prion-like, q-rich domains that are strong suppressors in yeast. intriguingly, the q-rich domains of these proteins, and several other q-rich domains, suppress toxicity when expressed in isolation. these suppressors are also efficacious in mammalian cells and, strikingly, one suppressor was independently shown to alleviate polyq-expanded ataxin- toxicity in a drosophila model. in yeast, the suppressors co-aggregated with an otherwise highly toxic glutamine expanded huntingtin exon protein (htt q), resulting in a non-toxic aggregate and eliminating populations of diffusible oligomeric species. using a transcriptional sensor for protein coaggregation, we determined that yeast and human proteins that normally co-aggregated with htt q did not co-aggregate with these hetero-aggregates. thus, these q-rich domains may suppress htt q toxicity by two complementary mechanisms: trapping potentially toxic oligomers in larger aggregates and by limiting the interactome of the larger htt q aggregates. structuring disorder: the case of the intrinsically disordered unique domain of c-src mariano maffei about two thirds of eukaryotic proteins contain large intrinsically disordered regions. they represent a change of paradigm from "structure-function" to "information-function" (uversky, ; babu et al., ). structured proteins are information rich, but the current challenge is to discover how information is stored in disordered protein. regulation of c-src activity, the first discovered oncoprotein, by its intrinsically disordered n-terminal region has been recently demonstrated (perez et al., ). functional studies have revealed that mutations in the ulbr cause strong phenotypes when introduced in fulllength c-src and expressed in xenopus laevis oocytes (perez et al., ) or in human sw colorectal cancer cells (unpublished). however, the connection with the classical regulatory mechanisms is still missing. c-src domain structure consists of four "src-homology" domains: sh , sh , sh and sh , arranged in this order from the n-terminus to the c-terminus, with the intrinsically disordered "unique" domain separating the sh and sh domains. classically, the sh and sh domains are involved in regulation and the sh domain is the membrane anchoring site. we will present our recent results showing that the unique domain is part of a long loop closed by the interaction of the sh and sh domains (maffei et al., ). the conformational freedom of this disordered region is further restricted through direct contacts between the rt-loop of the sh domain and, primarily, residues located within the recently discovered unique lipid binding region (ulbr). the interaction between the unique and sh domains is allosterically modulated by a poly-proline ligand binding to the canonical binding site of the sh domain (maffei et al., ) . these results demonstrate a direct connection between classical c-src regulation involving the sh domain and the new regulation mechanisms involving the intrinsically disordered regions and provide new evidence of the functional importance and the underlying mechanism behind regulation of signalling pathways by intrinsically disordered domains. in mammalian cells, the golgi reassembly and stacking proteins (grasp and grasp ) are involved in the stacking of golgi apparatus cisternae and in the formation of the golgi ribbon. since grasps have been identified in many organisms, other roles for grasps have already been pointed out, such as chaperoning and transport of other proteins, involvement in cell apoptosis, cell migration, unconventional secretion, and in mitosis. in saccharomyces cerevisiae, it is observed that only % of the golgi cisternae are in stacks and do not form ribbon structures. this build yeast contains a single grasp, called grh , that is analogue to grasp . the structural differences of the golgi apparatus and the functional repertoire of grasps suggest a structural dynamic of these proteins. here, we used a combination of biophysical/biochemical methods to investigate the behavior of grh . bioinformatics and circular dichroism (cd) analyses of grh indicated a high percentage of either flexible regions or extended loops. the partial unfolded grh structure in solution folded into more ordered structures under temperature increasing, dehydration onto a surface and nonaqueous solvents as reported also by cd. hydration of the dehydrated folded protein is a reversible process that is accompanied by unfolding. furthermore, grh showed slow migration in sds-page, high susceptibility to proteases and low cooperativity of the chemical-induced unfolding process. fluorescence of trp residues along with cd data showed grh preserves a considerable amount of residual secondary structure, and the unfolding transition monitored by trp presented higher cooperativity. another cooperative transition was also reported by the extrinsic hydrophobic fluorescence probe ans upon chemical denaturation. these set of experiments indicate that grh behaves as a protein containing intrinsically disordered regions (idrs), characterized by unstructured regions of high polypeptide mobility experiencing many conformations. these findings suggest that an idp-like behavior may be the solution found by nature to account for grh functional need for interactions with several different partners in the cell. conformational changes governing dengue virus capsid protein function and its inhibition by pep andr e f. abstract dengue virus (denv) infection affects millions of people and is becoming a major global disease for which there is no specific treatment available. the interaction of denv capsid (c) protein with host lipid droplets (lds) is essential for viral replication. pep - , a peptide designed based on a denv c intrinsically disordered conserved region, inhibits this crucial interaction. combining bioinformatics and biophysics we determined pep - structure and ability to bind different phospholipids, in the context of denv c function. pep - becomes a-helical upon binding to anionic phospholipids. structure prediction of denv c n-terminal intrinsically disordered region reveals orientations that alternatively shield or expose denv c hydrophobic pocket, supporting a novel autoinhibitory role for this region. these findings pave the way for similar studies to understand disordered proteins and improved peptidomimetics drug development strategies against flaviviruses. topics intrinsically disordered proteins protein-lipid interactions pf- developing mechanistic insight into modulators of tau aggregation eri nakatani-webster , hannah baughman , shaylin higgins , abhinav nath the pathological self-association of microtubule-associated protein tau is implicated in a range of neurodegenerative disorders collectively called tauopathies, perhaps the most prominent of which are alzheimer's disease (ad) and chronic traumatic encephalopathy (cte). tau aggregation in vitro shares many features in common with fibril formation by other amyloid-forming proteins: a nucleationdependent polymerization reaction progressing via oligomeric intermediates into b-sheet-rich fibrillar aggregates, characterized by a distinctive sigmoidal kinetic. over the years, many investigators have advanced our understanding of how these time-courses might best be characterized and interpreted. in particular, elegant analytical and numerical approaches have been developed that supersede the empirical sigmoidal equations typically used to fit fibril formation traces. these modern approaches have enabled more rigorous insight into the mechanism of amyloid formation, and into how small molecules, protein chaperones, and other binding partners can modulate the process. an understanding of a modulator's effects on amyloid formation mechanism is necessary in order for us to predict and engineer its effects on amyloid pathology in a biological context. a given modulator may affect rates of primary or secondary nucleation, elongation, or fibril fragmentation to different extents. each of these perturbations, individually or in combination, can alter the kinetics of aggregation, the final state of the amyloid fibrils, and the sampled ensemble of oligomeric intermediates. unfortunately, fitting of mechanistic models to amyloid formation kinetics is an example of an "ill-posed problem", in that dramatically different combinations of elementary parameters can nevertheless generate very similar sigmoidal kinetic traces. this has typically necessitated global analysis of amyloid kinetic traces collected over a broad range of protein concentrations -a substantial expenditure of time, effort and material that must then be repeated in the presence of a modulator in order to gain insight into its effects. we propose an alternative approach: to fit amyloid formation traces to a large distribution of parameter sets, and determine how various aggregation modulators affect the distribution of parameters. this socalled "parameter distribution analysis" enables the inference of mechanistic effects from measurements at a single protein concentration. parameter distribution analysis based on numerical modeling has been made tractable by advances in computer hardware and software, and can be easily extended to include additional mechanisms or phases relevant to a protein or modulator of interest. here, we illustrate how parameter distribution analysis, complemented by fluorescence correlation spectroscopy (fcs), electron microscopy (em) and other biochemical techniques, can shed light on fundamental aspects of tau amyloidogenesis. we examine the disparate effects that natural products, pharmacotherapies and protein chaperones can have on the mechanism of aggregation, and also discuss the effects of heparin (widely used as an inducer of tau aggregation). these insights demonstrate the value of parameter distribution analysis as applied to amyloid formation and other ill-posed biochemical problems. new insights into amyloidogenesis of tau protein induced by enantiomers of polyglutamic acid amyloidogenesis of tau protein leads to the formation of amyloid fibrils (ordered fibrillar protein aggregates) which are accumulated in neurons of central nervous system during the course of neurodegenerative diseases called tauopathies. studying tau (a typical intrinsically disordered protein) amyloidogenesis has been challenging for many reasons. positive charge on the tau molecule must be compensated (e.g. in the presence of polyanions) in order to initiate the process. heparin (glycosaminoglycan) has been the most intensively studied charge-compensating agent in this context. on the other hand induction of tau aggregation by polyglutamic acid is poorly characterized. mechanisms responsible for the propagation of tau conformations has become an interesting research objective. prion-like features of tau amyloid can be studied in vitro also in the seed-induced regime of aggregation. tau amyloid seeds can act as nuclei for amyloidogenesis. such seeds can be obtained by fragmentation of amyloid fibrils by means of sonication. given that amyloidogenesis can proceed through various assembly pathways resulting in distinct amyloid 'strains' (self-propagating structural variants of amyloid) we have used poly-l-glutamic acid (plga) and poly-d-glutamic acid (pdga) to direct tau onto different amyloidogenic pathways. we have hypothesized that the chirality of the inducers could lead to fibril polymorphism. in our studies, we have used a recombinant human n r tau isoform. we have been using transmission electron microscopy (tem), sedimentation and kinetic measurment. firstly, we have characterized unseeded plga-/pdga-induced tau aggregation to find out that corresponding kinetics were significantly different. secondly, we have used sonicated fibrils to characterize the kinetics of seeded processes. both plga-/pdga-induced amyloid seeds were able to efficiently seed tau aggregation in the presence of plga, whereas in the presence of pdga the aggregation was much less effective. surprisingly, we found that pdgainduced amyloid seeds were able to catalyze fibrillogenesis of tau more clearly in the presence of soluble plga than in the presence of pdga -the primary inducer. we could not induce aggregation of tau in the absence of polyglutamic acids which indicates that positive charge on tau molecules must be unconditionally compensated in order to promote amyloidogenesis. thirdly, using tem we have characterized different morphologies of tau amyloid fibrils generated in unseeded and seeded processes. finally, to further characterize properties of the fibrils we have performed sedimentation experiments. fibrils induced by plga, pdga and heparin revealed different sedimentation properties. heparin-induced fibrils underwent sedimentation more readily than pdga-induced fibrils, whereas plga-induced fibrils remained in the supernatant. these results indicate distinct physicochemical properties of these fibrils. we believe that our findings will contribute to the current understanding of the molecular dynamics of tau amyloidogenesis. self-organizing structures of alpha-synulceins and its aggregates by a coarse-grained monte carlo simulation ras pandey , peter mirau , barry farmer alpha-synuclein (asn) consisting of residues, an intrinsically disordered protein, is linked to such neurodegenerative diseases as parkinson's disease (pd) and alzheimer disease via toxic clumping into abstract amyloid fibrils. we investigate the structure and dynamics of an asn chain as a function of temperature by a coarse-grained approach where a residue is represented by a node. in our coarse-grained approach, a residue is represented by a node. the basic idea is borrowed from the 'united atom' approach in polymer chain modeling that has been used extensively where the benefits and pitfalls of the method is explored for decades. such coarse-grained method has also been used protein chain modeling in recent years (e.g. aip advances , ( )). although the atomic scale structural resolution is sacrificed its specificity is captured via a set of unique knowledge-based residue-residue interactions matrix (e.g. classic miyazawa-jernigan matrix, macromolecules , ( )). a number of local and global physical quantities are analyzed such as contact map, neighborhood and mobility profiles, mean square displacement of protein, its radius of gyration and the structure factor. based on the mobility profile, we are able to identify three distinct segment of asn along its contour, i.e. sluggish nterminal ( - ) and c-terminal ( - , least mobile) separated by the central region ( - ), the nonamyloid component (nac) with higher mobility. contact profile shows that the probability of intrachain residue aggregation (clumping) is higher in the n-terminal region than the c-terminal with least aggregation in the nac region. we find that the radius of gyration (rg) decays monotonically with the temperature, consistent with the finding of allison et al. (jacs, , ( ) ). from the detail analysis of the structure factor we are able to predict the variation of the spatial mass distribution with the temperature as the residues in asn chain organize and disperse by evaluating its effective dimension d. we find the protein conforms to a globular structure (d ) at the low temperatures and to a random coil (d ) at high temperatures which is consistent with the estimates of uversky et al. (j. biol. chem. , ( )). in addition, we provide the estimates of d ( d ) for the intermediate structures as the protein chain makes a transition from globular to random coil. questions under-investigation includes what are the effects of mutations (e.g. b-and g-synuclein), how does the structure of an isolated asn chain change in presence of many interacting protein chains, and how do they organize over the multiple length scales? attempts will be made to address some of these issues as the data become available. tear down the wall: dismantling the biofilm scaffold of e.coli cesyen cedeno , nani van gerven , wim jonckheere , imke van den broek , han remaut , peter tompa csga is the major subunit of the so-called curli fiber system. this is an amyloid structure formed in the outer membrane on e.coli and acts as a scaffold for the biochemical machinery/matrix in the extracellular milieu (biofilms). extracellular matrices of this nature are robust platforms helping bacteria colonization; in this context csga becomes a key target in order to break the architecture within bacterial biofilms. chaperones are molecular machines able to stabilize misfolding prone proteins or even retrieve proteins trapped in non-physiological states. here we show how erd acts as a molecular chaperone inhibiting the formation of csga amyloid fibers in vitro. this work illustrates an alternative approach towards biofilm treatment at a molecular level. coupled folding and binding of transcription factors sarah shammas , alexandra travis , jane clarke intrinsic protein disorder is ubiquitous in transcription, particularly within transcription factors, which frequently fold into structures upon binding to partner molecules (dna or protein). the coupled folding and binding reactions that take place between individual transcription factors and the key hub co-activator proteins are crucial in determining the expression profile of the cell, and hence its phenotype. these interactions have been well studied by structural and equilibrium methods. here we present mechanistic insights into the process, gained through complementary kinetics experiments, for the binding of five separate transcription factors to a single prototypical co-activator (cbp kix). the transcription factors investigated belong to cellular (cmyb, mll, creb, e a) and viral (htlv- blz) classes. these reactions are remarkably fast; after removing the effect of long-range electrostatic rate enhancement the association rate constant is still approximately x m- s- , which is just above the typically quoted upper limit for diffusion-limited reactions between pairs of proteins ( - m- s- ), and is also the highest such value we have found reported. this, combined with the apparent insensitivity of the association rate to residual structure within the unbound state, indicates that binding preceeds folding (induced fit mechanism). interactions between kix and its transcription factors are additionally modulated by allostery between its two binding sites. we investigate the basis for this, finding it to be mediated by changes in protein flexibility. alternative hit finding strategies for intrinsically disordered proteins, exemplified by forkheadbox transcription factors harm jan (arjan) snijder , maria saline , tomas jacso , frank janssen , mattias rohman , tyrrell norris astrazeneca r&d, discovery sciences, se- ,pepparedsleden forkhead box o (foxo) proteins are emerging as key transcription factors in insulin and glucose metabolism, regulation of immune responses, and to balance cell proliferation, apoptosis and senescence. foxo proteins are predicted to be intrinsically disordered proteins (idps); idps are largely unstructured and often function as hubs mediating multiple interactions. idps are considered to be largely evasive from classical small molecule interference and lead-generation approaches, as they lack defined binding pockets. the available methods for addressing these targets have been lagging behind and needs to be developed to assess tractability of this target class. here we have evaluated the tractability of fragment screening on various domains of a forkhead box o member. we could confirm the intrinsically disordered character of foxo and used nmr screening to identify fragments that interact with foxo. one of these fragments was subsequently confirmed as a direct foxo binder in d hsqc-nmr spectroscopy and this fragment showed an effect in a foxo reporter gene assay. these results demonstrate that fragment screening may be a valuable approach for intrinsically disordered proteins although challenges remain to expand these fragments into more potent hits in the absence of detailed structural data. the characterization of amyloid-beta peptide (abeta) oligomer samples is critical to advance in the field of alzheime rs disease (ad). here we report a critical evaluation of two methods used for this purpose, namely sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page), extensively used in the field, and electrospray ionization ion mobility coupled to mass spectrometry (esi-im-ms), an emerging technique with great potential for oligomer characterization. to evaluate their performance, we first obtained pure cross-linked abeta and abeta oligomers of specific order. analysis of these samples by sds-page revealed that sds affects the oligomerization state of abeta oligomers, thus providing flawed information on their order and distribution. in contrast, esi-im-ms provided accurate information, while also reported on the chemical modifications and on the structure of the oligomers. our findings have important implications as they challenge scientific paradigms in the ad field built upon the sds-page characterization of abeta oligomer samples. coarse-grained simulation of protein association: application to rate prediction and implication for association mechanisms yinghao wu , the kinetics of protein binding is of paramount importance for understanding cellular functions. for instance, the binding kinetics between membrane receptors and their ligands control the speed of signal transduction after cells are exposed to stimulation. the experimentally measured association rates of protein binding span ten orders of magnitude, a range that was divided into two regimes. it was proposed that a fast association regime is limited by protein diffusion, while the other side of the spectrum is controlled by conformational changes. consequently, all previous simulation methods neglected conformational changes when calculating the association rate of a diffusion-limited regime. however, the most updated theory of protein binding suggests that a protein remains in a pre-existing equilibrium of unbound conformations. binding shifts the equilibrium toward its bound state. this highlights the importance of conformational factors for regulating protein binding. enlightened by this conformational selection model, we hypothesize that the conformational flexibility of protein structures regulates association more widely than previously anticipated. we develop a new coarse-grained model to simulate the process of protein association via the kinetic monte carlo (kmc) algorithm. each residue in this model is represented by its ca atom and a side-chain functional site. a simple physically based potential is used to guide the relative diffusion of two interacting proteins. given the size of the simulation box and the length of the simulation, the association rate constant can be derived by counting the frequency of dimerization among a large number of simulation trajectories. we further designed a prediction strategy that accounts for both the conformational and energetic factors of binding. our method is able to predict rates of protein association that are highly correlated with experimentally measured values. due to the coarse-grained feature, our model was further applied to several special cases of protein association. in one example, we studied the binding kinetics of proteins with flexible linkers. the interaction between thrombin and its functional inhibitor, rhodniin, was used as a testing system. we captured the conformational changes of flexible linkers from the all-atom molecular dynamic simulations. we found that the association with full-length flexible rhodniin was faster than its two individual domains and that their dissociation was more difficult, supporting a "flycasting" mechanism in which partial structures of an intrinsic disordered protein (idp) dock to the target first, while the remaining segments undergo conformational searches and sequentially coalesce around the target. in another example, we studied the binding kinetics of membrane receptors from cellular interfaces. the interaction between membrane proteins cd and cd , cell adhesion molecules known to mediate the activation of t cells and natural killer cells, was used as a testing system. the diffusive properties of these proteins on lipid bilayer were captured from all-atom molecular dynamic simulations. we showed that both d and d association rates could be simulated quantitatively with our method. the calculated values were close to the experimental measurements. we also provided detailed analysis of how molecular diffusions and membrane fluctuations affected d association. pf- (un)structure-function relationships on the ureg enzyme in the nickel-dependent urease system barbara zambelli , francesco musiani , stefano ciurli urease is an essential enzyme for many pathogens and soil microorganisms. its activity relies on the presence of nickel in the active site ( ) . the incorporation of this metal ion into the enzyme requires the formation of a supra-molecular chaperone involving four accessory proteins, named ured, uref, ureg and uree. uree is a metallo-chaperone involved in nickel binding and delivery into the enzyme active site. ureg is a gtpase essential for providing energy to the process of nickel site assembly. uref and ured form a complex that regulates the gtpase activity of ureg. the present work focuses on ureg, which exists in solution as an ensemble of inter-converting conformations ( ) . this observation made this protein the firstly discovered natural enzyme with an intrinsically disordered behavior, possibly allowing it to interact with different protein partners, such as uree ( , ) and uref ( ) and cofactors, such as metal ions ( ), in the urease activation network. ureg folding was studied perturbing protein conformation with temperature and denaturants, and investigating its folding response using circular dichroism, nmr and fluorescence ( ). a combination of light scattering, calorimetry, mass spectrometry, and nmr spectroscopy shed light on the effect of metal ion binding onto the conformational equilibrium of ureg ensemble ( ) . the results suggest that metal binding and solution conditions modulate affect the protein-protein interactions and enzymatic activity of ureg. nuclear inclusion protein a-protease (nia-pro) is a protease involved in processing of pepper vein banding virus (pvbv) encoded polyprotein to generate various intermediates and mature proteins at different stages of the viral life-cycle. nia-pro has two domains-n-terminal viral protein genome linked (vpg) and the c-terminal protease domain (pro).vpg belongs to the group of proteins that are intrinsically disordered, but attain stable structures upon interaction with other globular proteins. such proteinprotein interactions have a regulatory role on the function of the interacting partners. previously, the influence of vpg domain on the activity of pro was studied and it was shown that there was a substantial increase in the protease activity upon interaction with vpg (both in cis and in trans). in the present investigation, several deletion mutants of vpg and nia were constructed with a view to delineate the domain of vpg involved in interaction with pro. it was observed that deletion of residues from nterminus of vpg resulted in a decrease in the activity of pro in cis and in trans probably because of the abrogation of interaction between the two domains. interaction studies using spr (surface plasmon resonance) and elisa confirmed that the n-terminal residues of vpg are important for interaction with pro. the n-terminal residues of vpg are a part of the disordered region of vpg and their deletion resulted in the change in the secondary structure of the vpg and its oligomeric state. the ser and trp residues of pro domain were shown to be important both for the interaction of the two domains and for the activity of protease by mutational analysis earlier. these residues were identified to be a part of wc loop (w -c ) which relay the conformational changes to the active site catalytic triad (his , asp and cys ) leading to activation. however, mutations of these residues did not completely abolish the protease activity as well as the interaction with vpg. therefore, in the present study h and h which are observed to interact with trp and c (via non-covalent interactions) were mutated to alanine and the h a and h a mutants showed a drastic reduction in the activity of protease. molecular dynamics simulations of the wild type pro and the mutants revealed that trp -his -his -cys interaction pathway of the wild type pro was disrupted in the mutants and additional residues were involved in the interaction pathway, such alterations in the network of interactions could be responsible for the loss of activity. however, a change in the oligomeric status of these mutants was also observed as compared to the wild-type pro, suggesting that these residues are important for both the structural and functional integrity of pro and its interaction with vpg. thus, these results provide a molecular insight into the vpg-pro interactions and the modulation of their structure and function upon mutation of residues that are part of the interaction interface. transthyretin (ttr) is one of many proteins that are capable of forming amyloid fibrils in vivo. this protein is associated with two distinct amyloidosis: familial cardiac amyloidosis (fca) that causes a restrictive cardiomyopathy and familial amyloid polyneuropathy (fap) that affect peripheral nerves, they are hereditary and caused by mutations in the ttr gene. the non mutated protein can also aggregate in cardiac tissue in advanced age patients. the diagnosis was established at university hospital since due to a collaborative between our group and the center of amyloidosis antônio rodrigues de mello (ceparm). the only mutation found in brazil was v m in patients diagnosed in france. our group discovered new mutation not described in brazil and a novel mutation not described yet a d. the diagnosed patients are registered in transthyretin amyloidosis outcomes survey (thaos). the novel mutation a d causes a severe restrictive cardiomyopathy that is certainly related to a higher profile of aggregation observed for this mutant if compared to others amyloidogenic mutants of ttr. structural predictions using a bioinformatics tool called foldx showed that the insertion of the mutation cause a electrostatic clash that facilitates the dissociation and aggregation of protein. this mutant was purified heterologously and biophysical studies revealed that this protein is a dimer and not a tetramer as commonly the ttr structure. the crystallographic structure indicates that this mutant is structurally identical to wild type. biophysical studies revealed that this protein is a dimer and not a tetramer as commonly the ttr structure. the thermodynamic stability of a d is lower than the wild type ttr. the aggregation profile showed us that this protein can aggregate in a higher manner and with a fast kinetic to that observed for others amyloidogenic mutants of ttr, forming fibers in two hours of aggregation. heterotetramers of a d and wt are able to aggregate in the same fiber structure. the analysis of interface interaction of this mutant using the pdbsum showed modifications in the profile of hydrogen bonds and non bonded contacts. in addition the oligomers of a d are toxic for primary culture of cardiomyocytes from murine heart. the amyloidogenic profile displayed by this new mutant can be directly correlated with the aggressiveness observed in the disease developed by the identified patient. furthermore the recent consolidation of ttr diagnosis in our university hospital led to the identification of the rare a d variant in a brazilian patient, suggesting that other new, uncharacterized mutants could be identified in the coming years. multiple cellular proteins interact with ledgf/p through a conserved unstructured consensus motif [ ] . the ledgf/p -mll -menin complex was structurally characterized, but only partially [ ] . using nmr spectroscopy, we identified and mapped a novel mll -ledgf/ p interface. colony forming assays in mll -af leukemic cells expressing mll interactiondefective ledgf/p mutants revealed that this additional interface is essential for leukemic transformation. interestingly, the newly defined interface overlaps with the binding site of known ledgf/p interactor, the hiv integrase [ ] . while the pathophysiological interactions of ledgf/p are intensively studied, its physiological role remains unclear. since ledgf/p contributes to hiv integration and leukemic transformation and has become a new therapeutic target for drug development, it is crucial to study its physiological interactions. in addition to hiv in and mll -menin, the ledgf/p integrase binding domain (ibd) also interacts with several other proteins [ , ] . our recent data (manuscript accepted in nat. commun.) revealed structural details of ledgf/p interactions with physiological binding partners. the interaction with the ledgf/p ibd is maintained by an intrinsically disordered ibd-binding motif (ibm) common to all known cellular partners. based on the knowledge of this motif, we identified and validated iws as a novel ledgf/p interaction partner. naturally occurring single mutants, i t, f i, w r and d h of lysozyme in human, have been known to form abnormal protein aggregates (amyloid fibrils) and to accumulate in several organs, including liver, spleen and kidney, resulting in familial systemic amyloidosis. these human pathogenic lysozyme variants are considered to raise subtle conformational changes compared to the wild type. here we examined the effects of the aberrant mutant lysozymes i t, f i,w r and d h, each of which possesses a point mutation in its molecule, on a cultured human cell line, hek , in which the genes were individually integrated and overexpressed. western blot analyses showed lesser amounts of these variant proteins in the medium compared to the wild type, but they were abundant in the cell pellets, indicating that the modified lysozyme proteins were scarcely secreted into the medium but were retained in the cells. immunocytochemistry revealed that these proteins resided in restricted regions which were stained by an endoplasmic reticulum (er) marker. moreover, the overexpression of the mutant lysozymes were accompanied by marked increases in xbp s and grp /bip, which are downstream agents of the ire _ signaling pathway responding to the unfolded protein response (upr) upon er stress.rnai for the mutant lysozymes' expression greatly suppressed the increases of these agents. next, we addressed the interaction between amyloidogenic lysozyme and grp /bip as the former proteins were obtained by immunoprecipitation with the latter protein as well as colocalization of both proteins in the er. lysozyme composes of a-domain rich in helices and b-domain rich in sheet. two helices of a and a in the n-terminal region arrange in parallel and face to face where hydrophobic amino acids at the f, l , l , l , l and l allocate with equal interval there. in the back of dock, there is a core region of amyloid fibril formation, of which the side chain of i is exposed on the protruding. probably, these hydrophobic amino acids might be crucial for lysozyme folding. although mutated lysozymes undergo folding by grp /bip in such environment, the dissociation of the grp from lysozyme by failure of folding is likely inhibited and both proteins remain bound to, resulting in staying to the er. a part of aberrant lysozymes seem to remain bound to grp /bip during folding and insolubilize with aggregation, thus accumulate in the er accompanied with er stress. lysozyme amyloidosis might be caused by long-term accumulation in the endoplasmic reticulum of the abnormal protein. structural characterization of toxic oligomers that are kinetically trapped during alpha-synuclein fibril formation the accumulation of abnormally aggregated proteins within the body is a common feature of several medical disorders, such as alzheimer's disease, parkinson's disease and diabetes mellitus type . while the specific protein found to be the major component of such deposits varies from one disease to another, the formation of the pathological aggregates seems to occur via a common process of misfolding and self-assembly of a normally soluble polypeptide chain into a series of oligomeric intermediates and, ultimately, into insoluble amyloid fibrils that accumulate within specific organs and tissues. increasing evidence indicates that certain oligomeric protein species generated during the self-assembly of specific proteins into ordered fibrillar aggregates can be highly cytotoxic and are likely to be key players in the initiation and spreading of neurodegenerative diseases. however, little detailed structural information is currently available for these oligomeric species due to their often transient nature and, more importantly, because of their variability in terms of size and structure. we report here the isolation and detailed characterization of an ensemble of stable toxic oligomers of alpha-synuclein, the protein whose deposition is the hallmark of parkinson's disease. by defining and minimizing the degree of heterogeneity of these isolated alpha-synuclein oligomers which have accumulated during the process of amyloid formation, we have identified distinct subgroups of oligomers and determined their structural properties and three-dimensional molecular architectures. this characterization has been achieved by the application of a set of complementary biophysical techniques, including a variety of spectroscopic techniques along with analytical ultracentrifugation, atomic force microscopy, and electron microscopy. although these oligomers exist in a range of sizes, with different extents and nature of beta-sheet content and exposed hydrophobicity, all the oligomeric subgroups possess hollow cylindrical architectures with marked similarities to amyloid fibrils. this suggests that these types of oligomers are kinetically trapped during protein self-assembly and that the accumulation of at least some forms of amyloid oligomers is likely to be a consequence of very slow rates of rearrangement of their beta-sheet structures. our findings reveal the inherent multiplicity of pathways of protein misfolding and the key role the beta-sheet geometry acquired in the early stages of the self-assembly process plays in dictating the rates of structural conversions, and thus the kinetic stabilities and pathological nature of different amyloid oligomers. the results of this study provide the basis for a more complete understanding of the nature of the self-assembly of polypeptides into beta-sheet rich amyloid aggregates, and potentially contributes to efforts to identify specific targets for drug discovery. fish otoliths and mammalian otoconia, biominerals composed of calcium carbonate and organic matrix, are involved in the functioning of the inner ear, the sensory organ that plays an important role in hearing and balance [ ] . however, their developmental origins, growth, and the role of the matrix, especially the protein component, are still poorly understood. it has been shown that proteins involved in the formation of biominerals are usually very acidic. they often belong to the group of intrinsically disordered proteins (idps), a class of proteins devoid of a rigid tertiary structure [ , ] . the shape and polymorph selection of calcium carbonate otolith in danio rerio is controlled by the starmaker (stm) protein [ ] . recently, a gene was identified encoding the starmaker-like (stm-l) protein from oryzias latipes, a putative homologue of stm. it has been suggested that stm-l has a similar function as stm, although there is no sequence similarity between stm and stm-l [ ] . several methods, such as size exclusion chromatography, cd spectroscopy and analytical ultracentrifugation demonstrated that stm-l is an coil-like idp, with the tendency to form locally ordered structures [ ] . because stm-l was suggested to play a crucial role in calcium carbonate mineralization, it is possible that calcium ions may influence its conformation, as was previously shown for stm [ ] . however, other ions may also be involved in this process. the aim of this study was to investigate the effect of mono and divalent metal ions on the conformation of stm-l. we used single molecule f€ orster resonance energy transfer (smfret) and fluorescence correlation spectroscopy (fcs), which have shown that calcium ions compacts the proteins most efficiently, followed by magnesium and the monovalent ions. the difference in the effect of monovalent and divalent ions on the protein dimensions is likely to result from the different properties of the ions, like charge density and radius. cd experiments have shown that a high excess of calcium ions caused the formation of ordered secondary structure in stm-l, which may be crucial for the formation of calcium carbonate crystals, when the ratio of building ions to protein is high. it has been demonstrated that dmp is proteolytically processed into fragments, including k n-terminal region and k c-terminal region. as many proteins characterized to be engaged in biomineralization, dmp and its fragments belong to the group of intrinsically disordered proteins (idps). it has been suggested that dmp and its fragments can take a part in otoconia mineralization, as the protein is present in mouse otoconia, but the role of dmp and its fragments in the mineralization of calcium carbonate has not been examined until now. to determine the influence of the dmp fragments for otoconia development, k dmp protein was expressed in bacterial expression system, purified and used in in vitro biomineralization test of calcium carbonate. in particular, immobilized metal anion affinity chromatography (imac) was applied as a first step of purification procedure. because of high content of acidic amino acids, ion exchange chromatography with a mono q column was used as a next step. the development of insects is regulated by the combined action of ecdysteroids and juvenile hormones (jh). pulses of -hydroxyecdysone ( e) initiate each step of metamorphosis, while jh modulates its action and prevents precocious differentiation. the biological and molecular mechanism of e action is well described. in contrary, the way of the jh activity is still poorly understood. in wilson and fabian [ ] reported that drosophila melanogaster mutants lacking met are resistant to toxic doses of jh and its analogue methoprene. it has been proved, that met binds jh at physiological conditions. therefore met is believed to be a putative jh receptor. met may also be involved in a cross-talk between two hormonal signalling pathways, involving e and jh. the detailed structure of met is still unknown. therefore our main aim is to characterize structural properties of met. in silico analysis performed on a full-length met suggested, that n-terminal part of met contains three conserved domains characteristic for bhlh-pas transcription factors, whereas c-terminal part is most probably unstructured. )). capitalizing on self-and cross-amyloid interactions, we designed highly effective, peptide-based inhibitors of amyloid self-assembly of abeta and iapp. due to their favourable properties the designed peptides are promising leads for targeting protein aggregation in ad, t d or both diseases while the inhibitor design strategy should be applicable to other amyloidogenic polypeptides and proteins as well. apoptosis, the process of programmed cell death, must be carefully regulated in multi-cellular organisms to ensure proper tissue homeostasis, embryonic development and immune system activity. the bcl- family of proteins regulates the activation of apoptosis through the mitochondria pathway. dynamic interactions between pro-and anti-apoptotic members of this family keep each other in check until the proper time to commit to apoptosis. the point of no return for this commitment is the permeabilization of the outer-mitochondrial membrane (omm). translocation of the pro apoptotic member, bax, from the cytosol to the mitochondria is the molecular signature of this event. molecular interactions and conformational changes associated with this event have been difficult to obtain due to challenges associated with taking subtle measurements in the complex environment of live cells. to circumvent these challenges, we developed a novel method to reliably detect f€ orster resonance energy transfer (fret) between pairs of fluorophores to identify intra-molecular conformational changes and inter-molecular contacts in bax as this translocation occurs in live cells. in the cytosol, our fret measurements indicated that the c-terminal helix is exposed instead of tucked away in the core of the protein. this coincided with measurements using fluorescence correlation spectroscopy (fcs) that showed that cytosolic bax diffuses much slower than expected, suggesting possible complex formation or transient membrane interaction. we propose that this exposed helix allows for this contact to occur. cross-linking the c-terminal helix (a ) to helix a reduced the instances of these interactions while at the same time yielded fret measurements that are consistent with the a helix tucked into the core of the protein. after translocation, our fret measurements showed that bax molecules form homo-oligomers in the mitochondria through two distinct interfaces involving the bh domain (helix a ) and the c-terminal helix. these findings provide insight into the molecular architecture that may involve possible contacts with other bcl- proteins to permeabilize the omm, which would also be necessary for the regulation of apoptosis. abstract spatial resolution is especially advantageous for bacterial cells because of their small sizes. in the past few years the spatial organization and dynamics of a variety of bacterial cellular structures and protein macromachineries have been revealed with unprecedented details. as the field matures, it is now time to focus on the functional aspect of the observed spatial organizations and dynamics. are they essential in carrying out a specific cellular function? do they play a regulatory role in controlling the on and off of a certain cellular process? in this work i will present a few examples from our laboratory that examine the spatial and functional organization of macromolecules involved in bacterial cell division. transcription factors (tf) exert their function by interacting with other proteins and binding to dna. the nucleus is a compartmentalized space, and the spatial organization of tfs and their partners represents other step of gene expression regulation. we used the glucocorticoid receptor (gr) as a model of tf's mechanism of action. gr is a ligand-activated tf with a relevant role in physiology and a great variety of effects. it can be recruited to specific response elements on dna or interact with other tfs. also, the activity of gr is modulated by different co-regulators, e.g. tif /grip . gr and tif do not distribute homogeneously within the nucleus but accumulate in distinctive clusters. the functional role of this particular intranuclear organization remains unknown. we used advanced fluorescence microscopy techniques to study the dynamics of gr and tif in the nucleus of living cells with high spatial and time resolution. gr and tif fused to fluorescent tags were transiently expressed in newborn hamster kidney (bhk) cells and visualized by a confocal microscope. fluorescence correlation spectroscopy (fcs) experiments were carried on to measure the intranuclear mobility of both proteins. the method is based on the analysis of fluorescence intensity fluctuations due to the movement of fluorescent molecules in and out the confocal volume. the data could be fitted with a model that considers a free diffusion of tif and gr in the nucleus and their binding to fixed targets. we also studied the dynamics of different gr mutants in the presence of different ligands and our results suggest that the binding depends on dna. both gr and tif autocorrelation curves reveal an increase in the bound population upon gr activation by its agonist dexamethasone (dex). a cross-correlation analysis showed that, as expected, dex-stimulus increases the population of gr-tif complexes. without hormone, gr shows a homogeneous distribution and tif forms large clusters in the nucleus. upon dex-binding, gr accumulates in the nucleus, is rapidly recruited to tif foci and there is an important re-distribution of both proteins, that co-localize in the same pattern of small intranuclear clusters. the dynamics of gr and tif molecules at these clusters were studied by performing orbital-scanning measurements, tracking the clusters position in silico and analyzing the intensity fluctuations of the clusters along time. a positive cross-correlation between both channels indicates that dex-bound gr and tif interact at these foci and dissociate from them forming tif -gr hetero-complexes. in conclusion, advanced fluorescence microscopy methods allowed obtaining a dynamical map of gr distribution and function in the nucleus of mammalian living cells. assembly of membrane pores as a mechanism for amyloid cytotoxicity by the bacterial prionoid repa-wh cristina fern andez , rafael n uñez-ramirez , mercedes jimenez , germ an rivas , rafael giraldo amyloid fibril formation is associated with human neurodegenerative diseases. prefibrillar oligomers formed during the fibril assembly process, rather than mature fibrils are known to be central to disease abstract and may be responsible for cell damage. a commonly proposed mechanism for the toxicity of small oligomers is their interaction with the lipid bilayer of cell membranes, leading to loss of membrane integrity [ ] . recent studies from our laboratory have shown that repa-wh , a winged-helix domain from a bacterial plasmid replication protein, can assemble into amyloid fibrils in vitro. when expressed in escherichia coli repa-wh functions as a cytotoxic protein that shares features with the mammalian amyloid proteinopathies. these features have proved repa-wh to be a suitable synthetic model system to study protein amyloidosis [ , , ] . in this work, using the repa-wh bacterial model system, we have studied the interaction between the protein and model membranes (large and giant unilamellar lipid vesicles, luvs, and guvs respectively). repa-wh shows association and aggregation to membranes composed of anionic phospholipids. protein association in guvs did not result in lysis of the vesicles, suggesting the assembly of discrete protein pores as the mechanism for repa-wh membrane damage. to investigate the formation of pores we analyzed by electron microscopy the aggregation of repa-wh in the presence of a pre-formed e. coli lipid monolayer. the em images show the presence of pore-like particles on the monolayer. amyloid pores formation explains the permeabilization effect of repa-wh in vesicle models and is in agreement with observations for human amyloidogenic proteins. the approaches presented here provide a deeper insight into amyloid cytotoxicity towards membranes and will make possible the assay of inhibitors and effectors of amyloidosis under controlled conditions. references: b -adrenergic receptor (b ar) is a member of g protein-coupled receptors, which represent the single largest family of cell surface receptors involved in signal transduction. b ar recognizes a variety of ligands and communicates with cytoplasmic g-proteins by transmitting signals through the cellular membrane. thus, investigation of communication pathways for b ar may give important insights for understanding its allosteric mechanisms and identifying new target sites for more specific and efficient drug molecules to be used in the treatment of pulmonary and cardiovascular disease. in this study, various conformations from ms molecular dynamics (md) simulations and available crystal structures of human b ar were investigated to reveal alternative signaling pathways between its extra and intracellular regions. specifically, shortest communication paths connecting key residues (more than Å apart) at the orthosteric ligand binding site (d , s , t , f , n ) to either l or s located near the g-protein binding site were investigated. the conformers from previous md simulations [ ] include the intracellular loop (icl ), which especially affects the transmembrane collective dynamics but is lacking in x-ray structures. the protein was described as a graph composed of nodes linked by edges. nodes were placed at the alpha-carbon atoms and the edges were calculated based on the number of atom-atom interactions within a cut-off distance . Å for each residue pair. twenty shortest pathways were revealed using k-shortest path algorithm [ ] on the coarse-grained network. our results indicated that distinct signaling paths progressed most frequently on tm but alternative paths were also present, which passed partially through tm , tm , tm or tm depending on the conformation. among the critical residues that transmitted the signal between distant sites, f and n were detected, whose functional roles were reported in previous experimental studies. pathway shifting was observed depending on the open-to-closed transition of icl during md simulations. the sulfonylurea receptor (sur ) is an atp binding cassette (abc) protein that forms the regulatory subunit in katp channels found in the pancreas and the brain. mgatp binding and hydrolysis at the two cytosolic nucleotide binding domains (nbd and nbd ) in sur control gating of the katp channel pore. , proper regulation of katp channel gating by sur is critical. over mutations that lead to diabetes, hyperinsulinism and developmental delay have been identified in different domains of sur , including the nbds. therefore, molecular-level understanding of the structure and function of the nbds is essential for designing improved treatments for sur-related diseases. here we present biophysical and biochemical studies aimed at understanding the effect of disease-causing mutations on the conformation and nucleotide binding of sur nbd . specifically, we are investigating sur nbd mutations that cause neonatal diabetes (r w and h t) or congenital hyperinsulinism (c d, g v, r g, r d and k t). our nuclear magnetic resonance (nmr) data shows that the hyperinsulinism mutation k t causes chemical shift changes throughout the spectrum of nbd , implying overall changes in protein conformation that may affect mgatp binding and inter-domain interactions with other domains in the sur protein. size-exclusion data show that the other hyperinsulinism mutations (c d, g v, r g, r d) produce mostly aggregated protein, likely as a result of misfolding of nbd . misfolding of nbd may be the underlying cause of reduced katp trafficking seen with these mutations and hence decreased katp channel gating observed in hyperinsulinism. in contrast to the k t mutations, the congenital diabetes-causing mutations (r w and h t) cause few nbd nmr spectral changes. however, the congenital diabetes mutation r w decreases the affinity of nbd for mgatp, which is unexpected for congenital diabetes mutations. our fluorescence, circular dichroism and microscale thermophoresis data corroborate the results that we have obtained by nmr spectroscopy. our data provide molecular-level details on the effects of disease causing mutations in human sur . egfr increased stability: rmsf of the ca atoms during the md simulations suggest that glycosylation is associated with dampened motions, suggesting that the glycans stabilize the structure. subdomain iii is the most stabilized while subdomain i is stabilized largely in the proximity of the ligand. both dimer interfaces including the dimerization arm from domain ii and the tip of domain iv fluctuate less upon glycosylation. hydrogen bonding; persistent interactions seen for protein-glycan: in the disaccharide-containing system, we observed three highly occupied hydrogen bonds between the glycans and domain iii and iv of egfr. hydrogen bonds of domain iii involve the residue asp in which a sidechain oxygen interacts with oxygen atoms of the n-acetylglucoseamine linked to asn . in domain iv a hydrogen bond is seen between the cys backbone amide and the oxygen atom of n-acetylglucosamine linked to asn . in the oligosaccharide-containing system hydrogen bonds observed between the glycan attached to asn and domain ii. these hydrogen bonds form between the gln sidechain oxygen atom and cys backbone oxygen atom and the mannose linked to asn . the reduction in the mobility of these amino acids suggests that hydrogen bonds impart stability to both the sugars and to the interacting egfr. insects possess a complement-like immune response utilizing thioester-containing proteins, or teps. the only arthropod tep of known structure is anopheles gambiae tep , which is a key component in the natural immunity of this mosquito to malaria parasites (genus plasmodium). unlike vertebrate complement factors, agtep does not contain an anaphylatoxin domain which acts to regulate a massive conformational change accompanying activation of the protein. the mechanism of agtep must therefore involve an alternative mechanism for allosteric regulation of thioester activation. in place of a small internal domain, a large, heterodimeric complex of two leucine-rich repeat (lrr) proteins, lrim and apl c, have been shown to specifically bind and stabilize the active conformation of agtep . i will present my group's most recent work in this area. we have shown that different alleles of tep , which are known to influence the vectoral capacity of wild mosquitoes, differ significantly in their susceptibility to thioester hydrolysis. allelic variation is centered on residues at the protein-protein interface within tep containing the thioester bond. the lrim /apl c heterodimer is shown to form an extended and flexible ensemble in solution. two closely-related genes to apl c, apl a and apl b, can also form a complex with lrim , and apl b lrr domain can form a homodimer. we propose that a flexible and heterogeneous group ensemble of lrim /apl dimers interact with the active conformation of tep , thereby producing an array of immune complexes to protect mosquitoes from a diverse set of pathogens. human flap endonuclease- (hfen ) is an essential metallo-nuclease involved in okazaki fragment maturation and long-patch base excision repair. during these processes, bifurcated nucleic acid intermediates with ssdna '-flaps are generated by polymerase strand displacement synthesis and then cleaved one nucleotide into the downstream duplex by fen to create a nicked-dna that is a suitable substrate for ligase. until recently, how hfen achieves tremendous catalytic power (rate enhancements > exp ) and exquisite selectivity for the scissile phosphate had been understood poorly ( ) . in , the grasby and tainer labs solved the structures of hfen in complex with product and substrate. this study revealed that scissile phosphate selectivity is largely due to the substrate dna undergoing a novel di-nucleotide unpairing (dnu), which places the scissile phosphate diester in contact with the requisite divalent metal ions. in addition, by comparing the structures of hfen alone ( ) and in complex with substrate and product dnas ( ), grasby and tainer proposed a model, whereby protein conformational changes occur upon binding substrate resulting in placement of key basic residues that position and/or electrophillically catalyse hydrolysis of the scissile phosphate diester. further work using a cd-based assay showed that metals are absolutely required for dnu, whereas the key basic residues in the active site are not. surprisingly, perturbations to the protein structure that are much more distant from the fen active site (i.e., helical cap) prevent dna unpairing, implying that the fen protein actively participates in the unpairing process ( , ); however, how it does remains a mystery. the maximal multiple turnover rate of hfen reaction is rate-limited by enzyme product release, whereas hfen kinetics under substrate-limiting conditions ([e]<[s]<km) suggest that the enzyme is approaching catalytic perfection (i.e., almost diffusion controlled - exp m- s- ) ( ) . previous work has shown that a paralogue of hfen (i.e., t fen ) is not ratelimited by chemistry under single turnover (st) conditions, but rather some physical limitation such as conformational change ( ) . to ascertain whether the protein and/or dna conformational changes of hfen mentioned above are rate limiting under st conditions, we have initiated kinetic and nmr studies to determine the role of conformational dynamics in the catalytic cycle of hfen . owing to the latest advance in the computational technologies of microprocessor, high-speed inter-processor connection, and parallelization algorithm, all-atom molecular dynamics (md) simulations of microsecond time scale are getting popular to study the pharmaceutical target proteins. an antibody binds to its antigen with structure changes. a small molecule moves around the target protein and finds an entrance to get into the binding site. these phenomena can be observed in microsecond simulations, but the vital issue is whether the adopted force field is enough accurate to correctly describe the phenomena. we are developing a force field (fuji) based on general amber atom types and amber van der waals parameters in order to describe arbitrary organic molecules in a unified manner including proteins and nucleic acids. we use the first principle theoretical method to refine the force field in contrast to common empirical fittings to experimental data. the dihedral torsion parameters of protein backbone were determined to agree with the torsion energy profiles calculated by high-level quantum mechanical theory for the model systems of protein backbone. conformational preferences of dipeptides in water were measured by vibrational spectroscopy. comparing with the experimental distribution of ramachandran angles of dipeptides, how accurately molecular calculations predict the conformation distribution of dipeptides were investigated for various force fields and semiempirical quantum methods. fuji force field gave the best prediction score among the various methods (tzanov et al, ) . in this work we examine dynamical structure behaviors of pharmaceutical target proteins by microsecond md simulations with fuji force field. epiregulin (epr) is a transmembrane protein with residues belonging to the epidermal growth factor family. mature epr ( residues) binds to epidermal growth factor receptor (egfr), which stimulates the proliferative signaling in cancer cells. our epr antibody has a proline at the residue in the third complementarity-determining region (cdr) of the heavy chain, which has cis peptide bond in free state and trans one in the bound state with epr according to x-ray crystallography analysis. in order to clarify the structure changes in binding we performed extensive md simulations of our antibody (fab; residues) and epr. the total simulation time was about microseconds. we also performed md simulations for a protein kinase and a small molecule. we show how the complex system reaches thermal equilibrium states in simulations from the stiff x-ray crystal structure. the calculations are compared with experiments such as x-ray crystal structures and thermodynamic quantities measured by isothermal titration calorimetry (itc) and surface plasmon resonance (spr). this research has been supported by mext spire supercomputational life science (hp , hp ) and first kodama project in japan. structure-based recombination of drug resistance enzymes: structural and functional tolerance to new dynamics in artificially-evolved enzymes our understanding of the contribution of protein dynamics to function is still emergent. in a protein engineering context, do we need to take into account the dynamics in order to maximize the fitness and function of the resulting proteins? using high resolution crystal structures, nmr relaxation dispersion and ms molecular dynamics simulations, we compare two naturally evolved homologous class a b-lactamases, tem- and pse- which share a high degree of structural and functional conservation. we observed a conservation of dynamics on a catalytically relevant timescale. this is consistent with dynamics being an evolutionarily conserved feature. however, laboratory-engineered chimeric enzymes obtained by recombination of the two homologs exhibit striking dynamic differences, despite the function and structure being conserved. the laboratory-engineered chimeras are thus functionally and structurally tolerant to modified dynamics on the timescale of the catalytic turnover. this tolerance of blactamases to dynamic changes could be linked to the high fitness of the naturally evolved proteins and implies that maintenance of native-like protein dynamics may not be essential when engineering functional proteins. conformations solution. an immense advantage of our recombinant system is the possibility to perturb it by introducing unnatural amino acids for subsequent labelling with fluorescent dyes or to omit subunits from the multisubunit rnap. comparing free, nucleic acid-and factor-bound rnap complexes we found that the position of the clamp is modulated during the transcription cycle in a fashion exceeding the simplified closed-open picture drawn from crystallographic studies. we show that the clamp adopts at least two distinct conformations in both initiation and elongation complexes. moreover, we were able to link conformational states to the catalytic activity of the rnap. transcription factors tfe and spt / , which both bind to the rnap clamp domain and stimulate the activity of the enzyme, shift the equilibria towards one of the main conformations suggesting that both prompt an allosteric switch that influences the structure of the rnap. hence, our data provide a mechanistic rationale for the function of these factors and provide new insights into the complex dynamic behaviour of the transcriptional machinery. solvent models for protein simulations -the good, the bad and the applications duy hua , amitava roy , he huang , carol post the solvent environment plays a crucial role in determining the structure, dynamics and function of a biomolecule. in order to utilize molecular dynamics (md) simulations to examine the structural changes abstract of biomolecules, it is imperative that the solvent environment be accurately modeled. implicit solvent models (isms), in which water molecules are absent and the solvent effect is estimated using an energy function, offer a low-cost approach to describe the solvent environment in md simulations. isms have been used for a variety of biological studies; yet, the performance of implicit solvation methods in capturing the structural characteristics of proteins has not been rigorously demonstrated. in this work, three isms, namely gbmv ii, facts, and scpism, were evaluated for their abilities to emulate the solvent environment and its effect on the structures, dynamics and electrostatic interac- thioredoxins (trx) are ubiquitous proteins that play a key role in redox state regulation of the cell. they interact with multiple targets in the cell. our group solved the structure of s. cerevisiae (ytrx ) and measured its dynamics in different timescales (pico to seconds). it is well established that the asp is the proton acceptor in the reduction process of the target protein. we proposed that asp also works to couple hydration and motion of the interacting loops. we studied conformational motions of the residues of water cavity and interacting loops. the water molecules in water cavity of the protein play a vital role in the redox activity of thioredoxin. the degree of mutational frustration explains in a great deal the multiple timescales observed in the protein dynamics in ytrx and the mutant d a. the ytrx displays a complex equilibrium between the ground state and several excited hydration states. this excited states are more permeable to water and it is key to understand the proton transfer and activation of cys . in this current study, we examined the mutant d a, and observed that this small hydrophobic residue induces conformational exchange in residues exposed to the water cavity, such as cys . along with molecular dynamics simulations and calculation of mutational frustration levels, we were able to describe conformational details of the water cavity. it is formed by three independent contiguous lobes which we called lobes a, b and c. lobe b is the central lobe that contains the catalytic important residues cys and asp . it closed upon mutation d a. lobe a and c remains open upon mutation, meaning that their hydration are independent. the nmr structures for the oxidized and reduced state of the mutant d a has been studied. in-vitro and in-silico studies of ligand binding to the nuclear receptor ppargamma using fret and md narutoshi kamiya , gert-jan bekker , takuma shiraki , haruki nakamura ipr, osaka university, kinki university the nuclear receptor ppargamma is a ligand-dependent transcription factor which regulates gene expression related to glucose homeostasis and insulin sensitization. it is a potential therapeutic target for metabolic syndrome, cancer, and inflammatory diseases. crystal structures have revealed that its ligands bind deep inside the pocket. however, how the ligand recognizes ppargamma and penetrates inside the pocket remains unclear. we have measured the binding kinetics using the fret method and have calculated potential dissociation paths via molecular dynamics (md) simulations. the binding kinetics were measured using fret between the ligand, c -bodipy, which is a fluorescent pigment structurally similar to one of its agonists -oxo-eicosatetraenoic acid, and cyan fluorescent protein (cfp) which is fused to ppargamma. fluorescence of c -bodipy is observed when it is close to cfp when cfp is excited by light at nm. the ligand was simultaneously added to the ppargamma-cfp solution using the stopped-flow method. time-resolved fluorescence signals of c -bodipy and cfp were also monitored. two time constants were observed by the fret experiment, which correspond with the landing and docking processes. we used our new md program, psygene-g [ ] , which utilizes the gpu for acceleration of the non-bonded interactions, such as electrostatic interaction which uses our original zero-dipole summation method [ , ] . previously we were able to attain similar thermodynamics with respect to the particle mesh ewald method in membrane proteins and dna-water-ion systems [ , ] and have applied it to the thermodynamic study of dynein [ ] . we have introduced random accelerated md (ramd) into the psygene-g program to predict a ligand's dissociation path from the initial crystal structure (pdb id: zk ). in ramd, an external random-directional constant force is added to the ligand. we executed -ramd simulations with different random seeds for the initial pull directions. twelve ramd trajectories managed to dissociate within ns, while the remaining simulations did not manage to disassociate within this time-frame. in all twelve cases the ligand went past r , which is located at the surface of ppargamma. interestingly, a somatic mutant (r h) is related to colon cancer. we concluded that r plays a role of a gate keeper to assist ligand binding. sumoylation is a post-translational modification involved in various cellular processes, such as nuclearcytosolic transport, transcriptional regulation, apoptosis, protein stability, response to stress, and progression through the cell cycle. sumoylation is a conjugation of glysine residue of sumo protein with lysine residue in target(rangap ) protein through the influence of enzyme e (ubc ) and e (ranbp ). sumo proteins modify the function of target protein in the cell. but the kinetic mechanism of sumoylation is not well-understand. we traced out the full kinetic process of sumoylation of the sumo -ubc -rangap -ranbp complex in atomic detail via molecular dynamic simulation. we uncovered the kinetics of sumoylation mechanism and verified the important residues which play a key role in the sumoylation process. the comparison of our results with the experimental one are also discussed. human potassium channel kcnq is expressed in several tissues including inner ear, heart muscle, lung, intestine, and stomach, each requiring a unique current profile for proper function. to tune its output current, kcnq complexes with several accessory proteins from the kcne family, each kcne family member modulating kcnq distinctly: kcne causes the channel to delay opening and become more conductive in the open state, kcne causes the channel to be constitutively conductive, and kcne closes the channel. the purpose of this study is to uncover the atomic-scale, mechanical mechanism of how kcne modulates kcnq . to do this we modeled the interaction between kcnq and kcne with a hybrid experimental-computational approach. our strategy is to determine the nmr structure of kcne alone in bilayer-mimicking bicelles, build a homology model of the open-state kcnq , and dock the structure of kcne onto the model of kcnq using electrophysiology-based restraints to validate and refine the resulting model complexes. paramagnetic relaxation enhancement, residual dipolar coupling and sequence analysis suggest the single span transmembrane helix of kcne is significantly curved. hydrogen-deuterium exchange, nuclear overhauser effect cross peaks, and amber simulation suggest kcne carries a significant amount of water in the c-terminal side of the transmembrane helix. the h- n two dimensional nuclear magnetic resonance spectra of a known phenotype-changing mutation in the center of the transmembrane helix, v t, suggests the helix adopts multiple conformations when the extra side-chain hydroxyl group is present. a docking funnel corroborates a binding pocket suggested by the in vivo electrophysiology data where kcne wedges between a helix near the potassium pore and two helices in the voltage sensitive element of the channel. within this pocket, we believe the conformational sampling and structural rigidity-or flexibility, in the context of the v t mutant-of the accessory protein kcne directly influence the modulation profile. watching conformational changes in proteins by molecular dynamics simulations kresten lindorff-larsen proteins are dynamical molecules and their ability to adopt alternative conformations is central to their biological function. examples include motions that underlie allosteric regulation or ligand binding, or protein dynamics in enzymes that can modulate the overall catalytic efficiency. protein motions can often be described as an exchange between a dominant, ground state structure and one or more minor states. the structural and biophysical properties of these transiently and sparsely populated states are, however, difficult to study, and an atomic-level description of those states is challenging. in an attempt to determine how well molecular dynamics simulations can capture slow, conformational changes in protein molecules we have studied two different protein systems which are known to undergo conformational exchange on the millisecond timescale, and for which structural information is available for both major and minor states. using enhanced-sampling all-atom, explicit-solvent molecular simulations, guided by structural information from x-ray crystallography and nmr, we show that current force fields and sampling methods allow us to sample experimentally-determined alternative conformations with surprisingly high accuracy. in particular, we find that we can reversible sample both the ground state and minor state, and that the simulations capture the structure of the minor states also. our simulations enable us to calculate the conformational free energy between the two states, and comparison with relaxation dispersion nmr experiments demonstrates a high accuracy. thus, we show for two distinct proteins that we can map the free energy landscapes and that both the structure and energetics are in excellent agreement with nmr experiments. our simulations provide insight into the structural and biophysical properties of transiently populated minor states, and help reinterpret previous experimental measurements. further, our results demonstrate that, at least in the two cases we have studied, modern simulation methods enable us to examine these otherwise "invisible" states of proteins and describe their structural, functional and thermodynamic properties. our results in this way help demonstrate how simulations can now add to our knowledge of transiently formed, "hidden" states of proteins. cell signaling is a fundamental process for all living organisms. g protein-coupled receptors (gpcrs) are a large and diverse group of transmembrane receptors which convert extracellular signals into intracellular responses primarily via coupling to heterotrimeric g proteins. in order to integrate the range of very diverse extracellular signals into a message the cell can recognize and respond to, conformational changes occur that rewire the interactions between the receptor and heterotrimer in a specific and coordinated manner. by interrogating the energetics of these interactions within the individual proteins and across protein-protein interfaces, a communication network between amino acids involved in conformational changes for signaling, is created. to construct this mapping of pairwise interaction energies in silico, we analyzed rhodopsin gpcr coupled to a gai b g heterotrimer. the structure of this g protein complex was modeled in the receptor-bound and unbound heterotrimeric states as well as the activated, monomeric ga(gtp) state. from these tertiary structural models, we computed the average pairwise residue-residue interactions and interface energies across ten models of each state using the rosetta modeling software suite. here we disseminate a comprehensive analysis of all critical interactions and create intra-protein network communication maps. these networks represent nodes of interaction necessary for g protein activation. structure and dynamics of the polymyxin-resistance-associated response regulator pmra in complex with the promoter dna yuan-chao lou , yi-fen kao , tsi-hsuan weng , yi-chuan li , chwan-deng hsiao , chinpan chen institute of biomedical sciences, academia sinica, institute of molecular biology, academia sinica pmra, an ompr/phob family response regulator (rr), takes part in the two-component system that manages genes for polymyxin resistance through a phosphorylation-dependent regulation. phosphorylation of ompr/phob rr induces the formation of a two-fold symmetric dimer in the n-terminal receive domain (rec), promoting c-terminal dna-binding domains (dbds) to recognize tandem repeat dna sequences on the promoter to elicit adaptive responses. recently, narayanan et al. presented the complex structure of active-like kdpe in complex with dna, which reveals a unique asymmetric rec-dbd interface that is necessary to form stable complexes for transcription activation. in this study, we report the . Å resolution crystal structure of bef -activated pmra in complex with promoter dna as well as its dynamics in solution by nmr. unlike the case of kdpe, pmra-dna complex structure reveals a different asymmetric rec-dbd interaction. interestingly, nmr assignments and dynamics study suggest that rec and dbd do not form stable contacts in solution; instead, domains tumble separately in the absence or presence of dna. relaxation dispersion experiments on methyl groups further show that several rec-dbd interfacial residues exhibit similar slow dynamics in the presence of dna. it is hence highly possible that the slow dynamics observed on these interfacial methyl groups are related to the formation of asymmetric rec-dbd interaction, which reduces the mobility of pmra-dna complex and promotes crystallization. in solution, two domains tumble independently and have diverse orientations, which together with the dbd-dbd interface may facilitate searching best interactions with rna polymerase holoenzyme for transcription activation. time-resolved x-ray observations of nano-scale protein assembly networks yufuku matsushita , hiroshi sekiguchi , noboru ohta , keigo ikezaki , yuji goto , yuji sasaki , the university of tokyo, graduate school of frontier science, advanced materials science, spring- , osaka university, institute for protein research protein aggregation and network formation in diverse protein species had been studying in various experimental approaches such as absorbance spectrophotometry, dynamical light scattering and x-ray scattering. however, these conventional methods are only able to observe averaged information on bulk conditions and also these techniques have a limitation of time resolved observations. recently, development of single molecular observation techniques are remarkable. diffracted x-ray tracking (dxt) is one of the notable single molecular observation methods to capture detailed intramolecular dynamics of an individual single protein molecule by the labeling x-ray method. in this study, we present an innovative method for observing the protein assembly networks of nano-scale size on the bulk solution using a dxt that possess pico-meter scale positional accuracy and micro-second time resolution. this method is founded by detecting angular rotational displacement of a coexisted and dispersed single gold nanocrystal (approximately nm) on target solution. for target sample, we choose a crystal precursor metastable state of mg/ml lysozyme solution (hen egg white lysozyme: . g/ mole on . m sodium acetate - w/v nacl, ph . buffer) and mg/ml of lysozyme solution such as stable condition. for reference sample, we prepare mg/ml and mg/ml of ribonuclease a solution (ribonuclease a (bovine): . g/mol, . m sodium acetate - w/v nacl, ph . buffer). the experiment was carried out at spring- bl xu. from dxt analysis, we obtained logarithmic rotational velocity distribution of mg/ml, mg/ml lysozyme solution and mg/ml, mg/ml of ribonuclease a solution during ms and we processed regression analysis of gaussian fitting in each distribution. from this result, mg/ml of lysozyme solution (stable) and mg/ml, mg/ml (reference) have definitely regressed by single peak normal gaussian distribution that are positional peaks at . mrad and . mrad and . mrad, respectively. in contrast, mg/ml of lysozyme solution consisted of double peak logarithmic distribution at . and . mrad. this peak separation tendency from dxt are typically observed in a supersaturated condition of the sodium acetate solution which coexisting nano-scale solute networks (dxt and small angle x-ray scattering (saxs) experiment). from this study, dxt measurement results and detailed analysis process for a protein solution such as crystal precursor metastable state of lysozyme solution are concerned that this technique is a powerful tool for observing nano-scale protein network's existence and its local dynamics in bulk solution. at the present time, we will demonstrate the detailed analysis and processing from dxt raw data and the conformity of the results from another experiment confirmation such as saxs and darkfield microscopy. finally, we present a detailed protein network model of lysozyme metastable solution from dxt result. increasing evidence suggests that conformational fluctuations experienced by proteins play a key role in promoting function. however, the experimentally derived dynamic behavior of discrete protein systems has yet to provide clear evidence on the evolutionary conservation of motional events between structural and functional protein homologues. in this work, we used a statistical coupling analysis (sca) approach to analyze the role of co-evolving amino acids in protein function and flexibility. sca facilitates the identification of distinct residue sectors and provides an understanding of the correlation between amino acid co-evolution and biological function. as a model system, we used the ribonuclease a (rnase a) family, which includes canonical members that are structurally similar, but perform diverse activities such as angiogenesis, anti-pathogenicity and neurotoxicity, while preserving the common ribonucleolytic function. analysis of sequences with sca . allowed the determination of five independent components (ics) corresponding to amino-acid sectors with distinct functional, structural and/ or dynamical roles within the family. while most ics correspond to residues critical for protein stability and catalytic function, ic correlates with residues experimentally determined to be highly dynamic on the catalytic timescale in several rnase a homologues, as we confirmed by nmr n-cpmg experiments. additionally, ic residues form a hydrogen-bonding network shown to allosterically modulate and coordinate catalytically productive motions. these results provide a direct observation between the role of residue sectors in stability and motions relevant to function in the rnase a family. this study highlights the role of concerted action of multiple residues towards a common function, which can guide experiments for engineering proteins to enhance function such as catalysis. halophilic archea are a type of extremophiles that thrive in hypersaline conditions. in order to avoid the osmotic shock, their cytoplasm is maintained with higher ionic strength up to - m. such a high ionic strength of intracellular environment protects the cells from desiccation or salting out. however, one wonders how such high salt concentrations keep the cell functional and active throughout all the cellular reactions (i.e. enzyme catalysis). the answer lies in the evolution of the halophiles that has left the halophilic proteins with a certain preference for amino acids composition [ ] (prefers more acidic residues and overall decrease in hydrophobic side-chains). it's also known that the salt concentration can affect the activity and the underlying mechanism [ ] of the halophilic enzymes. in order to further understand the haloadaptation, we have chosen to study a model system, dihydrofolate reductase (dhfr) from haloferax volcanii (hvdhfr). dhfr is a very well understood enzyme and known to bound variety of ligands that can modulate the dynamics and the energy landscapes of the enzyme [ ] . we have probed the ls-ms dynamics of hvdhfr bound to the cofactor nadph (holoenzyme) and other substrates (dhf, thf) using relaxation dispersion nmr. in order to understand the salt contribution to the dynamics and catalytic mechanism, the experiments were done in the increasing order of salt concentration, shedding light on the conformational ensembles involved in the catalytic mechanism. the nmr relaxation dispersion analysis of different enzyme complexes in the presence of m and m kcl has shown more conformational fluctuations than compared to the enzyme with m kcl. this result suggests that the enzyme preferably is highly active & samples more flexible conformers in the hypersaline environment. further, quantification of these conformers/invisible-states and their salt-dependence will provide new insights into the mechanism of the enzyme-catalysis. also, it will facilitate designing novel enzymes that can be used in the bioprocess industrial set-up under extreme conditions. . tadeo we have identified the extracellular protein hbps in the cellulose degrader streptomyces reticuli and shown that it specifically binds iron ions, heme and aquo-cobalamin. based on d crystal structures, structural alignments, sequence comparisons, mutagenesis, and comparative biochemical investigations, we identified the coordination sites for iron, heme and aquo-cobalamin, and binding kinetics were elucidated [ , ] . hbps interacts with the membrane-embedded sensor kinase sens. while under nonstressed conditions hbps inhibits sens autophosphorylation, under oxidative-stressing conditions activates it. sens in turn phosphorylates the response regulator senr that activates the transcription of anti-oxidative genes [ ] . we crystallized hbps and solved its d crystal structure that revealed an octomeric assembly which is required for interaction with sens [ ] . using mutagenesis, fret, cd spectroscopy, fluorescence spectroscopy and site-directed spin labelling combined with pulse electron paramagnetic resonance spectroscopy, we demonstrated that ironmediated oxidative stress induces both secondary structure and overall intrinsic conformational changes within hbps. we additionally showed that hbps is oxidatively modified, leading to the generation of highly reactive carbonyl groups and tyrosine-tyrosine bonds [ ] . we concluded that these molecular events are responsible for the hbps-mediated activation of the sensor kinase sens. this presentation will focus on the hbps structure and dynamics within the protein. indian institute of technology bombay for (fop-related protein of kda), a centrosomal protein, is conserved across all ciliated eukaryotes. it has been shown to be involved in ciliogenesis in rpe cells and in the s-phase progression in hela cells. it localizes to the pericentriolar satellite and at the centrosome. the localization of for at the centrosome has been found to be throughout the cell cycle. in paramecium, for is involved in docking of the basal body to the cell membrane and in the assembly of the transition zone. we found that for colocalizes with ciliated microtubules in nih t cells. since, the localization of for to the pericentriolar satellite is dependent on microtubules and the cilium itself is a microtubule-based structure; we sought to investigate the role of for in microtubule assembly. the over-expression of gfp-for in hela cells led to the depolymerization of microtubules while only the gfp expressing hela cells showed typical microtubule network. in addition, the microtubules of gfp-for expressing hela cells were found to be more sensitive towards nocodazole, a microtubule-depolymerizing agent, suggesting that for destabilizes microtubules. the effect of for on the polymerization of tubulin was monitored by light scattering, sedimentation assay and electron microscopy. in vitro, for inhibited the rate and extent of polymerization of tubulin. for example, , , and mm for inhibited the polymerization of purified tubulin by , , and %, respectively. further, the sedimentation assay suggested that for inhibited the polymerization of tubulin. in addition, electron microscopic analysis of the assembly mixture showed that for inhibited microtubule formation in vitro. the binding of for to purified tubulin was monitored by several complimentary techniques. using size exclusion chromatography, for was found to be co-eluted with tubulin indicating that for binds to tubulin. further, the interaction between for and tubulin was analyzed by surface plasma resonance technique and the result showed that for binds to tubulin with a modest affinity. the data together suggested that for regulates microtubule assembly through a direct interaction with tubulin. amide hydrogen exchange (hx) is widely used in protein biophysics even though our ignorance about the hx mechanism makes data interpretation imprecise. notably, the open exchange-competent conformational state has not been identified. based on analysis of an ultra-long molecular dynamics trajectory of the protein bpti, we propose that the open (o) states for amides that exchange by subglobal fluctuations are locally distorted conformations with two water molecules directly coordinated to the n-h group. the hx protection factors computed from the relative o-state populations agree well with experiment. the o states of different amides show little or no temporal correlation, even if adjacent residues unfold cooperatively. the mean residence time of the o state is ps for all examined amides, so the large variation in measured hx rate must be attributed to the opening frequency. a few amides gain solvent access via tunnels or pores penetrated by water chains including native internal water molecules, but most amides access solvent by more local structural distortions. in either case, we argue that an over-coordinated n-h group is necessary for efficient proton transfer by grotthuss-type structural diffusion. differences in redox reactions with nadp /h between ferredoxin-nadp oxidoreductases from bacillus subtilis and rhodopseudomonas palustris daisuke seo , hidehiro sakurai , pierre s etif , takeshi sakurai graduate school of natural science and technology, kanazawa univ., research institute for photobiological hydrogen production, kanagawa university, ferredoxin-nad(p) oxidoreductase (fnr) is a ubiquitous enzyme that catalyze the redox reaction between soluble small iron-sulfur protein ferredoxin and nadp /h. fnrs from photosynthetic green sulfur bacterium chlorobaculum tepidum (ctfnr), low-gc content gram-positive bacterium bacillus subtilis (bsfnr) and purple non-sulfur bacterium rhodopseudomonas palustris (rpfnr) are homodimeric proteins containing one fad prosthetic group per subunit. crystal structure analyses of ct-and bsfnrs have revealed their significant structural homology to nadph-thioredoxin reductase from eschelicia coli, which is distinct from the monomeric fnrs from plastids of higher plants, cyanobacteria, g-proteobacteria and putidaredoxin reductase. previous studies on steady-state reaction of bs-and rpfnrs with nadp /h by diaphorase assay have demonstrated that nadph oxidation rates of bsfnr and rpfnr were comparable to those of the fnrs from plastid and cyanobacteria. but affinities toward nadp /h and rates for oxidation of nadph differed significantly between bsfnr and rpfnr, despite their high amino acid sequence homology. in this work, we report pre-steady state reactions of bsfnr and rpfnr with nadp /h by a stopped-flow spectrophotometry. mixing oxidized bsfnr with nadph yielded a rapid formation of charge transfer complexes (ctcs) followed by a reduction of the enzyme. bsfnr was almost fully reduced at equilibrium. mixing photochemically reduced bsfnr with nadp also rapidly provided an absorption spectrum of ctc but followed reoxidation of reduced bsfnr was very slow. the amount of oxidized bsfnr after equilibrium depended on nadp concentration. kinetic analyses indicated that the rate-determining steps were the hydride-transfer reactions in both directions and the rate for the forward direction was much faster than that for the reverse direction. mixing oxidized rpfnr with nadph exhibited a rapid ctcs formation followed by a slower reduction of the enzyme. increase in nadph concentration reduced the observed rate, suggesting redox potential of rpfnr was similar to that of nadp /h couple in the presence of excess nadph. mixing photochemically reduced rpfnr with nadp rapidly provided ctcs. the decay corresponding to the reoxidation of reduced rpfnr with nadp involved two distinctive kinetic phases, which would be due to the similarity in hydride transfer rates of forward and reverse directions, and the presence of excess amount of nadp . kinetic analyses indicated that the rate-determining steps were the hydride-transfer reactions in both directions and the rate for the forward direction was close to that for the reverse direction. obtained data suggested bsfnr and rpfnr are optimized for different direction of the reaction and may play different physiological roles. stephan k€ ohler , , friederike schmid , giovanni settanni , institute of physics, johannes gutenberg university mainz, germany, graduate school materials science in mainz, fibrinogen is a serum multi-chain protein which, when activated, aggregates to form fibrin, one of the main components of a blood clot. fibrinolysis controls blood clot dissolution through the action of the enzyme plasmin, which cleaves fibrin at specific locations. although the main biochemical factors involved in fibrin formation and lysis have been identified, a clear mechanistic picture of how these processes take place is not available yet. this picture would be instrumental, for example, for the design of improved thrombolytic or anti-haemorrhagic strategies, as well as, materials with improved biocompatibility. here, we present extensive molecular dynamics simulations of the fibrinogen complex which reveal large bending motions centered at a hinge point in the coiled-coil region of the complex. this feature, likely conserved across vertebrates according to our analysis, suggests an explanation for the mechanism of exposure to lysis of the plasmin cleavage sites on fibrinogen coiled-coil region. it also explains the conformational variability of fibrinogen observed during its adsorption on inorganic surfaces and it is supposed to play a major role in the determination of the hydrodynamic properties of fibrinogen. in addition the simulations suggest how the dynamics of the d region of fibrinogen may contribute to the allosteric regulation of the blood coagulation cascade through a dynamic coupling between the a-and b-holes, important for fibrin polymerization, and the integrin binding site p . membrane curvature -the assembler of proteins mijo simunovic , , gregory voth , patricia bassereau chemistry department, the university of chicago, physico-chimie curie, institut curie many biological phenomena require the membrane to change its shape. this process is often mediated by curvature-generating proteins, most notably by those containing one of many bar domains. at the same time, membrane curvature controls the way proteins interact with one another and so it acts as a vital signaling mechanism in the cell. we combine theoretical modeling with microscopy imaging techniques to study the driving force underlying the reshaping of biological membranes induced by n-bar proteins. in particular, we employ a combination of coarse-grained molecular dynamics with field-theoretical simulation methods to study the assembly of proteins on the membrane at molecular resolution. this approach allowed us to elucidate the precise mechanism by which cell membranes rapidly and from large distances recruit proteins to membrane-reshaping sites. it also let us identify a surprising role of membrane tension in directing the strength and geometry of protein-protein interactions. by complementing our simulations with fluorescence and atomic force microscopies, we study the way molecular assembly of proteins affects the membrane morphology at a more macroscopic level. we demonstrated how largescale ordering of the proteins on the membrane is mediated by a strikingly long-range interaction that is driven by membrane fluctuations. in sum, our combined theoretical and experimental approach gives vital clues on how the mechanical properties of the membrane may regulate protein dynamics in living cells. adnan sljoka , alexandr bezginov kyoto university, university of toronto understanding how a protein functions depends in critical ways on predicting which parts are rigid and which are flexible. using rigidity-theoretical techniques, a number or programs such as first, proflex or abstract kinari can rapidly decompose a protein into flexible and rigid regions. in this study we extend this technique and develop a novel computational approach for detecting protein allosteric interactions. it is widely believed that the binding of a ligand at the allosteric site triggers a conformational change that is transmitted through the protein to cause a rearrangement and alteration of the shape of the active site. allostery was first described more than years ago; however, the underlying allosteric mechanism is still not well understood. we will introduce a rigidity-based allosteric mode of communication together with an algorithm which can detect transmission of rigidity and shape changes between two (or more) distant binding sites in allosteric proteins. our algorithm is also used to predict and identify regions in the protein that are critical for the coupled communication between distant sites (i.e. allosteric pathways). starting with a set of known gpcr -dimensional structures, we apply our methods and show how binding of an activating ligand (i.e. agonist) triggers small rigidity changes which propagate to the critical g-protein binding regions. in contrast, in the inactive gpcr structures no such rigidity allosteric communication is observed. detailed predictions and analysis on activated (agonist-bound) and inactive adenosine receptors is discussed and results are compared with experimental evidence. these results show that rigidity-based allosteric model and algorithm is a powerful new tool for detecting allostery in gpcrs. comparing the intrinsic dynamics of multiple proteins using elastic network models reveals global similarities based on their overall shape sandhya tiwari , , nathalie reuter , with increasing protein structural data, we find ourselves with the opportunity to study the dynamical similarities within large ensembles in order to understand the role of protein dynamics at various levels. is there evolutionary pressure to conserve dynamics? is this necessary to retain functionality, as it has been suggested? we performed strategic comparative analysis using the elastic network model, which has proven to be highly efficient and informative (fuglebakk et al., bmc bioinformatics, ) to investigate how the dynamics of related proteins change when their functional or oligomeric state shifts, and if it is conserved within protein families. in our work, we have found that proteins with different ligandbinding states, as in adenylate kinase (tiwari et al., bmc bioinformatics, ), or oligomeric states, as in the pyrr family (perica et al., science, ), can be classified based on the global similarity of their intrinsic dynamics. moreover, our recent analysis on functionally distinct protein families with the tim barrel fold indicates that the overall similarity in shape dictates similarity in intrinsic dynamics regardless of sequence and functional conservation or evolutionary links. we suggest that since shape dictates a large part of dynamical similarity in proteins, the changes in local flexibility play a strong role in differentiating various functional states. oxygen-affinity and cooperativity of hemoglobin introduction: the widely-held mechanism of allostery of hb has been that the changes from the r-quaternary/tertiary structures of oxy-hb to the t-quaternary/tertiary structures of deoxy-hb exert certain constraints on the coordination structure of the heme group, leading to a lower o -affinity of the hemes and thus that of hb [ ] . however, we found that there is no causal correlation between the static t/r-quaternary structures and the low/high o -affinities of hb, respectively [ ] . we explore an alternative mechanism of the regulation of the ligand-affinity and cooperativity in hb. results and discussion: the o -affinities of free fe[ii]-protoporphyrin ix-nitrogenous base complexes in organic solvents are very low (p > torr), whereas the apparent o -affinities of these metalloporphyrins, which are incorporated in apo-myoglobin, apo-hb, serum albumin, etc., increase substantially to p < - torr, though their coordination structures are apparently unchanged [ ] . such substantial increases in the apparent ligand-affinities of metalloporphyrin-containing proteins are accomplished by preventing/inteferring with the dissociation of the ligand by protein matrix, since the interior of globin is nearly fully packed by protein matrix. in hb, the dissociation process of the ligand proceeds through the "caged" state [ ] [ ] [ ] , which can be produced by cryogenic photolysis of the ligated-states at . k and in which the metal-ligand bond is broken and the un-bonded ligand is trapped near the bonding site within the globin moietiy. this "caged" state has spectral features distinct from those of either deoxyor ligated states of the respective hemoproteins. the apparent ligand-affinities of hb are regulated by heterotropic effectors without detectable changes in either static quaternary/tertiary structures of the globin moiety or the coordination/electronic structures of the metalloporphyrin moiety and thus the ligand-affinity of the metalloporphyrins themselves [ ] [ ] [ ] . the reduction of the apparent ligand-affinities of hb may be caused by increases in the migration rate of ligands through globin matrix from the "caged" state to solvent, resulting from the effector-linked, enhanced high-frequency thermal fluctuations which increase the transparency of the globin matrix toward small diatomic ligands [ ] [ ] [ ] . conclusion: the ligand-affinity of hb is regulated through protein dynamics by heterotropic effectors, rather than static quaternary/tertiary structural changes. thus, the "caged" state of hb acts as a critical transition state in regulation of the affinity for small diatomic ligands in hb [ ] . the role of metal ions in the regulation of life processes is extremely important. they act as signal transducers, protein configuration stabilizers, enzymatic cofactors, oxygen transport supporters and many others. for example, subtle perturbations in calcium homeostasis may lead to mental disabilities and are linked to diseases such as autism spectrum disorders (asd). in this study we focus on complex protein systems, mainly those present in the brain. we search for dimers mediated by the presence of metal ions, and determine the impact of the presence or absence of the latter on the structure and energetic properties of the complex in the protein-protein interface. we investigate ions' influence on the interface stability using classic molecular dynamics methods (md), including steered md. moreover, we apply a novel suite of enhanced md-based methods recently developed by our team (rydzewski & nowak) to explore ion diffusion pathways in protein fragments of the synapses. finally, we describe specific inter-protein ion binding motifs with the most important interactions, collating them with various structures deposited in the protein data bank [ ] . the binding of integrins to collagen plays a critical role in numerous cellular adhesion processes including platelet activation and aggregation, a key process in clot formation. collagen is an unusually shaped ligand, and its mechanism of recognition and role in selectivity and affinity are unique, and at this stage not well understood. the i-domain of the integrin protein binds to collagen specifically at multiple sites with variable affinities, however the molecular mechanism of integrin i-domain (ai) regulation remains unknown. using nmr, along with isothermal titration calorimetry, mutagenesis, and binding assays we are developing a novel integrated picture of the full recognition process of the integrin a i binding to collagen. the adhesion of the a b integrin receptors to collagen is cation-dependent with collagen binding a mg(ii) ion that is located at the top of the extracellular integrin a i-domain (a i). our results show evidence for a regulatory effect of the mg(ii) ion on a i affinity, by inducing allosteric ms-ms motions of residues distant from the binding site. we propose a novel model of a i recognition to collagen, comprising a two-step mechanism: a conformational selection step, induced by mg(ii) coordination, and an induced-fit step caused by collagen binding. hydrogen-deuterium exchange experiments show that the induced-fit step is facilitated by the reduced local stability of the c-terminus. we propose that the conformational selection step is the key factor that allows discrimination between high and low affinity collagen sequences. cytochromes p (cyp) are heme containing enzymes involved in the metabolism of endobiotics and xenobiotics, such as drugs or pollutants. [ ] in humans, cyps are attached to the biological membranes of endoplasmic reticulum or mitochondria by n-terminal transmembrane anchor and they are partially immersed by their catalytic domain to different level. [ ] generally, the composition of lipid membrane may significantly affect behavior of protein embedded in respective membrane e.g. the cholesterol in membrane alters membrane properties such as: thickening of the membrane, changing the stiffness or enhancing ordering of the membrane. furthermore, the increasing amount of cholesterol in membrane may also alter interaction with membrane proteins and affect solute partitioning between membrane and water molecules. [ ] cholesterol is also known to noncompetitively inhibit the most typical drugmetabolizing cyp -cyp a , [ ] however the mechanism was unknown. for this reason, we prepared the set of simulations of cyp a embedded in dopc lipid bilayers with various cholesterol concentrations ( , , , and % wt; figure ) and the ns long md simulations were carried out. md simulations showed the formation of funnel-like shape of the lipids close to the catalytic domain of cyp. in addition, the cholesterol molecules have tendency to accumulate in the vicinity of membrane-attached f/g loop. the catalytic domain sunk deeper into the membrane with cholesterol and also the number of amino acids in contact with membrane was bigger than in the pure dopc bilayer. in contrast, the presence of higher amount of cholesterol affected the pattern of channel opening effectively blocking the access to the active site from the membrane, which in turn may affect the substrate preferences and catalytic efficiency. [ ] finally, we study the effect of different lipid types on membrane-attached cyp a . anti-( -hydroxy- -nitrophenyl)acetyl (np) antibodies are one of the most widely analyzed type of antibodies, especially with respect to affinity maturation [ ] [ ] [ ] . affinity maturation is a process in which b cells produce antibodies with increased affinity for the antigen during the course of an immune response, and is like "evolution" in term of increasing antigen-binding affinity. during the course of affinity maturation, the structural dynamics of antibodies, which are closely correlated with the binding function, can change. to analyze the structural dynamics at atomic resolution and the single-molecule level, we tried to express and purify single-chain fv (scfv) antibodies against np. using scfv antibodies, we can also analyze the effects of key residues on affinity maturation via site-directed mutagenesis. as the first step, we have succeeded in generating a sufficient quantity and good quality of scfv of affinity-mature anti-np antibody, c , with a linker composed of four repeats of gggs. the scfv protein was expressed in the insoluble fraction of e. coli, and solubilized using m urea, followed by refolding by step-wise dialysis to decrease the urea concentration. the final step of purification using an antigen column indicated that approximately % of the solubilized protein was correctly refolded and possessed antigen-binding ability. the analytical ultracentrifugation (auc) analysis showed that the purified c scfv exists in the monomeric state with little oligomeric contamination. the secondary structure and thermal stability of c scfv were analyzed using circular dichroism (cd). the far-uv cd spectra of c scfv indicated typical b-sheet-rich structures. upon antigen binding, the far-uv cd spectrum remained unchanged, but the thermal stability increased by approximately oc. the antigen-binding function of c scfv was analyzed using a surface plasmon resonance (spr) biosensor, biacore. the binding affinity and kinetics of c scfv for np conjugated to bovine serum albumin immobilized on the sensor chip were similar to those of intact c . taken together, the results of auc, cd, and spr indicated that c scfv could be refolded successfully and would possess its functional structure. next, to analyze the structural dynamics of c scfv in the absence or presence of antigen, experiments involving diffracted x-ray tracking (dxt) were performed [ ] . c scfv with an n-terminal his-tag was immobilized on substrate surfaces using tag chemistry, and au-nanocrystals were labeled on the surface of scfv as tracers. the motions of c scfv were analyzed in two rotational directions representing tilting (u) and twisting (v) mean square displacement (msd) analysis from more than trajectories showed that the slope for c scfv without antigen, especially in the u direction, was greater than that for c scfv with antigen, suggesting that the motion of scfv was suppressed on antigen binding. the antibiotic resistance enzyme aph( '')-ia confers antimicrobial resistance to aminoglycoside antibiotics in staphylococci and enterococci. this kinase phosphorylates aminoglycosides such as gentamicin and kanamycin, chemically inactivating the compounds. we have determined multiple structures of the enzyme in complex with nucleoside and aminoglycoside substrates and cofactor magnesium. introduction of aminoglycoside to crystals of aph( '')-ia induce gross conformational changes in crystallo, illustrating several important stages of the catalytic cycle of the enzyme. an interaction between nucleoside triphosphate and an amino acid residue on a conserved loop has also been identified that appears to govern a conformational selectivity and modulates the enzyme activity when no substrate is present. comparisons between multiple protein molecules both within and between crystal structures allow us to infer functional states of the enzyme as it carries out catalysis. these structures collectively highlight an enzymatic flexibility that not only allows the binding of diverse aminoglycosides, but also appears to transition from a stabilized, inactive enzymatic state to a catalytically active enzyme with an active site geometry identical to distantly-related eukaryotic protein kinases. mechanistic insight gained from these studies begin to demystify a widespread staphylococcal resistance factor, and provide a starting point for the development of anti-infectives toward this important antimicrobial resistance machine. ryan godwin , william gmeiner , freddie salsbury wake forest university -department of physics, wake forest university health sciences -department of cancer biology the zinc-finger of the nf-jb essential modulator (nemo) is a ubiquitin binding domain, and an important regulator of various physiological processes including immune/inflammatory responses, apoptosis, and oncogenesis. the nominally functioning residue monomer ( jvx) is represented by a bba motif, with a cchc active site coordinating the zinc ion. here, we investigate the effects of a single point mutation that has been linked to the disease states associated with ectodermal dysplasia. the single mutation of the last binding cysteine (residue ) to a phenylalanine ( jvy) distorts the available conformation and dynamics of the protein, as shown via microsecond, gpuaccelerated molecular dynamics simulations. we examine these two proteins in various states of zinc-binding and coordinating cysteine protonation. in addition to destabilization of the alphahelix induced by the cysteine to phenylalanine mutation, prominent conformations show the bsheets turned perpendicular to the alpha-helix, providing a possible mechanism for the induced disease state. , catalytic ( - aa) and c-terminal ( - aa)) were expressed in e. coli. several truncated in variants containing amino acids - , - , - and - were also prepared. a full-size ku with a gst-tag on its n-terminus was purified from e. coli. all the experiments performed showed that neither n-terminal nor c-terminal domains of hiv- in are essential for its binding with ku despite a weak binding capacity retaining to the c-terminal domain. the catalytic core ( - aa) as well as the mutant lacking c-terminal domain ( - ) both demonstrated affinity to ku comparable to the affinity of the full-size in, whereas its truncated variant ( - aa) bound to ku protein only weakly. we also expressed a c-terminal ha-tagged full-length in and its - variant in hek t cells together with a wt ku - flag and showed that both in variants are stabilized by co-expression with ku by approx. twofold. we hypothesize that the binding surface within in lies in the region from to a.a. that is a long a-helix. we have shown that a homologous integrase from prototype foamy virus that lacks this structural element does not bind to ku . it is worth noting that ku does not affect the interaction of in with its major cellular partner -ledgf/p as well as its interaction with the dna substrate. this work was supported by an rfbr grant - - and by an rscf grant - - . the nadph-dependent cytochrome p oxidoreductase (cypor) is large amino-acid long microsomal multidomain enzyme responsible for electron donation to its redox partner cytochrome p (cyp) involved in drug metabolism. electron transfer (et) chain is mediated by two riboflavin-based cofactors -flavin mononucleotide (fmn) and flavin adenine dinucleotide (fad) within their respective domains and nicotinamide adenine dinucleotide phosphate (nadph). during this electron transfer cypor undergoes several structural changes in open and closed state of both domains in different degree of contact. in spite of the fact that cyp-cypor complexes play a key role in drug metabolism, the atomistic mechanism of structural rearrangements during complex electron transfers is still lacking. here, we present the results of our study on structural changes during cypor multidomain complex movement between individual electron transfers using classical molecular dynamics (md) and metadynamics (mtd) simulations with cofactors of nadph, fad and fmn in resting state. homology model of human cypor in both forms (opened and closed) were embedded into pure dioleoylphosphatidylcholine (dopc) bilayer. after system equilibration (figure ), structural changes of protein, anchor and cofactor movement were studied. we were able to select possible cypor-membrane orientation which would allow interaction with cytochrome p . in addition, spontaneous closing of open cypor was observed. however structural changes between crystal structures and structures obtain from md simulations lead us to the use of metadynamics in order to speed up the process. fmn and fad cofactor remained in close van der waals contact during the -ns long simulation stabilized by p stack interaction of fad with trp , whereas continual movement of nadph continually weakens its p stack interaction with fad. after ns of classical md additional metadynamics simulations were performed in order to investigate internal motion of cofactors during electron transfer. atoms c n (nadph) and n (fad) which are responsible for et were able to move closer to the distance of Å after adding biasing potential. this distance is more than sufficient for electron transfer to occur. after switching back to classical md cofactors got into resting positions ( Å) again. our results show that cypor undergo several structural changes and internal motions of cofactors in order to transfer electrons to its redox partner -cyp. research & utilization div., jasri/spring- , grad. school frontier sci., univ. tokyo, grad. sch. sci., univ. hyogo, japan, national institute of advanced industrial science and technology, japan, pentameric ligand-gated ion channels (plgics) are a major family of membrane receptors that open to allow ions to pass through the membrane upon binding of specific ligands. plgics are made up of five identical (homopentamers) or homologous (heteropentamers) subunits surrounding a central pore. structural information about their multiple allosteric states, carrying either an open or a closed channel, has become available by recent studies by x-ray crystallography. however, dynamic information are needed to understand their mechanism of gating, notably the long-range allosteric coupling between the agonist binding site and the ion channel gate. here we used the diffracted x-ray tracking (dxt) method ( ) to detect the motion of the extracellular and transmembrane domain two plgics: the nicotinic acetylcholine receptor (nachr) and a proton-gated bacterial ion channel from gloeobacter called glic. dxt is a powerful technique in biological science for detecting atomic-scale dynamic motion of allosteric proteins at the single molecular level and at tens of micro seconds timescale resolution. the dynamics of a single protein can be monitored through trajectory of a laue spot from a nanocrystal which is attached to the target protein immobilized on the substrate surface ( , ). dxt detects two kinds of rotational motions of nanocrystal, tilting and twisting, based on x-ray incident beam axis. dxt analysis with . ms/f time resolution showed that tilting motion of the transmembrane domain of glic and both tilting and twisting motions of the extracellular domain of glic and nachr were enhanced upon application of agonists (lowering the ph for glic, and binding of acetylcholine for nachr). the detailed dynamic information, including size effect of gold nanocrystal to the motion of them, is discussed. [ proteins possess unique structure-encoded dynamics that underlie their biological functions. here, we provide experimental evidence for an evolutionary mechanism driven solely by long-range dynamic motions without significant backbone adjustments, catalytic group rearrangements, or changes in subunit assembly. crystallographic structures were determined for several ancestral gfp-like proteins that were reconstructed based on posterior sequence predictions, using members of the stony coral suborder faviina as a model system. the ancestral proteins belong to the kaede-type class of gfps, a group of proteins that undergoes irreversible green-to-red photoconversion and is therefore frequently employed in superresolution microscopy. surprisingly, we find that the structures of reconstructed common green ancestors and evolved green-to-red photoconvertible proteins are very similar. therefore, we analyzed their chain flexibility using molecular dynamics and perturbation response scanning. we find that the minimal number of residue replacements both necessary and sufficient to support lightinduced color conversion provide for increased fold stiffness at a region remote from the active site. at the same time, the allosterically coupled mutational sites appear to increase active site conformational mobility via epistasis. these data suggest that during evolution, the locations of fold-anchoring and breathing regions have been reversed by allosteric means. therefore, we conclude that the green-tored photoconvertible phenotype has arisen from a common green ancestor by migration of a knob-like anchoring region away from the active site diagonally across the beta-barrel fold. based on titration experiments, we estimate that at ph , . % of the protein population harbors neutral side chains for his and glu , residues that form an internal salt bridge near the chromophore. we propose that this reverse-protonated subpopulation constitutes the catalytically competent state. in the electronically excited state, light-induced chromophore twisting may be enhanced, activating internal acid-base chemistry that facilitates backbone cleavage to enlarge the chromophore. in this way, a softer active site appears to be coupled to a mechanism involving concerted carbon acid deprotonation and betaelimination. dynamics-driven hinge migration may represent a more general platform for the evolution of novel enzyme activities by tuning motions in the active site. the binding of an agonist to a gpcr causes a conformational change in the receptor that leads to its activated functional state. rhodopsin, the membrane receptor responsible for photoreception in the vertebrate retina, is a prototypical gpcr and has been extensively used in structural, biochemical and biophysical studies of this class of receptors. different small molecules have been described to be capable of binding to rhodopsin. in addition, mutations in rhodopsin have been associated with retinal diseases and efforts have been carried out in order to find potential ligands that can offset the effect of these mutations. cyanidins, a group of flavonoids within the larger family of polyphenols, have been reported to stimulate chromophore regeneration of rhodopsin by means of the formation of regeneration intermediates. the aim of the current study was to evaluate the effect of the flavonoid quercetin on the conformational properties of both native bovine rhodopsin and heterologously expressed recombinant rhodopsin. rhodopsin was purified from bovine retinas by immunoaffinity chromatography, and photobleaching, thermal stability, metarhodopsin ii decay and chromophore regeneration assays were carried out in the absence or in the presence of mm quercetin. for recombinant rhodopsin, a plasmid encoding wild-type opsin was transfected into mammalian cos- cells, in the absence or in the presence of mm quercetin, harvested, regenerated with -cis-retinal, or -cis-retinal, and subsequently purified in dodecyl maltoside solution. no differences in photobleaching behavior, upon illumination, could be detected in the purified quercetin-containing samples compared to those in the absence of this flavonoid. in the case of rhodopsin, and the recombinant wild-type protein regenerated with -cis-retinal, quercetin did not significantly alter the thermal stability and rate of regeneration of the purified proteins under our experimental conditions. however, a two-fold increase in the thermal stability and a % increase in chromophore regeneration were observed for the recombinant wild-type protein regenerated with -cis-retinal in the presence of quercetin. in contrast, the presence of quercetin did not alter the electrophoretic and basic spectroscopic properties of rhodopsin, or those of the recombinant wild-type protein, suggesting no important structural alterations as a result of quercetin binding to the receptor. the positive effect of quercetin on the stability, and chromophore regeneration of rhodopsin, could be potentially used to counteract the effect of naturally-occurring misfolding mutations in rhodopsin. thus, quercetin could help stabilizing rhodopsin mutants associated with retinal diseases such as retinitis pigmentosa. furthermore, docking of the ligand, carried out on the crystallographic structure of rhodopsin (entry gzm), reveals several favorable sites for quercetin binding. one of this would be compatible with -cis-retinal suggesting a complementary binding to the receptor of this isomer which would not be compatible with -cis-retinal binding. identification of prospective allosteric sites of p by computational methods protein function is intrinsically associated with structural flexibility, so that understanding the functional properties of proteins requires going beyond the static picture produced by x-ray diffraction studies. structural flexibility can also be interpreted as a dynamic exchange between different conformational states with low energy barriers at room temperature. allosterism is a mechanism to regulate protein function associated with the plasticity exhibited by proteins. allosteric sites can be considered transient cavities that can be occupied by a small molecule with the subsequent modulation of the protein plasticity. occupation of these sites may modify the affinity of the protein for its native substrate that can be positive when the affinity increases or negative when the affinity decreases. allosterism can be used for the design of non-competitive ligands as new therapeutic agents. this mechanism of activity modulation is particularly interesting for those targets that use a common substrate for activation, like in the case of kinases to search for selective compounds. proteins can be viewed in solution as an ensemble of diverse energy accessible conformations. binding of an allosteric ligand produces a redistribution of the population of the diverse conformational states, which at the end modulate the affinity of the native substrate. allosteric sites can be characterized using computational methods by ensemble docking. it consist of characterize a set of structures that represent the accessible conformations of a protein that can then be used to perform virtual screening. in the present work we have studied prospective allosteric sites of p using computational methods. the protein is a member of the mitogen-activated protein kinases (mapks), a highly regulated group of enzymes that control a variety of physiological processes, including mitosis, gene expression, apoptosis and metabolism movement among others. the conformational profile of p was assessed using a us trajectory of accelerated molecular dynamics as sampling technique in explicit solvent. we used as starting structure the apoform of p in its inactive conformation (entry p ). the conformational features of the protein were assessed through the analysis of the variance of the most flexible regions of the protein using principal component analysis. the snapshots of the trajectory were projected onto the two principal components. subsequent cluster analysis permitted us to select a few structures for further studies. specifically, prospective biding sites were identified using a hydrophobic probe as implemented in the sitemap program. the results show previously described regulatory sites and some new prospective ones. hydrogen/deuterium exchange-mass spectrometry provides clues on the mechanism of action of min e maria t. villar , kyung-tae park , joe lutkenhaus , antonio artigues cell division in most bacteria is initiated by the formation of the z ring, an essential cytoskeletal element that serves as a scaffold for the cytokinesis machinery, at the mid body of the cell. in e coli the spatial location of the z ring is regulated by the min protein system, comprised by three major proteins: minc, mind and mine. the dynamic interaction between these proteins results in the formation of an oscillating protein gradient between the poles of the cell. this oscillation determines the position of the formation of the z ring. many aspects of this simple mechanism are beginning to be understood. in particular, the conformational changes associated with the interaction of the three min proteins between them and with the cell membrane, are of especial interest. hydrogen/deuterium exchange mass spectrometry (hdx ms) is a sensitive technique for the detection of changes in protein conformation and dynamics. the main advantages of this methodology are the ability to study native proteins in solution, the requirement for low protein concentrations, the potential to discriminate multiple coexisting conformations, and the lack of an upper limit to the size of protein to be analyzed. here we use hdx ms to analyze the dynamics of the wild type mine and of its inactive double mutant d a d a. our results show significant differences in the rates of exchange and in the total amount of deuterium exchanged at the end of the reaction between these two forms of mine. the wild type protein exchanges most of the amide hydrogen during the first few seconds of initiation of the exchange reaction. on the other hand, the mutant protein exchanges only % of the total amide hydrogen atoms during the first seconds of initiation of the exchange, and the remaining % amide hydrogen atoms are exchanged more slowly during the next few minutes of the reaction. our data are consistent with the existence of a highly flexible structure for the wild type protein and the coexistence of at least two rigid conformations for the double mutant that are undergoing a cooperative transition. interestingly, the central b-sheet forming the interface between the two subunits is protected against exchange on both proteins. these results provide insights into the conformational changes that mine undergoes during its interaction with mind. biased signalling and heteromization of the dopamine d receptor in schizophrenia and parkinson's disease pablo herrera nieto , james dalton _ , jes us giraldo _ universidad aut onoma de barcelona biased signalling and heteromization of the dopamine d receptor in schizophrenia and parkinson's disease as a significant component of dopamine signalling in the brain, the dopamine d receptor (d r), a member of the class a gpcr family, is an important target in the treatment of neurological conditions such as schizophrenia and parkinson's disease. d r shows a variety of signalling pathways through g proteins, including adenylyl cyclase inhibition, gbgpotentiation of adenylyl cyclase , and erk kinase activation, in addition to b-arrestin recruitment,. these pathways are differentially activated by some agonists and it has been suggested that d r ligands with gai/o antagonist and b-arrestin agonist activity may have anti-psychotic behavioural activity with reduced extra-pyramidal side effects. d r has also been found to form homodimers or higher-order hetero-oligomers with other gpcrs, which may modulate d r conformation and activity, thus constituting an additional form of allosteric receptor regulation. based on these findings, we have computationally modelled the full-length structure of d r, including its long intracellular loop (icl ) that is residues in length and absent in all homologous gpcr crystal structures. using state-of-the-art tools, such as rosetta for ab initio protein folding and acemd for micro-second molecular dynamics (md) simulations we have successfully de novo folded icl , which primarily consists of extensions to transmembrane helices (tmh) and and an intervening disordered histidine/proline-rich region, which is highly flexible. the latter is observed to interact with other receptor intracellular loops (icl and icl ) and appears to restrict access to the g-protein binding-site. in addition, we have docked a structurally diverse collection of ligands (biased agonists, antagonists and allosteric modulators) into our d r model and observed characteristic binding patterns suggestive of different biased signalling mechanisms. finally, through protein-protein docking with rosettadock, we have generated a complete heterodimer model of d r with the adenosine a a receptor (aa ar), where a mutual interface is formed between their respective tmhs and , as well as an association between the c-terminus of aa ar and icl of d r. this may be a particularly relevant biological complex in the treatment of parkinson's disease where antagonists of aa ar have been shown to ameliorate disease effects, potentially through direct interaction with d r. bis-ans as a tool to monitor conformational changes upon assembly of binary and ternary complexes of eif e, e-bp inhibitory protein, and the mrna 'cap specific recognition of the mrna ' terminal cap structure by the eukaryotic initiation factor eif e is the first and rate-limiting step in the cap-dependent translation. small e-binding proteins, e-bp , e-bp , and e-bp , inhibit the translation initiation by competing with eif g initiation factor for the same binding site, and by blocking the assembly of the translation machinery [ ] . our recent studies revealed intricate cooperativity between the cap and e-bp binding sites of eif e [ ] . here, we applied a fluorescent dye, , '-dianilino- , '-binaphthyl- , '-disulfonate (bis-ans) to investigate conformational changes upon assembly of binary and ternary complexes composed of human eif e, e-bp , and the mrna 'cap analogue, m gtp. the fluorescence quantum yield of bis-ans increases significantly upon binding to hydrophobic sites of proteins, making the probe a convenient tool to determine the accessibility to hydrophobic surfaces, and to monitor structural reorganisation of macromolecules [ ] . we characterised the interaction of bis-ans with eif e and e-bp by fluorescence titration. the association processes takes up to several hours until the saturation of the fluorescence signal is achieved, reflecting high flexibility of the protein structures. the association constants kas of eif e/bis-ans complexes are very high for the non-specific interaction. the kas values for eif e/bis-ans and eif e/ e-bp /bis-ans are similar ( m ), whereas the presence of m gtp results in ca. -fold weaker binding of the probe to eif e. the affinity of bis-ans for e-bp is -fold lower than that for eif e. we found no effect of either m gtp or e-bp on the fluorescence of bis-ans in complex with eif e, thus indicating lack of conformational changes around the probe on eif e/m gtp or eif e/ e-bp complex formation. it also testifies that bis-ans does not bind to the cap-binding site, despite the hydrophobic nature of this eif e region. on the contrary, addition of m gtp to the eif e/ e-bp /bis-ans complex causes an increase of the probe fluorescence, which indicates differences in the structural reorganisation in the binary, m gtp/eif e, compared with the ternary, m gtp/eif e/ e-bp , complexes, and confirms the spatial cooperation between the cap and e-bp binding sites. we also observed an increase of fluorescence for bis-ans bound to e-bp in the presence of eif e, pointing out that e-bp partially folds upon association with eif e. in summary, our results provide a deeper insight into the structural aspects of the molecular interaction at early stages of the cap-dependent translation. acknowledgements: this work was supported by the bst /bf project from university of warsaw background: beta -glycoprotein (b gpi) is a protein abundantly present in human plasma and highly conserved in all mammals. b gpi has been identified as the major antigen in the antiphospholipid syndrome (aps), a severe thrombotic autoimmune disease. despite its importance in the pathogenesis of aps, the physiological role of b gpi is still elusive. in a previous work we have demonstrated that b gpi significantly prolongs the clotting time in fibrin generation assays, and inhibits aggregation of gel-filtered platelets (ic . um), either isolated or in whole blood, by inhibiting cleavage of par on intact platelets (ic . um) and in solution. importantly, b gpi does not alter the ability of thrombin (fiia) to generate the anticoagulant protein c, with or without thrombomodulin added. hence, we concluded that b gpi inhibits the key procoagulant properties of fiia, without affecting its unique anticoagulant function. we also proposed that b gpi, together with other more efficient anticoagulant pathways such as thrombomodulin-fiia -protein c and antithrombin iii-fiia, may function as a mild anticoagulant in vivo especially in those compartments were the efficacy of thrombomodulin is limited, as in the large vessels, or is even absent, as in the brain vasculature. aims: lacking the threedimensional structure of b gpi-thrombin complex, the aim of this work is to identify the peptide regions either on thrombin and b gpi involved in complex formation. results: data obtained by fluorescence and surface plasmon resonance (spr) indicated that b gpi interacts whit fiia whit physiological affinity (kd nm). kd values calculated by reverting the interacting systems are very similar to each other (kd nm), suggesting that b gpi in the mobile phase has a conformation which is competent for the binding to immobilized fiia. the affinity of fiia for immobilized b gpi is markedly decreased by increased ionic strength (i.e. kd increases by -fold going from . m to . m), suggesting the electrostatic interactions play a key role in fiia -b gpi recognition. filling/inactivation or perturbation of fiia active site does not alter the affinity of fiia for immobilized b gpi, confirming that the active site is not involved in the interaction. mapping of thrombin binding sites with specific exosite-directed ligands (i.e. hirugen, gpibalpha, hd aptamer) and thrombin analogues having the exosites variably compromised (i.e. prothrombin, prethrombin- , alpha-thrombin), reveals that the positively charged exosite-ii of fiia plays a key role in b gpi binding. from the docking model of the bb gpi-thrombin complex, we identified a highly negatively charged segment - in domain v of b gpi interacting with positively charged pathes in thrombin exosite ii. the synthetic peptide b gpi( - ) was able to bind to fiia with an affinity (kd nm) comparable to that of full-length b gpi, deduced from fluorescence or spr measurements and to compete in spr measueremnts with the binding of full-length b gpi to thrombin. hence, combining experimental and theoretical data, we obtained a reliable model of the b gpi-thrombin complex. metalloproteases are one of the most diverse types of proteases, presenting a wide range of folds and catalytic metal ions. in the case of the merops ma clan, where most of the known metalloproteases are grouped based on the consensus hexxh sequence motif, a single catalytic zinc ion and common fold architecture [ ] . despite these common features, members from distinct families present distinct domain composition and topology. given our interest in developing new tailor-made metalloproteases for bioengineering applications, an in-depth understanding of the factors governing their function is required. protein internal dynamics includes the space of functionally-relevant structural changes occurring during an enzymatic reaction, and there is an increasing understanding on how it relates with protein sequence and structure evolution. therefore, we have recently assessed how the structural heterogeneity of metalloproteases relates with the similarity of their dynamical profiles [ ] . first, the dynamical profile of the clan ma type protein thermolysin, derived from the anisotropic network model, was evaluated and compared with those obtained from principal component (pc) analysis of a set of crystallographic structures and essential dynamics (ed) analysis of a ns molecular dynamics simulation trajectory [ ] . a close correspondence was obtained between normal modes (nm) derived from the coarse-grained model and experimentally-observed conformational changes (rmsip between nm -nm and pc of . ), corresponding to functionally-relevant hinge bending motions that were shown to be encoded in the internal dynamics of the protein (cumulative overlap of ed -ed and pc of . ). next, dynamics-based comparison methods that employ a related coarse-grained model (b-gaussian elastic network model) was made for a representative set of ma clan members [ ] , allowing for a quantitative description of its structural and dynamical variability. although members are structurally similar ( % pairs with dalilite z-score > . ), they nonetheless present distinct dynamical profiles ( % of pairs with aladyn p-value > . ), with no identified correlation between structural and dynamical similarity. for cases where high dynamical similarity was observed, the respective modes corresponded to hinge-bending motions encompassing regions close to the active site. further inspection of the produced alignments indicates that for ma clan metalloproteases, conservation of internal dynamics has a functional basis, namely the need for maintaining proper intermolecular interactions between the protein and respective substrate. previously unnoticed dynamical similarity between clan members botulinum neurotoxin type a, leishmanolysin and carboxypeptidase pfu was also found. together, these results suggest that distinct selective pressure mechanisms acted on metalloprotease structure and dynamics through the course of evolution. this work shows how new insights on metalloprotease function and evolution can be assessed with comparison schemes that incorporate additional information of protein dynamics. glucokinase from antarctic psychrotroph pseudoalteromonas sp. as- (psgk) has a higher specific activity at low temperatures and a higher thermal stability than its mesophilic counterpart from e. coli (ecgk). in order to elucidate the structural basis for cold-adaptation and thermal stabilization of psgk, we have determined the crystal structure of psgk at . Å and compared it with the ecgk structure. psgk is a homodimer of the subunit of amino acid residues. each subunit consists of two domains, a small a/b domain (residues - and - ) and a large a b domain (residues - ). the active site is located in a cleft formed between the two domains. the identity of amino acid sequence between psgk and ecgk was %, but three dimensional structures of them are very similar to each other, having the conserved catalytic residues and substrate-binding residues. the analysis of the mainchain temperature factors revealed that the regions of small domain and the hinge region connecting two domains of psgk showed higher temperature factors with a lower number of intramolecular hydrogen bonds and ionic interactions than the corresponding regions of ecgk. however, the large domain regions of psgk showed lower temperature factors with a higher number of intramolecular hydrogen bonds than ecgk. furthermore, the atomic temperature factors of catalytic asp on the small domain were higher, but those of glucose-binding glu , his , and glu on the large domain were lower than ecgk. these results suggest that highly flexible hinge region and the catalytic residue on the small domain of psgk may contribute to its cold-adaptation, namely higher activity at low temperatures, whereas a more rigid structure of the large domain of psgk stabilizes its overall structure more strongly than ecgk. nowadays non-waste technologies in synthetic chemistry become more and more popular. such processes are often carried out using different enzymes. dehydrogenases represent the large group of enzymes, which are widely used in synthesis of chiral compounds and other useful molecules. such enzymes need nadh or nadph as a cofactor and due to high cost of reduced coenzymes a cofactor regeneration system is an obligate part in such kind of processes. it was shown that formate dehydrogenase (fdh, ec . . . .) is one of the best enzymes for nad(p)h regeneration. fdh catalyses the reaction of formate oxidation to carbon dioxide coupled with reduction of nad(p) to nad(p)h. the main advantages of fdh are the irreversibility of catalyzed reaction, low price of formate ion and wide ph optimum of activity. our laboratory has the largest collection of formate dehydrogenases from different sources. many fdh genes from bacteria, yeasts and plants were cloned and enzymes were expressed in active and soluble forms. mutant formate dehydrogenases from bacterium pseudomonas sp. show the highest thermal stability as well as activity in comparison with other reported formate dehydrogenases. now we have focused on eukaryotic genes. the recombinant enzymes from soya glycine max (soyfdh), arabidopsis thaliana (athfdh), moss physcomitrella patens (ppafdh) and yeast ogataea parapolymorpha (opafdh) were obtained by genetic engineering methods. it was revealed, that soyfdh has the best michaelis constants among all known fdhs, but it's less thermally stable compared to other fdhs. new mutant forms of soyfdh with excellent catalytic characteristics and high thermal stability were obtained by protein engineering. other enzymes (athfdh, ppafdh and opafdh) are comparable in their stability with majority of bacterial enzymes (but not with psefdh), so all the new obtained fdhs can be successfully used for cofactor regeneration. marmara university, wellesley college, antibiotics are essential therapeutic drugs widely used in the treatment of bacterial infections. unfortunately, misuse of these drugs resulted in the development of bacterial defense mechanisms. blactamase synthesis is among these mechanisms that renders b-lactam antibiotics ineffective. understanding the dynamic behavior of this enzyme is an important step in controlling its activity. in a former study, the importance of highly conserved w in modulating the hinge type h motion was reported. in the light of this information, mutant tem- b-lactamase enzymes with w a, w f and w y substitutions were constructed. wild-type and mutant tem- b-lactamases purified with ni affinity chromatography were subjected to enzyme assay using centa as the substrate. with w f and w y mutations, the remaining activity was approximately % of the initial activity. however with the w a mutation, activity was totally lost. structural studies of the w a mutant with cd and florescence spectroscopy indicated that there was no major change in the overall structure. however this mutation disrupted the interactions of w which resulted in an increase in the flexibility of this region of the protein. this project was supported by t € ub _ itak project no m . light-switchable zn binding proteins to study the role of intracellular zn signaling stijn aper , maarten merkx zn plays an important catalytic and structural role in many fundamental cellular processes and its homeostasis is tightly controlled. recently, free zn has also been suggested to act as an intracellular signaling molecule. to get increased understanding of the signaling role of zn we are developing light-switchable zn binding proteins to perturb the intracellular zn concentration using light. these protein switches consist of two light-responsive vivid domains and the zn binding domains atox and wd , linked together with flexible peptide linkers. in the dark, zn is tightly bound in between the two zn binding proteins. light-induced dimerization of the vivid proteins disrupts this interaction and thus results in zn release. the fluorescent proteins cerulean and citrine were attached to the vivid domains to allow the different conformational states of the protein switch to be monitored using fret. zn titrations revealed a -fold decrease in zn affinity going from dark-to light-state for the initial design, which was further improved to -fold by optimizing the linkers between the protein domains. in addition, the zn affinities of both states were tuned to be optimal for intracellular applications. switching between the high affinity dark-state and the low affinity lightstate was found to be reversible for at least two light-dark cycles. following the in vitro characterization, we are currently assessing the performance of this genetically encoded 'caged' zn in mammalian cells. proteins as supramolecular building blocks: engineering nanoscale structures school of biological sciences, university of auckland, school of biological sciences, victoria university proteins hold great promise in forming complex nanoscale structures which could be used in the development of new nanomaterials, devices, biosensors, electronics and pharmaceuticals. the potential to produce nanomaterials from proteins is well supported by the numerous examples of self-assembling proteins found in nature. we are exploring self-assembling proteins for use as supramolecular building blocks, or tectons, specifically the n-terminal domain of a dna binding protein (nterm-lsr ) and a typical -cys peroxiredoxin (hsprx ). non-native forms of these proteins have been designed undergo selfassembly into supramolecular structures in a controllable manner. self-assembly of nterm-lsr is initiated via proteolytic cleavage, thereby allowing us to generate supramolecular assemblies in response to a specific trigger. we will show that the degree of oligomerisation can be controlled by variations in environmental conditions such as ph and protein concentration. furthermore, via protein engineering, we have introduced a new "switch" for oligomerisation via enteropeptidase cleavage. the new construct of nterm-lsr can be activated and assembled in a controlled fashion and provides some ability to alter the ratio of higher ordered structures formed. hsprx has been shown to oligomerise into dimers, toroids, stacks and tubes in response to specific triggers such as ph and redox state. in this work we have utilised the histidine tag to further control the assembly of this versatile protein tecton. we will show that minute variations in ph can induce oligomersation of hsprx toroids into stacks and tubes. furthermore, by utilising the histidine tag as a ligand we can bind divalent metals to these supramolecular structures. this not only drives the formation of higher ordered oligomers but also provides a facile route which may facilitate the functionalisation of these protein nanoscale structures after they have been assembled. danielle basore , , rajesh naz , scott michael , sharon isern , benjamin wright , katie saporita , donna crone , christopher bystroff , , biological sciences, rensselaer polytechnic institute, cbis, rensselaer polytechnic institute, chemical and biological engineering, rensselaer polytechnic institute, computer science, rensselaer polytechnic institute, obstetrics and gynecology, west virginia university, unintended pregnancy is a worldwide public health concern, with million pregnancies being classed as unintended in . the magnitude of this number clearly indicates an unmet need in terms of contraception. methods that are currently available are effective, but exhibit many problems. side effects, ease of use, cost, and availability are all concerns. we propose a contraceptive vaccine that would be safe, effective, long-lasting, cheap, and reversible. our vaccine would prevent pregnancy by targeting sperm with antibodies raised in the woman's body. several approaches have been taken to developing a contraceptive vaccine in recent years. the most successful so far has been using human chorionic gonadotropin (hcg), a hormone produced during pregnancy, as an antigen . the hcg vaccine progressed to phase clinical trials, but only displayed an % efficacy, which is insufficient for a contraceptive. our lab uses a structure based approach to the design of an anti-sperm antigenic protein. we believe this will raise a more vigorous immune response that will produce a longer lasting titer. the catsper complex is a heterotetrameric calcium channel found in the tail region of sperm . each subunit of the complex contains an exposed loop known as the p-loop. the p-loop is unique on the surface of sperm because it is not glycosylated, allowing antibodies to potentially recognize and bind it. ylp is a twelve residue peptide that mimics the glycans in the glycocalyx of sperm . ylp is a member of the flitrx library, and in mice, produced protective titers that were reversible both voluntarily and involuntarily. our designs will introduce these two potential antigens into a loop of the l protein of human papilloma virus. l spontaneously assembles into virus like particles, and will aid in the production of a robust immune response. protein carriers for passage of the blood-brain barrier sinisa bjelic medical solutions that help protein therapeutics accumulate into the brain are crucial for future treatment of neurological disorders. biodrugs have a tremendous potential to treat disorders of the nervous system, but their efficiency has been severely restricted. to reach the brain all drugs must traverse the blood-brain barrier (bbb) -a permeable wall that separates blood from the brain -whose main function is to protect the nervous system from environmental influences of bacteria and toxins. unfortunately the bbb is also the culprit that effectively blocks access to therapeutics required for treatment of neurological diseases. a way to boost exposure of therapeutics across the bbb is to piggyback onto the transferrin receptor, a multidomain protein anchored in the membrane, which is involved in the physiological facilitation of iron uptake. here i present research that aims at successfully developing potent protein carriers for transferrin receptor-mediated passage of the bbb by using computational protein design in combination with yeast display methodology for hit validation and optimization. the longterm goal is to couple therapeutics -as for example drugs against alzheimer's -to the designed carriers to increase the brain uptake and cure neurological disorders. medium-throughput multistep purification of coagulation factor viia jais r. bjelke , gorm andersen , henrik Østergaard , laust b. johnsen , anette a. pedersen , tina h. glue there is a need of medium-to-high throughput purification of low-titre recombinant protein variants for screening to identify the final biopharmaceutical lead. such proteins include coagulation factors to be used for treatment of haemophilia and other bleeding disorders. at novo nordisk we have established a platform for production of recombinant coagulation factor viia variants, which include a spectrum of single-point mutations to large domain insertions. the variants were produced using transiently transfected hek f, hkb or choebnalt (qmcf technology) suspension cells. harvest cultivations were typical in the range of . -to l. a -step continuous, multistep purification method was implemented on € aktaxpress systems (ge healthcare). the interlinked process steps include capture using an immunoaffinity column, polish, concentration and buffer exchange using an anion-exchange column and proteolytic activation of the zymogen variant forms using a coagulation factor xaimmobilized column. buffers were designed such that elution from the capture column was aligned with binding conditions on the polish column to avoid a desalting step in-between. the following and final enzymatic activation was optimized with regards to flow rate to ensure full conversion while minimizing unwanted secondary cleavages in factor viia. the final products were fractionated in sharp chromatographic peaks ready for characterization. hplc and sds-page analyses showed a solid quality of the produced variants and more than variants have been produced in sub mg scale using the outlined method. biomimetic sequestration of co : reprogramming the b domain of protein g through a combined computational and experimental approach esra bozkurt , ruud hovius , thereza a. soares , ursula rothlisberger ecole polytechnique f ed erale de lausanne, federal university of pernambuco protein engineering is a powerful tool to generate highly specific enzymes for biomimetic production of chemicals. among many applications, the development of enzymes to accelerate carbon dioxide fixation is a possible route to limit co emission. in this project, we are inspired by the ancient enzyme carbonic anhydrase which efficiently catalyzes the reversible hydration of carbon dioxide in the presence of a zinc ion active site. to create an efficient biocatalyst, the engineered gb domain containing a his cys zn (ii) binding site was used as a starting point. in subsequent work, b domains comprising of his wat zn (ii) binding sites have been rationally designed to produce carbonic anhydrase mimics. the re-engineering was accomplished through a series of mutations to orient the zinc bound reactive species to form a hydrogen bond network in the active site while retaining the native secondary structure. we performed classical molecular dynamics (md), quantum mechanics/molecular mechanics (qm/ mm) simulations and metadynamics, with the aim to explore potential catalytic roles of the reengineered b domains and to elaborate the reaction mechanism. briefly, we introduced novel zn (ii) binding sites into thermostable b domain. in parallel, experiments are underway. wild-type protein was expressed and purified. structural and mutagenesis studies are ongoing. the results emphasize the power of theoretical work to enable the mimicking of nature's enzymes for desired catalytic functions. the roles of entropy and packing efficiency in determining protein-peptide interaction affinities diego caballero , , corey o'hern , , , , lynne regan , , physics, yale university, integrated graduate program in physical and engineering biology, yale university, mechanical engineering and materials science, yale university, applied physics, yale university, molecular biophysics and biochemistry, yale university, chemistry, yale university despite many recent improvements in computational methods for protein design, we still lack a quantitative and predictive understanding of the driving forces that control protein stability, for example, we do not know the relative magnitudes of the side-chain entropy, van der waals contact interactions, and other enthalpic contributions to the free energy of folded proteins. in addition, we cannot reliably predict the effects of point mutations on enzyme specificity or sequence tolerance in ligand binding sites. the tetratricopeptide repeat (tpr) motif is a common and versatile protein system that has been used as a model to study protein-protein interactions. for example, recent studies have experimentally measured the binding affinity and specificity for different tpr binding pockets and peptide ligands and generated a ranking of the protein-peptide pairs with the highest affinity. to gain a fundamental understanding of the interplay between atomic close packing and fluctuations of side-chain conformations in protein-peptide binding pairs, we performed all-atom langevin dynamics simulations of key residues near the binding interface of tpr proteins and their cognate peptides. the langevin dynamics simulations enabled us to calculate the entropy and potential energy of side chain conformations in the presence of backbone fluctuations for each protein-peptide pair. we compile rankings of the stability and affinity of mutant tpr-peptide structures to those obtained from experimental studies. this research has enhanced our ability to rationally manipulate protein-peptide interfaces. advances from this research will enable the design of tpr modules that specifically recognize biologically important proteins. monitoring protein-protein interactions using tripartite split-gfp complementation assays protein-fragment complementation assay (or pca) is a powerful strategy for visualizing protein-protein interactions in living cells. previously described split-gfp based sensors suffer from the poor solubility of individual pca fragments in addition to background signal originating from their spontaneous selfassembly ( ). we developed a new encoded genetic reporter called "tripartite split-gfp" for visualizing protein-protein interactions in vitro and in living cells. the assay is based on tripartite association between two twenty amino-acids long split-gfp tags, gfp and gfp , fused to interacting protein partners, and the complementary gfp - detector. when proteins interact, gfp and gfp selfassociate with gfp - to reconstitute a functional gfp ( ). using coiled-coils and frb/fkbp model systems we characterize the sensor in vitro and in escherichia coli. we extended our studies to mammalian cells and examine the fk- inhibition of the rapamycin-induced association of frb/fkbp . the small size of these tags and their minimal effect on fusion protein behavior and solubility should enable new experiments for monitoring protein-protein association by fluorescence and for screening modulators of complex formation in cell-based assays. aldehyde dehydrogenases (aldhs) catalyze the oxidation of aldehydes to their corresponding acids using nad(p) as coenzyme. these enzymes are responsible for the detoxification of lipid peroxidation products, which have been involved in the etiology and pathogenesis of different diseases involving increments in oxidative stress. recent data from our group, showed that aldh a is resistant to inactivation by lipid peroxidation products, even at concentrations - times higher than those required to inactivate aldh a and aldh . the amino acids sequence of the aldehyde-binding site of the three enzymes was analyzed, and it was found that the enzymes susceptible to the effect of lipid peroxidation products (aldh a and aldh ), have cys residues flanking the reactive cys (position ), based on this criteria and considering that these aldehydes react preferentially with cysteine, a mutant of aldh was generated changing the cys residues adjacent to cys . the mutant aldh -cys thr-cys val, was resistant to the inactivation by acrolein and -hne, even at concentrations -fold higher than those required to inactivate aldh . however, the mutant presented values of km , and -fold higher for acrolein, propionaldehyde and acetaldehyde, respectively, compared to the wild type enzyme, but showed a catalytic efficiency similar to the parent enzyme. these data revealed that cys residues near to the reactive cys in aldh are important in the inactivation process induced by lipid aldehydes, but also participate in determining the specificity for the substrates in this enzyme. small molecule-assisted shutoff: a widely applicable method for tunable and reversible control of protein production h. kay chung , conor jacobs , yunwen huo , jin yang , stefanie krumm , richard plemper , , roger tsien , michael lin department of biology, stanford university, department of pediatrics, stanford university, department of pharmacology, university of california san diego, department of pediatrics, emory university, institute for biomedical sciences, georgia state university, department of chemistry and biochemistry, university of california san diego, howard hughes medical institute, university of california san diego, the ability to quickly control the production of specific proteins would be useful in biomedical research and biotechnology. we describe small molecule-assisted shutoff (smash), a technique in which proteins are fused to a self-excising degron and thereby expressed in a minimally modified form by default. degron removal is performed by a cis-encoded hepatitis c virus (hcv) protease, so that applying clinically available hcv protease inhibitors causes degron retention on subsequently synthesized protein copies and suppresses further protein production. we find that smash allows reversible and dosedependent shutoff of various proteins with high dynamic range in multiple cell types, including yeast. we also successfully use smash to confer drug responsiveness onto a rna virus for which no licensed drug inhibitors exist. as smash does not require permanent fusion of a large domain, it should be useful when control over protein production with minimal structural modification is desired. furthermore, as smash only uses a single tag and does not rely on modulating protein-protein interactions, it should be easy to generalize to multiple biological contexts. top, a protein of interest is fused to the smash tag via a hcv ns protease recognition site. after protein folding, the smash tag is removed by its internal ns protease activity, and is degraded due to an internal degron activity. bottom, addition of protease inhibitor induces the rapid degradation of subsequently synthesized copies of the tagged protein, effectively shutting off further protein production. vaccine development has emerged, epitope-focused immunogens, but in the past these have failed to deliver the expected outcome. here, we employed a new computational design methodology (rosetta fold from loops or ffl) to design epitope-focused immunogens. ffl was devised to insert structurally defined functional sites into protein scaffolds. throughout the ffl stages the structure of the scaffold is folded and its sequence designed to stabilize the desired functional conformation of the inserted site. we used ffl to design epitope-focused immunogens for the respiratory syncytial virus (rsv), for which despite the intense research we are still lacking an approved vaccine. we designed three-helix bundles harboring an rsv epitope, that was previously co-crystallized with the neutralizing antibody motavizumab. the designs were thermodynamically stable (tm > ˚c) and showed extremely high affinities to motavizumab (kd pm). structural characterization through x-ray crystallography of antibodybound and unbound scaffolds showed good agreement to the computational models in the overall structure (rmsd - . Å) and exquisite mimicry of the epitope region (rmsd - . Å), when compared to the peptide-epitope in complex with motavizumab. the designed immunogens were used to immunize non-human primates (nhp), and approximately % of the cohort developed rsv neutralizing activity, in some instances with high potency. to evaluate the therapeutic relevance of the elicited neutralization activity, we compared the nhp neutralization titers to those of human sera after natural rsv infection, which generally yields protective levels of antibodies. the neutralization potency of the best nhp responders was comparable to that of the human sera. to better understand the features of the antibodies elicited, we isolated several rhesus monoclonal antibodies (rhmabs) from the animal that exhibited the most potent neutralization. two of the rhmabs bound to the immunogen with very high affinity (kd pm) and were potent rsv neutralizers. interestingly, these rhmabs were approximately fold more potent than the fda-approved prophylactic antibody palivizumab. our results provide the first proof-of-principle for epitope-focused vaccine design, and demonstrate the power of the ffl figure . schematic of nucleotide binding, exchange and hydrolysis in tubulin, and its coupling to mt assembly. exchange of gdp (orange) for gtp (magenta) at the e-site in b-tubulin (blue) happens in the unpolymerized dimer (left). the active, gtpbound tubulin dimer adds to a growing mt (right). interaction of the incoming a-tubulin (green) with the e-site nucleotide at the plus end of a mt (with b-tubulin exposed) results in gtp hydrolysis. the mt cartoon (bottom right) shows an oversimplified representation of a gtp cap as it first grows by tubulin addition and then shrinks by polymerization-coupled gtp hydrolysis (here b-tubulin that is bound to gtp is shown in red and that bound to gdp is shown in blue). cryo-em density map (emdb- ) and atomic model (pdb: jak) for an eb -decorated mt bound to gtpgs. a-tubulin, b-tubulin and eb are colored green, blue, and orange, respectively. computational methodology. we anticipate that ffl will be useful for a variety of other challenges in the computational design of functional proteins. designed repeat proteins as templates for photoactive molecules and fluorescent nanoclusters sara h. mejias , , antonio aires , , javier l opez-andarias , pierre couleaud , , begoña sot , , carmen atienza , nazario mart ın , , aitziber l. cortajarena , imdea nanoscience, c/faraday, , ciudad universitaria de cantoblanco , cnb-csic-imdea nanociencia associated unit "unidad de nanobiotecnolog ıa", departamento de qu ımica org anica i, facultad de qu ımica, universidad complutense self-assembly of biological molecules into defined functional structures has a tremendous potential in nanopatterning, and the design of novel bionanomaterials and functional devices. molecular selfassembly is a process by which complex three-dimensional structures with specified functions are constructed from simple molecular building blocks. we present first the study and characterization of the assembly properties of modular repeat proteins, in particular designed consensus tetratricopeptide repeats (ctprs), and their application as building blocks in order to generate functional nanostructures and biomaterials. ctpr proteins can be assembled into self-standing thin films, and thin nanometer fibers in solution. in this work, we show the use of the designed consensus repeat proteins as scaffolds to template: ( ) photoactive organic molecules, and ( ) fluorescent nanoclusters. .we explore the potential of ctpr proteins to arrange donor-acceptor pairs for electro-active materials. in particular, porphyrin rings arranged by ctprs in a defined distance and orientation for favoring face-to-face orientation which should lead to an improvement in the optoelectronic properties. our results confirm the successful ability of ctpr proteins to be used as scaffold for ordering organic chromophores, while preserving their structure. the unique self assembly properties of ctpr scaffolds have been exploited to generate ordered conductive films of the protein-porphyrin conjugates. these results open the door to fabricate hybrid protein-based solid devices. .we show results on the ability of ctpr to encapsulate and stabilize fluorescent gold nanoclusters. we investigated the influence of the protein sequence in the final properties of the nanoclusters. the structural and functional integrity of the protein template is critical for future applications of the protein-cluster complexes. therefore synthetic protocols that retain the protein structure and function have been developed. as a proof of concept, a ctpr module with specific binding capabilities has been successfully used to stabilize nano clusters. biohybrid photoelectrochemical cells have been developed by functionalizing the hematite photoanode with the light-harvesting cyanobacterial protein c-phycocyanin (pc) yielding a substantial enhancement of the photocurrent density. photoelectrochemical cells combining light-harvesting proteins and inorganic semiconductors have potential for the use in artificial photosynthesis. in this work we present processing routes for the functionalization of hematite photoanodes with pc, including in situ co-polymerization of pc with enzymatically-produced melanin and using a recombinantly produced pc . moreover, recombinant forms of the light-harvesting protein c-phycocyanin from synechocystis sp. pcc were engineered to carry a peptide with affinity for hematite. similarly, a bacterial laccase was engineered to acquire affinity for hematite. results obtained from the different approaches to hematite functionalization and the advantages offered by protein engineering will be presented. minimizing a suitable free energy expression is arguably the most common approach in (ab initio) protein structure prediction. the achieved accuracy depends crucially on the quality of the free energy expression in use. here, we present corrections to existing free energy expressions which arise from the thermal motion of the protein. we (i) devise a term accounting for the vibrational entropy of the protein, and (ii) correct existing potentials for 'thermal smoothing'. (i) vibrational entropy is almost always neglected in free energy expressions as its consideration is difficult. this practice, however, may lead to incorrect output because distinct conformations of a protein can contain very different amount of vibrational entropy, as we show for the chicken villin headpiece explicitly [ ] . for considering vibrational entropy, we suggest a knowledge based approach where typical fluctuation and correlation patterns are extracted from known proteins and then applied to new targets. (ii) at ambient conditions, timeaveraged potentials of proteins are considerably smoothened due to thermal motion where the strength of this effect varies strongly between atoms. distinguishing these inhomogeneities by introducing new atom species regarding their locale environment can therefore increase the precision of time-averaged potentials [ ] . extraction of general principles from the continually growing protein data bank (pdb) has been a significant driving force in our understanding of protein structure. atomistic or residue-level statistical potentials, secondary-structural propensities, and geometric preferences for hydrogen bonding are among the classical insights that arose from observations in the pdb. given the magnitude of structural data available today, it is likely that many quantitative generalizations remain to be made. here we hypothesize that the pdb contains valuable quantitative information on the level of local tertiary structural motifs (terms), with term statistics reflecting fundamental relationships between sequence and structure. we define a term to be the structural fragment that captures the local secondary and tertiary environments of a given residue, and put our hypothesis through a series of rigorous tests. first, we show that by breaking a protein structure into its constituent terms, and querying the pdb to characterize the natural ensemble around each, we can estimate the compatibility of the structure with a given amino-acid sequence through a metric we term "structure score." considering submissions from recent critical assessment of structure prediction (casp) experiments, we find a strong correlation (r . ) between structure score and model accuracy, with poorly predicted regions readily identifiable. this performance exceeds that of leading atomistic statistical energy functions. next, we show that by considering the terms of a structure that are affected by a given mutation, and mining the pdb to characterize sequence statistics associated with each, we are able to predict mutational free energies on par with or better than far more sophisticated atomistic energy functions. finally, we ask whether term statistics are sufficient to enable the design of proteins de-novo. we demonstrate that given a native backbone conformation, term considerations alone with no input from molecular mechanics correctly predict roughly the same fraction of amino acids from the corresponding native sequence as state-ofthe-art computational protein design methods. knowledge-based energy functions have already put pdb statistics to good use by parsing structural environments into geometric descriptors, generally assuming their conditional independence. our results suggest that it may now be possible to instead consider local structural environments in their entirety, asking questions about them directly. if this is the case, then the pdb is an even larger treasure trove of information than it has been generally known to be, and methods of mining it for term-based statistics should present opportunities for advances in structure prediction and protein design. comprehensive understanding of a protein fold is intertwined with successful design. recent advances in designing de novo structures have shown that proteins can be designed for a few globular and helical folds. however, designing all-b structures and barrels remains challenging because loops and intricate long range interactions that are important in these topologies are difficult to control. for designing novel catalysts, the (a/b) -barrel (or tim-barrel) fold is one of the most important examples, for it is the most common topology for enzymes. for almost year, attempts in designing de novo tim barrel structures have all resulted in poorly folded proteins. here we describe the successful design of a -fold symmetrical (a/b) barrel directly from geometrical and chemical principles. designed variants with a wide range of stabilities from being molten globules to cooperatively folded proteins were experimentally characterized, and the results revealed the importance of sidechain-backbone hydrogen bonding for defining the characteristic a/b-barrel. the residue tim barrel structure is among the smallest tim-barrels and has a fully-reversible melting temperature of c. the x-ray crystal structure shows atomic-level agreement with the design model. despite this structural similarity, psi-blast searches do not identify sequence similarities to known tim-barrel proteins. more sensitive profile-profile searches suggest that the design is sufficiently distant from other native tim-barrel superfamilies to be in a superfamily of its own, further implying that nature has only sampled a subset of the sequence space available to the tim-barrel fold. the ability to de novo design tim-barrels opens new possibilities for custom-made enzymes. university of texas southwestern medical center, biofrontiers institute, university of colorado creation of new molecular sensors and actuators based on fluorescent proteins relies on methods for identifying complex photophysical phenotypes and subsequently performing separations on cell populations. we developed a microfluidic flow cytometry approach tailored to interrogating the performance of genetically-encoded fluorophores and present the results of studies employing this technology. the system screens cell-based libraries on the basis of multiple photophysical parameters relevant to imaging, including brightness, photostability, and excited-state lifetime (i.e. a proxy for fluorescence quantum yield) at a rate of up to cells/sec. in a first generation of experiments, molecular dynamics-guided design was used to create a library of mcherry mutants that was screened with this system, resulting in the identification of a variant with a higher stability b-barrel and improved photostability but with a decreased brightness due to reduction in the fluorescence quantum yield. to avoid inadvertent decreases in this important performance criterion, subsequent rounds of selection were performed on the basis of both photostability and excited-state lifetime as sorting criteria. in these second generation selections, mutations were designed to target pathways of oxygen access through the bottom of the bbarrel in addition to a position that directly interacts with the chromophore. furthermore, subsequent rounds of screening were used to improve folding and maturation. the multiparameter sort identified multiple clones with up to -fold improved photostability and up to double the excited-state lifetime of the parent mcherry fluorescent protein. the best mutant we identified produces one order of magnitude more photons before photobleaching compared to mcherry, at excitation conditions characteristic of confocal fluorescence microscopy. our results demonstrate the utility of combining moleculardynamics-guided library design with technology for photophysics-based selections. we anticipate that the new fluorescent proteins obtained in this work will find use in low-copy-number and long-duration imaging live cell imaging applications in cell-lines created by genomic editing techniques. targeted protein degradation achieved through a combination of degrons from yeast and mammalian ornithine decarboxylase rushikesh joshi , ratna prabha c. the maharaja sayajirao university of baroda targeted protein degradation achieved through a combination of degrons from yeast and mammalian ornithine decarboxylase targeting the over accumulated protein in the cell for degradation using specific degrons is an emerging research area. the degradation of the vast majority of cellular proteins is targeted by the ubiquitin-proteasome pathway. but in the case of ubiquitin independent protein degradation, odc/az system is more effective in achieving targeted protein degradation than other types of degradation . ornithine decarboxylase (odc) is key regulatory enzyme in the biosynthesis of polyamines. the protein has two domains namely, n terminal a/b barrel domain and c-terminal b-sheet domain. degradation of odc is mediated by polyamine inducible protein, antizyme (az). antizyme interacts with odc on n-terminal region, which results in degradation of odc by proteasomes. in mammalian odc the c-terminal has an unstructured tail of residues, which pulls odc into proteasome for degradation. it was reported earlier by coffino's group that the unstructured tail acts as a degron in chimeric fusion with gfp . in yeast, same function is achieved by n-terminal residues . present study focuses on accomplishing targeted protein degradation in saccharomyces cerevisiae by adding these two degradation signals or degrons of yeast odc and mammalian odc as tags to a reporter protein. we have selected two degrons namely, n terminal a/b barrel domain of yeast odc and c-terminal residues of mouse odc and grafted them to n and c-terminus of the reporter protein yegfp. degradation of yegfp and yegfp fusion with degrons of odc (degron-yegfp) were monitored by western blot using anti-gfp antibody and fluorescence spectroscopy. initially, the amount of degron-yegfp fusion protein was very low compared to control yegfp. it means that the chimeric protein underwent rapid degradation in the cells. after inhibition of proteasome, increase in the level of degron-yegfp was observed, confirming that the degrons cause rapid degradation of reporter protein through proteasome. earlier, we have also tagged ubiquitin from yeast with last residues of modc and observed enhanced degradation of ubiquitin in saccharomyces cerevisiae. therefore, both the degrons of odc alone and in combination are capable of decreasing stability of reporter protein in the cells. however, the combination of degrons is more effective than either of them in isolation. enzymes fold into unique three-dimensional structures, which underlie their remarkable catalytic properties. the requirement that they be stably folded is a likely factor that contributes to their relatively large size (> , dalton). however, much shorter peptides can achieve well-defined conformations through the formation of amyloid fibrils. to test whether short amyloid-forming peptides might in fact be capable of enzyme-like catalysis, we designed a series of -residue peptides that act as zn dependent esterases. zn helps stabilize the fibril formation, while also acting as a cofactor to catalyze acyl ester hydrolysis. the fibril activity is on par with the most active to date zinc-protein complex. such remarkable efficiency is due to the small size of the active unit (likely a dimer of -residue peptides), while the protein is at least -fold larger in molecular weight. the observed catalytic activity is not limited to ester hydrolysis. we have designed copper binding peptides that are capable oxygen activation. these results indicate that prion-like fibrils are able to not only catalyze their own formation -they also can catalyze chemical reactions. thus, they might have served as intermediates in the evolution of modern-day metalloenzymes. these results also have implications for the design of self-assembling nanostructured catalysts including ones containing a variety of biological and nonbiological metal ions. rational design of the cold active subtilisin-like serine protease vpr with improved catalytic properties and thermal stability abstract proteinase vpr, from a psychrophilic vibrio species and its thermophilic structural homologue, aqualysin i (aqui) from thermus aquaticus, we set out to design a mutant of vpr which would be more thermostable, but would retain the high catalytic activity of the wild type enzyme. our starting protein template was a previously stabilized mutant containing two inserted proline residues close to the nterminus of vpr (n p/i p). this vpr_n p/i p mutant was shown to have a significantly increased thermal stability but displayed a concomitant tenfold loss of catalytic efficiency. from our previous studies we selected two mutations, one which increased catalytic activity (q k) of the enzyme significantly and another which stabilized the protein against thermal denaturation (n d). the n d mutation had been shown to introduce a salt bridge into the structure of the cold adapted proteinase, yielding higher stability but without negative effects on activity. the q k exchange had been shown to double the turnover number (kcat) to that of the wild type enzyme. insertions of these selected mutations into the vpr_n p/i p mutant were according to predictions; the q k increased the kcat tenfold, and the n d mutation increased the thermal stability. in the combination mutant, vpr_n p/i p/n d/q k, thermal stability was increased by c and c, in terms of tm and t %, respectively. furthermore, the catalytic activity of the mutant was somewhat higher than that of the wild type enzyme. critical peptide stretches may not serve as faithful experimental mimics for protein amyloidogenesis bishwajit kundu , dushyant garg certain amino acid stretches are considered critical to trigger the amyloidogenesis in a protein. these peptide stretches are often synthetically produced to serve as experimental mimics for studying amyloidogenesis of the parent protein. here we provide evidence that such simple extrapolation may be misleading. we studied the amyloidogenesis of full length bovine carbonic anhydrase ii (bcaii) and compared it with those formed by its critical amyloidogenic peptide stretch - (pepb). under similar solution conditions and initial monomeric concentrations, we found that while amyloid formation by bcaii followed aggregation kinetics dominated by surface-catalyzed secondary nucleation, pepb followed classical nucleation-dependent pathway. the afm images showed that bcaii forms short, thick and branched fibrils, whereas pepb formed thin, long and unbranched fibrils. atr-ftir revealed parallel arrangement of cross b sheet in bcaii amyloids, while pepb arranged into antiparallel b sheets. amyloids formed by bcaii were unable to seed the fibrillation of pepb and vice versa. even the intermediates formed during lag phase revealed contrasting ftir, far uv cd signature, hydrophobicity and morphology. we propose that for any polypeptide, the sequences flanking a critical region are equally effective in modulating the initial nucleation events, generating prefibrillar and finally fibrillar species with contrasting characteristic. the results have been discussed in light of amyloid polymorphism and its importance in the design of therapeutic strategies targeting such toxic regions. aksana labokha , ralph minter all approved biological drugs target extracellular proteins and not the majority of the expressed human genome, which resides within intracellular compartments. included in the latter category are many important, disease-relevant targets which cannot be easily addressed by small molecule approaches, such as the oncology targets c-myc and k-ras. although bacteria and viruses have evolved strategies to deliver biological material to the cell cytoplasm and nucleus, our ability to engineer recombinant proteins to replicate this is somewhat limited by (i) our nascent understanding of protein uptake and trafficking pathways and (ii) the ability to easily quantify cell delivery to the cytoplasm and cellular organelles. the aim of my project is to address these challenges by developing an effective assay for cytoplasmic uptake and then using it to measure the delivery efficiency of recombinant proteins which mimic natural delivery strategies e.g. cell penetrating peptides fusion, exotoxin mimics, and supercharged proteins (proteins with high surface charge which can enter cells). i also intend to explore the influence of the rab superfamily, which are the master regulators of protein trafficking, to influence and control both the kinetics and final subcellular destination of exogenous proteins. protein engineering: what's next? with the growing industrial need for engineering enzymes for the deconstruction and transformation of plant biomass in biorefineries, there is a want for the development of new approaches for designing special purpose biocatalysts. techniques, such as directed evolution, which mimic the natural selection process by evolving proteins towards the improvement of a given property, have unquestionably demonstrated their value and are routinely used in large industrial companies. nevertheless, the brute force employed in these methods, could significantly gain from an all-atom description of the underlying catalytic mechanisms, to center the efforts on more limited areas of the protein. in the last years, we have developed computational tools, which combine the electronic structure description of qm/mm methods with the potential to model long time scale processes of pele, to study the details of a variety of reactions. examples, which will be discussed, include rationalizing the selective oxyfunctionalization of steroids using fungal enzymes and the study of the effect of point mutations on the oxidation efficiency of laccases. these methods have shown their potential not only at the descriptive level but, more importantly, through their high predictive capability that opens many opportunities for their use in biotechnology. in this talk, we will show how recent advances in in silico approaches are setting new grounds for future computer guided directed evolution. several orthogonal bioreactions take place simultaneously within membrane bound organelles in eukaryotes and proteinaceous microcompartments in bacteria. these subcellular structures contain sets of enzymes co-involved in metabolic pathways. towards the goal of creating artificial protein microreactors, we seek to develop an artificial organelle that emulates the metabolic activity of the carbon fixating organelle of autotrophic bacteria, the carboxysome. here, we show that the two key carboxysomal enzymes, ribulose- , -bisphosphate carboxylase/oxygenase (rubisco) and carbonic anhydrase (ca), can be efficiently co-encapsulated using our previously reported encapsulation system which is based on a bacterial capsid formed from the protein lumazine synthase (aals- ). our preliminary results suggest that the enzymes can act in tandem and that the co-encapsulation of ca with rubisco in the capsid is necessary for enhanced rubisco activity in vitro. we attribute this observation to the high local concentrations of the rubisco substrate, co , produced by ca within the capsid. we are developing a theoretical model of a minimal carboxysome using the kinetic rate constants of our rubisco and ca variants and aals- as the shell to complement these experiments. next, we will incorporate our minimal carboxysome within an expression host such as e.coli, opening up the possibility of further optimization through directed evolution. in the past targeting and engineering of chemokines has led to several interesting drug candidates. [ ] amongst them, met-rantes, a met-ccl with high g protein-coupled receptor (gpcr) affinity but no subsequent signal transduction, as well as mutants addressing the interaction with the so-called glycosaminoglycans (gags) seem to be the most promising candidates. both, gag knockout as well as gag affinity matured chemokine isoforms have been considered as anti-inflammatory drug candidates, out of which an il- mutant with modifications reached clinical phase where it was profiled for acute neutrophil-related exacerbation in copd. [ ] cxcl (ip- ) is a proinflammatory chemokine released by various cells following stimulation by interferon g (ifn-g) . it is therefore considered as a late chemokine being responsible for the attraction of different lymphocytes. [ ] any therapeutic indication is consequently related to chronic and multiple applications. we have therefore engineered cxcl very conservatively at positions to ultimately generate dominant-negative mutants with a mildly improved gagbinding affinity and an entire knock off gpcr activity. the first steps of our engineering approach were in silico modelling of the mutants and the establishment of a suitable upstream-and downstreamprocessing protocol. next we generated a fluorescently engineered cxcl variant for our fluorescence-based affinity studies which was subjected to biocomparability investigations relative to the native, non-fluorescent protein. compared to the wild type, the fluorescently engineered mutant exhibited similar biological, chemotactic and gag-binding properties. next we started to produce sufficient amounts of the members of our nascent mutant library which were tested with respect to their biophysically behavior as well as to their knocked out chemotactic potency on cells. these experiments included gel electrophoresis and western blot analysis to determine identity and purity; circular dichroism (cd) and chaotrope-induced unfolding to approximate structure; isothermal fluorescence titration (ift); surface plasmon resonance (spr) and isothermal titration calorimetry (itc) to quantify gagbinding affinity and boyden chamber experiments to determine the chemotactic activity. our results show that we are able to tune the gag binding strength along with the gpcr activity of human cxcl which could lead to therapeutic applications in the future. nanodiscs are composed of a nanometer-sized phospholipid bilayer encircled by two a helical, amphipathic membrane scaffold proteins (msps). these particles provide a unique detergent free lipid bilayer model enabling biochemical and biophysical characterization of membrane proteins in a physiologically relevant medium. previously, the largest diameter reported of a nanodisc assembled using msps was about - nm. here we present a method to create large nanodiscs (up to nm in diameter) assembled with covalently circularized msps (cmsp). we can observe the homogeneity in nanodiscs diameter as a narrow distribution using negative-stain em. using our method, we have created nm nanodiscs and used them to study poliovirus ( nm diameter) entry and rna translocation. a nm nanodisc is sufficiently large to accommodate multiple copies of the cd receptor (also known as the poliovirus receptor), and has enough surface area to act as a surrogate membrane for the rna translocation complex during viral uncoating. the nm nanodiscs functionalized with the his-tagged ectodomain of poliovirus receptor, cd , were generated by adding lipids derivatized with a nta nickel- chelating head group to the lipid mixture during nanodisc assembly. cd receptor was added to the already assembled nanodiscs and incubated for minutes at room temperature. the receptordecorated nanodisc complex was purified by size exclusion chromatography. the purified complex was then incubated with poliovirus for minute at c, and then heated to c for minutes to initiate receptor-mediated viral uncoating. virus binding to nanodisc-cd complex and subsequent insertion of viral components into and across the membrane were confirmed by negative-stain electron microscopy (figure c) . to obtain a high-resolution structure for the rna translocation complex we conducted single-particle cryo-em studies using a polara f microscope. unlike liposomes, generating a reconstruction of samples containing nanodiscs is less complicated since the nanodiscs are more homogenous in size, and allow for thinner ice. also, the viral rna can be visualized more easily. the method for making large nanodiscs as well as the negative stain and cryo-em data will be will be presented and discussed. parametric design of alpha-helical barrels and pore-like assemblies with very high thermodynamic stabilities computational design of novel protein structures and enzymes with new functions is a promising tool to create superior biological materials with tailor-made properties, new pharmaceuticals, complex fine chemicals or renewable fuels. it also challenges our understanding of protein folding, protein evolution, molecular recognition and catalysis. here we present a procedure for designing proteins with backbones produced by varying the parameters in the crick coiled-coil generating equations [ ] . combinatorial design calculations using the software suite rosetta identify low energy sequences for alternative helix supercoil arrangements. after that, loop modeling is applied to connect the designs with lowest energy. the extent to which the designed sequences encode the designed structures is evaluated using large-scale structure prediction calculations, as well as symmetric and asymmetric protein-protein docking calculations. subsequently, synthetic genes are generated for sequences that converge strongly on the designed structure for experimental characterization. we applied this approach to monomeric three and four helical bundle structures as well as a pentameric five-helix bundle structure using idealized coiled-coil geometries [ ] . recently we expanded this approach to higher complexity backbones, which resulted in the de-novo design of monomeric, antiparallel six-helix bundles with untwisted, left-and right-handed geometries. circular dichroism (cd), size-exclusion coupled multi-angle light scattering measurements (sec-mals), negative stain electron micrographs (em) and small angle x-ray scattering (saxs) of these designs suggest that they indeed form the designed structures. in addition, we used rosetta protein-protein interface design functionality to computationally design oligomers out of our previously published three and four helix bundle structures to generate self-assembling pore-like structures with the potential use as channels or transporters. again, experimental validation of these designs by cd, sec-mals, em and saxs show that the designs are correct. we are currently undertaking further structural investigation of all these designs by x-ray crystallography. the designs described above can act as templates for protein or small molecule binding, holding a catalytic machinery or for scaffolding enzymes in reaction cascades. some of these applications are currently under investigation, including a self-sufficient redox system employing two copper-centers, binding of heme-moieties as a prosthetic group and tailoring the pore-like geometries to be used in nanopore sequencing. university of washington, university of california, san francisco, repeat proteins are an example of how evolution proceeds by building on existing structures and functions, but also a source of modular protein scaffolds for molecular recognition and biomaterials. however, it is unclear whether the limited number of folds and families that we know today is the result of the intrinsic limitations of polypeptide chains or the consequence of the path followed by evolution. we explored this hypothesis by computational design of repeat proteins based on modular units formed by two alpha helices and two loops of variable lengths, without relying on information from available repeat protein families. the automated sampling of the conformational space resulted in a large number of architectures from which de novo designs were selected for experimental characterization. % of the proteins were stable up to c and monodisperse and designs were structurally validated by small angle x-ray scattering. crystal structures were solved for of them, with root mean square deviation from the models between . Å and . Å. the designs differ from known proteins both at the sequence and structure levels and cover a broader range of geometries than observed in naturally occurring repeat protein families, indicating that existing architectures represent only a small fraction of what can be achieved. our results show that it is possible to expand the range of repeat protein architectures beyond the naturally occurring families, and that computational design can provide new scaffolds and enable the design of proteins tailored for specific applications. the serpin family of proteins consists of over members, all with a highly conserved native structure that is metastable ( ). serpins use this metastability to control the activity of proteases, via a specific inhibitory process. the serpin binds to its target protease through specific residues within the reactive centre loop, the protease cleaves the loop and results in a large conformational change causing the protease to become distorted and catalytically inactive whilst the serpin becomes much more stable ( , , ) . the metastable nature of aat is therefore required to facilitate the rapid and gross conformational changes required for its inhibitory function ( , ) . several disease-causing mutants of aat have been identified, the most common of them being the z-variant ( ). the z-variant has an increased propensity to polymerize in the endoplasmic reticulum of hepatocytes leading to cell death and liver damage ( ) . during the past fifteen years, many groups have unsuccessfully screened a number of serpins and a vast range of solution conditions to identify a combination of serpin and conditions that will enable the folding reaction of a serpin to be characterized. we have now taken an alternative approach and designed a synthetic "model" serpin that folds reversibly to its native state. in order to do this, we used a consensus design approach, analysing a sequence alignment of serpin sequences and determining the prevalent amino acid residue at each position, we termed this serpin conserpin (consensus serpin). here we present the structural, biophysical and functional characterisation of conserpin. combined crystallographic and folding studies reveal the characteristics of conserpin that likely dictate its unique stability and folding behaviour, whilst retaining activity as a serine protease inhibitor. the development of enhanced protein binding scaffolds is a key for engineering protein inhibitors and biosensors with advanced characteristics. utilizing the structural variability and designability of repeat proteins offers a means for designing protein binders where the overall shape is customized to optimally match a target molecule. we developed a computational protocol for the design of repeat proteins with a predefined geometry. by combining sequence optimization of existing repeats and de novo design of capping structures, we designed leucine-rich repeat (lrr) proteins where the building blocks assemble into a novel structure. the suggested design procedure was validated by engineering an artificial donut-like ring structure, which is constructed from ten self-compatible repeats. characterization of several designed constructs further suggests that buried cysteines play a central role for stability and folding cooperativity in certain lrr proteins. this effect could provide a means for selectively stabilizing or destabilizing specific parts of an lrr-based protein binder. the computational procedure may now be employed to develop repeat proteins with various geometrical shapes for applications where greater control of the interface geometry is desired. engineering apobec g enzymes for altered specificity and processivity louis scott , muhammad razif , aleksandra filipovska , , oliver rackham , harry perkins institute of medical research, school of chemistry and biochemistry, the university of western australia apobec g (a g) is a host-encoded protein involved in the defense against hiv- and other retroviral infections. a g is a cytidine deaminase with a ' to ' processive nature, causing targeted c to t mutations along a dna strand. the catalytic and processive activity of a g leads to the hypermutation of nascent retroviral cdna, resulting in premature termination codons and dysfunctional proteins. ultimately, the action of a g inhibits viral replication. the ability of a g to jump and slide along a dna strand, deaminating at targeted sequences, makes it an interesting candidate for protein engineering. engineered a g enzymes for increased activity, altered specificity, and altered processivity are attractive options for expanding the dna modifying enzyme toolbox. mutation of catalytic residues, residues thought to affect its processive nature and those thought to be involved in target recognition, can create novel a g enzymes. using structure guided selection, residues in key functional sites that are amiable to mutation will be chosen. individuals from the resulting libraries of mutants will be selected by directed evolution for desired characteristics. the resulting a g enzymes will be examined for the relationship between their structure and function. such engineered a g enzymes could be targeted to catalyse the reversion of deleterious genetic mutations. furthermore, engineered a g enzymes could be used in mutational studies that call for targeted deamination along a dna strand, or mutational studies that call for unspecific and high throughput dna deamination. engineering porous protein crystals as scaffolds for programmed assembly thaddaus huber , luke hartje , christopher snow a key motivation for nano-biotechnology efforts is the creation of designer materials in which the assembly acts to organize functional domains in three dimensions. crystalline materials are ideal from the validation perspective because x-ray diffraction can elucidate the atomic structure. relatively little work has focused on engineering protein crystals as scaffolds for nanotechnology, due to the technical challenges of coaxing typical proteins into crystallizing, and the likelihood of disrupting the crystallization process if changes are made to the monomers. we have circumvented these limitations by installing guest protein domains within engineered porous crystals ( nm pore diameter) that have been rendered robust using covalent crosslinks. the retention of the scaffold structure despite changes to the solution conditions and macromolecule uptake can be validated through x-ray diffraction. we have engineered scaffold crystals for the non-covalent and covalent capture of guest macromolecules. by controlling the reversible loading and release, we can prepare "integrated" crystals with spatially segregated guest loading patterns. as assessed using confocal microscopy, such host-guest crystals are highly stable. ultimately, the resulting crystals may serve as a robust alternative to dna assemblies for the programmed placement of macromolecules within materials. engineering ultrasensitive protein probes of voltage dynamics for imaging neural activity in vivo francois st-pierre , , michael pan , , helen yang , xiaozhe ding , , ying yang , , thomas clandinin , michael lin , department of bioengineering, stanford university, department of pediatrics, stanford university, nervous systems encode information as spatiotemporal patterns of membrane voltage transients, so accurate measurement of electrical activity has been of long-standing interest. recent engineering efforts have improved our ability to monitor membrane voltage dynamics using genetically encoded voltage indicators. in comparison with electrophysiological approaches, such protein-based indicators can monitor many genetically defined neurons simultaneously; they can also more easily measure voltage changes from subcellular compartments such as axons and dendrites. compared with genetically encoded calcium indicators, voltage sensors enable a more direct, accurate, and rapid readout of membrane potential changes. however, several challenges remain for in vivo voltage imaging with genetically encoded indicators. in particular, current voltage sensors are characterized by insufficient sensitivity, kinetics, and/or brightness to be true optical replacements for electrodes in vivo. as a first step towards addressing these challenges, we sought to develop new voltage indicators that further improve upon the performance of the fast voltage sensor accelerated sensor of action potentials (asap ). in asap , voltage-induced conformational changes in a natural voltage-sensing domain perturb the fluorescence emission of a covalently linked green fluorescent protein (gfp). using a structurebased approach to guide mutagenesis, we discovered several amino acids that tune the kinetics and voltage sensitivity of asap . these residues are not only located in the voltage-sensing domain, but also in the fluorescent protein and in the linkers bridging sensing domain and gfp. our most improved variant, asap , exhibits improved sensitivity to voltage transients such as neuronal action potentials and subthreshold depolarizations. we sought to characterize the ability of these new voltage sensors to monitor neural activity in vivo using laser-scanning two-photon microscopy, a technique that allows imaging with lower autofluorescence and deeper tissue penetration. we report that asap sensors were able report stimulus-evoked voltage responses in axonal termini of the fly visual interneuron l . asap sensors enabled voltage imaging with dramatically improved temporal resolution compared to three recently reported calcium and voltage sensors. overall, our study reports novel voltage indicators with improved performance and highlights how specific amino acids can tune the performance of a proteinbased fluorescent sensor. we anticipate that these results will pave the way for further engineering of voltage sensing proteins, and that our new sensor asap will facilitate current and future efforts to understand how neural circuits represent and transform information. assembly of armadillo repeat proteins from complementary fragments erich michel , randall watson , martin christen , fabian bumback , andreas pl€ uckthun , oliver zerbe demonstrated that complementary fragments of a designed consensus armadillo repeat protein (armrp) recognize each other [ ] . the two fragments ym : ma, in which y, m and a denote the n-cap, internal repeats and the c-cap, respectively, form a : complex with a nanomolar dissociation constant, which is essentially identical to the crystal structure of the continuous ym a protein. we further demonstrate that structurally intact armadillo repeat protein complexes can be reconstituted from fragments obtained at various split sites -essentially after every repeat but also within repeats. the fragments display variable affinities towards each other, depending on the split site. the low affinity of some complementary pairs can be dramatically increased upon addition of peptide ligands. while a number of proteins are known that can be reconstituted from fragments we believe that the fact that armadillo repeat proteins can be reconstituted from various complementary fragments is novel and opens new interesting perspectives and applications in biochemistry. a reliable method for generating optically controllable proteins would enable researchers to interrogate protein functions with high spatiotemporal specificity. we recently engineered a tetrameric fluorescent protein, dronpa n, that undergoes light-induced monomerization, then developed a general architecture for lightinducible proteins based on this light-induced transition. we created proteins whose active sites were blocked by fused dronpa n domains in the dark, but would become unblocked by light. here we present further two extensions to this concept that together enabled the generalization of this method to additional classes of proteins. first, we engineered a photodissociable dimeric dronpa (pddronpa) with tunable affinity, faster photoswitching speed, and decreased level of protein aggregation, enabling better performance of fusion proteins. second, we introduce the concept of caging a protein active site by insertion of dronpa domains into loops rather than strictly at the protein termini. we use the pddronpa system to impose optical control on kinases and the cas endonuclease. the resulting light-inducible mek kinase, raf kinase, and cas endonuclease showed high caging efficiency of protein activities in the dark, and robust protein activation upon light illumination. we believe that our efforts on further improving and generalizing this method would bring the power and benefits of light control to a broad community of biologists. exploring the evolution of folds and its application for the design of functional hybrid proteins saacnicteh toledo patiño , birte h€ ocker the structural diversity of proteins may appear endless, nevertheless even large protein complexes can be decomposed into protein domains and smaller sub-domain sized fragments. only recently, we could identify such fragments employing sequence-based comparisons of different folds, as the tim-barrel and the flavodoxin-like fold (farias-rico et al., ) . as an extension of this work, we compared all a/b proteins and identified several fragments shared by different folds illustrating how nature may have achieved structural and functional diversity from a reduced set of building blocks. inspired by this combinatorial concept, we searched for homologous fragments bearing active sites to engineer a functional fold-chimera. we extracted the vitamin-b binding part from methylmalonyl coa mutase, which belongs to the flavodoxin-like fold (fl) and used it to replace the corresponding fragment in uroporphyrinogen iii synthase, which belongs to the hemd-like fold (hdl). the new hybrid resulted in a stable and well-folded protein whose structure was determined by x-ray crystallography. moreover, cobalamin-binding function was successfully transferred to the new protein from the fl parent, which shows the advantage of using this approach for the design of new functional proteins. in addition, profile alignments revealed sequence and structural evidence that suggested an evolutionary path for hdl from fl by gene duplication. to test this hypothesis, we expressed a modified c-terminal half of uroporphyrinogen iii synthase and solved its structure by nmr spectroscopy, thereby confirming the predicted fl architecture. altogether, our approach facilitates the detection of common ancestry among different folds contributing to our understanding of protein development. furthermore, our results show how new complex proteins can be designed using fragments of existing proteins that serve as building blocks in a lego-like manner. we believe that combining fragments containing existing properties will provide a successful method for the design of novel functionalities in the future. [ ] . the active site cysteine plays a key role in the reaction mechanism and we investigated this residue in more detail by exchanging this moiety with selenocysteine (sec) and homocysteine (hcy). the sortase mutants were generated by semisynthesis using expressed protein ligation (epl). the resulting cys-, sec-and hcy-sortase enzymes were characterized and showed a moderate - -fold reduction of activity for sec-sortase. the activity of hcysortase was barely detectable with less than % of wildtype activity. the alkylation efficiency of the active site nucleophiles correlated with the expected pka values of sec, cys and hcy. analysis of the ph dependency of the transpeptidation reactions showed that the activity optimum of sec-sortase was shifted towards more acidic conditions. these investigations provide further insights into the reaction mechanism of sortase a and the semisynthetic enzymes may provide new tool for further biochemical studies. propanediol oxidoreductase from escherichia coli (fuco) uses nadh/nad as cofactors to catalyze the conversion of s-lactaldehyde to s- , -propanediol and vice versa. fuco is an attractive enzyme in the search for possible biocatalysts producing a-hydroxy aldehydes, which are important for the synthesis of natural products and synthetic drugs. enzymes catalyzing these types of reactions are unique in catalytic power and stereoselectivity. the usage of fuco in synthetic industry is limited by the restricted substrate scope, which makes fuco inactive with larger phenyl-substituted alcohols. we used reengineering and directed evolution to enable fuco to catalyze the regio-and enantioselective oxidation of arylsubstituted vicinal diols, such as phenylpropanediols, into a-hydroxy aldehyde products. we mutated amino acids considered to restrict the entry into the active site, and modeled the mutants that were most active with the substrates phenylacetaldehyde and s- -phenyl- , -propanediol and performed docking studies with them. as expected, our experimental and in silico results show that the mutations enlarge the active site cavity and enable the mutant enzymes to accommodate the new substrates. we also found specific amino acids in the active site, which need to be conserved to allow the substrates to make stabilizing interactions. interestingly, an asparagine residue makes the mutant enzymes able to discriminate between phenylacetaldehyde and s- -phenyl- , -propanediol. in conclusion, we successfully re-engineered the specialist enzyme fuco to accept also bulkier molecules as substrates, thereby making it more useful for industrial purposes. one way to gain insight into the sequence-structure-function relationship in proteins is to de novo design artificial proteins. despite impressive successes in de novo protein design, designing a folded protein of more than amino acids still remains a challenge. using this approach, an idealized (beta/ alpha) fold protein was designed leading to the production of a protein of amino acids (octarellin v). this protein showed a low solubility and stability. through directed evolution we produced a soluble variant, octarellin v. . the biophysical characterization of octarellin v. shows a well folded monomeric and thermostable protein with a tm over c. however, after several screenings, we could not find crystallization conditions for this protein. as an alternative, we decided to co-crystallize octarellin v. with a protein partner that helps the crystallization process. we used protein partners: alpha-reps and nanobodies. the first one is characterized to interact through a large surface contact, whereas the second is characterized to recognize an specific small epitope. crystallization of both complexes was performed successfully by vapor diffusion and the structures were solved. the experimental structures correspond to the first for an artificial protein of this size and it will allow to criticize the computational design of the octarellin v. generation of synthetic antibodies against membrane proteins in nanodiscs for use in structural biology methods. here, we describe a robust strategy for generating a class of high performance antibodybased affinity reagents that have proven useful in determining the structures of relevant functional states of membrane proteins. these reagents are fab fragments that are generated by phage display from fully synthetic libraries and are called synthetic antibody fragments, or sabs. we have developed phage display sorting strategies that can trap a desired conformational state, making it accessible to structural analysis, or target a particular epitope on the protein surface. however, to maximize this technology for membrane proteins, several limitations of phage display sorting in detergent formats had to be overcome, the greatest being that using detergents can produce non-native conformational biases. we sought to address these limitations by embedding membrane proteins into nanodiscs, soluble lipidfilled discoidal particles, to better mimic the native membrane environment. nanodiscs stabilize the membrane protein and allow it to respond to conformation-inducing stimuli such as ligands, ions and ph during phage display selections. we have established and validated an improved protocol using two membrane protein systems: ) mj , an archaeal membrane protein of unknown function, and ) cora, a pentameric magnesium ion channel. using mj , we compared the nanodisc protocol with the standard method performed in detergent, and as an important byproduct, we characterized the influence of the membrane protein environment on the apparent affinity of sabs to their cognate antigen. using cora, we developed a more sophisticated sorting strategy resulting in a variety of sabs specific to either the open or closed conformation of the channel. finally, using sabs as crystallization chaperones we obtained the structure of mj at . Å resolution, and crystallized cora in several new conditions. lipocalin-type prostaglandin d synthase (l-pgds) is a member of the lipocalin superfamily, and binds a large variety of small hydrophobic molecules. using this function of l-pgds, we have already reported the feasibility of l-pgds as a novel drug delivery vehicle for the poorly water-soluble drugs [ ] . sn- , -ethyl- -hydroxy-camptothecin, is a semi-synthetic analogue of anti-cancer alkaloid camptothecin that targets dna topoisomerase i. despite of the potent anti-tumor activity, however, sn- was not used directly in a clinical practice due to its poor water solubility. thus, irinotecan hydrochloride (cpt- ), which is the water-soluble prodrug of sn- , is used for the cancer treatment. however, cpt- shows approximately . % cytotoxic activity of sn- against the various cancer cell lines in vitro, and its metabolic conversion rate is % of the original volume of cpt- . here, we show the development of the drug delivery system utilizing l-pgds, which enables a direct clinical usage of sn- . first, we investigated the effect of l-pgds on the solubility of sn- . in the presence of mm l-pgds, the concentration of sn- was . mm, which was , -fold as compared with that in pbs. then, we carried out isothermal titration calorimetry measurements to investigate the detailed binding mode of sn- to l-pgds. as a result, it was revealed that l-pgds binds three molecules of sn- , and the dissocia- control over the sensitivity with which artificial biomolecular receptors respond to small changes in the concentration of their target ligand is critical for the proper function of many cellular processes. such control could likewise be highly useful in artificial biotechnologies in which highly responsive behavior is of value, such as biosensors, genetic logic gates, and "smart" materials and delivery devices. in nature, the control of molecular responsiveness is often achieved using "hill-type" cooperativity, a mechanism in which sequential binding events on a multivalent receptor are coupled such that the first enhances the affinity of the next, producing a steep, higher-order dependence on target concentration. here we use an intrinsic-disorder-based mechanism that can be implemented without requiring detailed structural knowledge to rationally introduce this potentially useful property into several normally noncooperative biomolecules. to do so we fabricate a tandem repeat of the receptor that is destabilized (unfolded) via the introduction of a long, unstructured loop. the loop spatially separates the two sets of the two halves of the binding sites, preventing a complete binding site that enables target molecule binding without prior closure of the loop. thus, the first binding event requires the energetically unfavorable closing of this loop, reducing its affinity relative to that of the second binding event, which, in contrast occurs at a pre-formed site. using this approach we have rationally introduced cooperativity into three unrelated aptamers, achieving in the best of these a hill coefficient experimentally indistinguishable from the theoretically expected maximum. the extent of cooperativity, and thus the steepness of the binding transition, are, moreover, well modeled as simple functions of the energetic cost of binding-induced folding, speaking to the quantitative nature of this design strategy. essential and non-essential amino acid species for an ancestral protein satoshi akanuma the translation system is an essential element for life because it links genetic information embedded in genes to functional molecules, proteins. the modern genetic code, which encodes the standard amino acids (and three terminations) using triplet codons, is shared by most of the extant organisms on the earth. a number of theories have been proposed for the origin and evolution of the genetic code, and these theories suggest that only a fewer amino acids were used in primitive proteins and later the amino acid repertoire gradually increased up to through the course of evolution. if so, one would wonder how many number of and which types of amino acids were involved in the primitive proteins. i have begun to address this issue experimentally. i first resurrected several ancestral proteins and then restricted the amino acid usage of one of the resurrected proteins. i targeted nucleoside diphosphate kinase (ndk) that catalyzes the transfer of a phosphate from a nucleoside triphosphate to a nucleoside diphosphate. ndk may have arisen early because at least one gene that encodes ndk is present in most extant organisms. the first step in the reconstruction of ancestral ndk sequences is to prepare multiple amino acid sequence alignments using homologous sequences of ndk from extant species. then, phylogenetic trees were built. ancestral sequences of ndk that represent the last common ancestors of archaea and of bacteria were reconstructed using the information contained in the predictive phylogenetic trees. the reconstructed ancestral kinases are extremely thermally stable [akanuma et al., ] . then, using the most thermally stable ancestral ndk, arc , as the starting molecule, i restricted its amino acid usage. arc does not contain any cysteine residue and therefore consists of amino acid species. i completely replaced one of the amino acid species by other amino acid species and thus created proteins each of which consisted of amino acid species. then, i evaluated the stabilities and activities of the resulting arc variants to assess the individual contributions of the amino acid species. as the result, i found that the amino acid species do not equally contribute to the stability and activity of arc and that some amino acid species can be easily lacked but others are important or essential for its stability and function. the result clearly shows that the full amino acid species are not necessarily essential and supports the hypothesis that proteins in the early stage of evolution were made from a reduced amino acid set. the protein surface recognition for protein-protein interactions (ppi) is involved in signal transduction, immune reaction, and creation of the nanostructures in living cells. the methods for rational designing of ppi that could provide non-antibody scaffolds and nanostructured materials are required for the therapeutic and nanotechnological applications. although there have been some successful rational designs with computational methods, it is still difficult to design freely the ppi onto arbitrary proteins. the reason for this limitation is decreased solubility in the designed protein due to the additional hydrophobic residues in order to drive ppi. another reason is a limited set of design modes by which proteins can interact, because the target proteins have individual surface structures. therefore, many methods of constructing an interface for numerous target scaffold proteins without loss of their solubility are necessary. surface exposed a-helices are often observed in natural globular proteins. moreover, there are many examples for naturally occurring oligomeric proteins where an a-helix from each subunit interacts to form an intermolecule coiled coil. further, the works related to designing of artificial helical bundle reported by the several other groups have provided information about how to generate and tune the interaction between a-helices. therefore, a surface exposed a-helix would be a good target for designing a de novo interface onto the scaffold protein. here we engineered two different proteins, sulerythrin and cys-larfh, to form the cys-larfh-sulerythrin dimer-cys-larfh heterotetramer via an intermolecular helix-helix interaction. wild-type sulerythrin forms a dimeric eight-helix bundle. cys-larfh is a designed monomeric protein that forms four-helix bundle containing interhelical s-s bonds. both sulerythrin and cys-larfh are extremely thermostable. to design protein-protein interfaces onto the individual proteins, we first introduced six leucines to the two a-helices of sulerythrin and three leucines to a a-helix of cys-larfh. as expected, the introduction of the hydrophobic amino acids reduced their solubilities. to recover the solubility, we then introduced six aspartates or glutamates around the hydrophobic surface of the sulerythrin (hereafter referred to as l d or l e). similarly, three arginines were introduced around the artificial hydrophobic surface of the cys-larfh (hereafter referred as iv- l r). the solubilities of the mutants with the hydrophobic interface and additional charged residues were recovered their solubility. in addition, the sulerythrin mutants l d and l e exist mainly as dimer. the cys-larfh mutants iv- l r, also exists as monomer. we then examined the interaction between l e or l d and iv- l r. a pull-down experiment, in which co beads bound to either his-tagged cys-larfh and iv- l r were used to pull down wild-type sulerythrin, l d, or l e, demonstrates that l d or l e specifically interacts to iv- l r. furthermore, when analysed by size exclusion chromatography, the dominant peaks of the mixture of l d and iv- l r appeared at the volume expected for the heterotetrameric complex. thus we successfully created the de novo ppi by using a very simple concept involving hydrophobic interaction in combination with charge interactions. in vitro selection of liposome anchoring peptide by cdna display naoto nemoto , ryoya okawa , yuki yoshikawa , toshiki miyajima , shota kobayashi a liposome-anchoring peptide (la peptide) was selected against liposomes composed of dioleoyl-snglycero- -phosphocholine (dopc) by in vitro selection using cdna display method. the selected peptide la peptide consists of the n-terminal region (hydrophobic) and the c-terminal region (basic) in a characteristic manner. thus, la peptide was synthesized chemically and the interactions between la peptide and particular types of liposomes were investigated and confirmed by confocal laser scanning microscopy. designing of a novel platinum-binding amino acid sequence on a protein surface asumi kaji , hiroya niiro , satoshi akanuma , tetsuya uchida , akihiko yamagishi designing of a novel interaction between a metal and a protein is a key to create hybrid materials between organic and inorganic materials. for example, in a glucose biosensor, which is widely used for measuring glucose concentration in blood, glucose oxidoreductase molecules are immobilized on a platinum electrode by polyacrylamide gel. a metal-binding tags that is added to the n-or cterminus of a protein is also used for fix the protein to a metal. however, a technique to create a metal binding site on a desired position of a protein has not been invent. if such a technique would be established, the technique would contribute to developing and improving biosensors and to producing new bionanoelectronic materials. in this study, we created a platinum-binding site on a loop located at a protein surface. we used an artificial protein, larfh, that had been synthesized by connecting four identical alpha helices originated from the c-terminal segment of the escherichia coli lac repressor with three identical loops. we randomized the ser, gly, gln, gly, gly, ser sequence within one of the inter-helical loops and then selected for binding to platinum by a t phage display system. most of the selected larfh variants contained the tyr, lys, arg, gly, tyr, lys (ykrgyk) sequence in the randomized segment. we then evaluated the affinity of the larfh variant to platinum by means of quartz crystal microbalance analysis. we found that the variant binds to platinum more strongly than does the original larfh. in the annual symposium, we will also report about the affinity of the isolated ykrgyk sequence to platinum and about the crucial role of the first tyrosine in binding to platinum. engineering of an isolated p a subunit of pi ka permits crystallization and provides a platform for structure-based drug design pi ka remains an attractive target for development of anticancer targeted therapy. a number of p a crystal structures in complex with the nsh -ish fragment of p regulatory subunit have been reported, including a few small molecule co-crystal structures, but the utilization of this crystal form is limited by low diffraction resolution and a crystal packing artifact that partially blocks the atp binding site. taking advantage of recent data on the functional characterization of the lipid binding properties of p a, we designed a set of novel constructs allowing production of isolated stable p a subunit missing the adapter binding domain (abd) and lacking or featuring a modified c-terminal lipid binding motif. while this protein is not catalytically competent to phosphorylate its substrate pip , it retains ligand binding properties as indicated by direct binding studies with a pan-pi ka inhibitor. additionally, we determined apo and pf- bound crystal structures of the p a ( - ) subunit at . Å and . Å respectively. comparison of isolated p a ( - ) with the p a/p complex reveals a high degree of structural similarity, which validates suitability of this catalytically inactive p a for iterative sbdd. importantly, this crystal form of p a readily accommodates the binding of non-covalent inhibitor by means of a fully accessible atp site. the strategy presented here can be also applied to structural studies of other members of pi kia family. identification of structural determinants involved in the differential conformational changes of ef-hand modules emma liliana arevalo salina , joel osuna quintero , humberto flores soto , gloria saab rinc on instituto de biotecnolog ıa, universidad nacional aut onoma de m exico identification of structural determinants involved in the differential conformational changes of ef-hand modules calcium signals are regulated by several proteins, most of which belong to the ef-hand superfamily. the ef-hand motif is formed by a helix-loop-helix that binds calcium through its loop . these motifs occur in adjacent pairs, forming a single globular domain which is the basic structural and functional ca binding unit. the proteins in this family can be classified as calcium sensors or modulators, according with their function. the first group undergoes a major conformational change upon calcium binding, while the second one remains practically unchanged , . to explain the biophysics behind the different behavior of these proteins upon ca binding, we have sought to identify structural determinants that could account for these features, especially for the difference in the conformational change. we examined the primary structure from two ef-hand motifs: a sensor ef-hand from chicken troponin c (sciii) and a modulator ef-hand from bovine calbindin d k (clbn). the main differences were in the binding ca loop and a group of charged residues in the h helix of the modulator ef-hand. then, we constructed chimeric clbn motifs containing the loop or the loop and h from sciii motif (h clbnsciii and h h clbnsciii). these constructs were analyzed using a reporter system that discriminates ef-hand-sensor motifs from signal-modulators at the single-motif level. this reporter is based on the fusion of genes codifying for the ef-hand and the prephenate dehydrogenase from e. coli (tyra), a protein which is active only as a dimer. isolated ef-hand motifs have the ability to homo-dimerize and in the fusion can stabilize and activate tyra. the sensor motif exhibits a conformational change by binding calcium and in doing so, destabilizes the dimeric conformation of tyra and virtually eliminates its activity. in the modulators, on the other hand, the rather small conformational change only gives rise to a decreased tyra activity. both constructed chimeric ef-hand fusions showed a loss of activity upon ca binding, indicating that the residues connector of the sensor ef-hand from sciii is sufficient to confer the conformational change. in addition we used cd and extrinsic fluorescence spectroscopies to analyze any conformational change in the h h clbnsciii and h clbnsciii isolated modules, not finding any difference between the ca free and ca bound chimeras, suggesting that the change in activity of the reporter protein is due to a change in the orientation of the helices in the ef-hands induced by calcium. the effect of ca binding of the chimeras in the context of the entire calbindin d k protein is under investigation. mapping side chain interactions at the n-and c-termini of protein helices nicholas e newell, independent researcher interactions involving one or more amino acid side chains near the ends of protein helices stabilize helix termini and shape the geometry of the adjacent loops, contributing to supersecondary structure. side chain structures that have been identified at the helical n-terminus include the asx/st n-caps, the capping box, and hydrophobic and electrostatic interactions. at the cterminus, capping is often achieved with main-chain polar groups, (e.g. the schellman loop), but here also particular side chain motifs clearly favor specific loop geometries. key questions that remain concerning side chain interactions at helix termini include: ) to what extent are helix-terminal motifs that include multiple amino acids likely to represent genuine cooperative interactions between side chains, rather than chance alignments? ) which particular helix-terminal loop geometries are favored by each side chain interaction? ) can an exhaustive statistical scan of a large, recent dataset identify new side chain interactions at helix termini? in this work, three analytical tools are applied to answer the above questions for both n-and c-termini. first, a new perturbative least-squares d clustering algorithm is applied to partition the helix terminal structures in a large ( , example), low-redundancy pdb dataset by loop backbone geometry. the clustering algorithm also generates a set of structural exemplars, one for each cluster, that is used to represent the most important loop geometries at each terminus. next, cascade detection (newell, bioinformatics, ), an algorithm that detects multi-amino acid cooperativities by identifying overrepresented sequence motifs, is applied to each cluster separately to determine which motifs are most important in each loop geometry. finally, the results for each motif are displayed in a capmap, a d conformational heatmap that depicts the distribution of motif abundance and overrepresentation across all loop geometries by projecting these quantities onto the structural exemplars generated by clustering. the capmap reveals the loop conformations most favored by a motif. actual structures from the clusters corresponding to these favored conformations are then examined in a structure browser to characterize the side chain interaction associated with the motif. this work identifies a 'toolkit' of side chain motifs which are good candidates for use in the design of synthetic helix-terminal loops with specific desired geometries, because they are used in nature to support these geometries. highlights of the analysis include determinations of the favored loop geometries for the asx/st motifs, capping boxes, big boxes, and other previously known and unknown hydrophobic, electrostatic, h-bond, and pi-stacking interactions. a goal of future work is to make these results available in a structurally-addressable database that would enable researchers to immediately retrieve the side chain interactions most compatible with a desired loop geometry. generation of fluorescent protein-tagged gp mutants to analyze the intracellular distribution of hiv- envelope protein shuhei nakane , zene matsuda green earth research center, green earth institute co., ltd., res ctr for asian infect dis, inst of med sci, the univ of tokyo, lab of struct virol and immunol, institute of biophysics, cas hiv- is a causative enveloped virus of aids. its envelope protein (env) has two non-covalently associated subunits, gp and gp , which are proteolytically processed from a gp precursor. the gp subunit is a surface protein and gp is a transmembrane protein. the gp and gp subunits are responsible for the receptor recognition and membrane fusion, respectively. the cytoplasmic tail (ct) of gp is about amino acids long and is believed to play a critical role in intracellular trafficking of env. to visualize dynamic trafficking, the c-terminus of gp has been tagged with fluorescent proteins such as gfp. however, tagging of ct may cause a concern to affect the interactions between the ct and cellular proteins that are involved in intracellular trafficking. to avoid this problem, here we tried to insert gfpopt, a gfp variant, into five variable regions of gp . we have analyzed the phenotypes of env mutants, such as the cell surface expression, processing of gp , membrane fusion activity, and virion incorporation. among variable regions of gp , the v region was most sensitive to insertion. v /v region was less sensitive than v . consistent with the recently revealed structure, exteriorly located v and v were highly tolerant to insertion. we used the mutant with the gfp insertion in the v region to analyze the intracellular distribution of env with and without ct. we found that deletion of ct increased the presence of vesicles colocalized with late endosome markers. this is consistent with the hypothesis that the ct region contains a motif regulating intracellular trafficking. our results showed that env with gfpopt insertion in its gp subunit is a useful tool for the study of intracellular dynamics of hiv- env. these mutants would also be useful to trace the fate of virus particles during infection. pi- ngs-guided phage panning: comparison to conventional panning strategy buyung santoso , dorain thompson , john nuss , john dwyer phage display is a powerful tool for generating binders to a target protein. multiple rounds of panning with conventional phage display strategies typically result in a number of hits, which are then individually screened using in vitro assays. clones screened at this stage are a combination of specific binders, sequences that are selected due to amplification bias, and non-specific binders. if the number of specific clones is low relative to the non-specific sequences, a larger number of clones have to be screened to ensure sufficient diversity of early leads. with the advent of next generation sequencing (ngs) technology, we aim to test whether we can increase the diversity of specific hits and decrease the number of non-specific sequences. in our experiment, four rounds of conventional panning produced ten peptide binders to target protein. ngs analysis after two rounds of panning was done in parallel, yielding more than ten thousand sequences, ranked by abundance. all ten binders from conventional panning were found in the top most abundant ngs hits. more importantly, additional hits were found in ngs analysis but not in conventional panning, highlighting this strategy as a promising alternative for hit discovery with the significant upside of more diverse and higher affinity leads. numerous processes in pharmaceutical development, including construct screening, structural genomics, protein engineering and expression optimization among others, require the use of higher throughput plasmid dna purification. the majority of issues encountered in mini, midi, and maxiprep purification kits involve flocculate removal following alkaline lysis, and there is currently no easy way to produce large amounts of plasmid dna without the addition of complicated and time consuming clarification steps. the existence of a hassle-free automated system that is not restricted by sample size would significantly help in cutting time and costs during the initial processing steps of plasmid purification. the autoplasmid mea instrument provides a fully automated solution to traditional problems faced in plasmid purifications, allowing mini, midi, and maxiprep plasmid purifications to be performed on a single instrument. the data presented here on plasmid yield, purity, and suitability for sequencing and transfection/transformation illustrate a new strategy for automated plasmid preps. by eliminating traditional clarification methods, cell culture volumes between - ml can be processed leading to yields ranging from - lg. this flexible system was developed in order to satisfy a wide variety of concentration and yield requirement, while eliminating the time consuming steps previously needed to obtain similar results. the ability to perform fully automated mini, midi, and maxi plasmid preps on one instrument allows for a customized all-in-one purification system that is not restricted by traditional clarification methods, eliminating manual intervention, and streamlining the purification process. the modular nature of protein architectures suggests that proteins have evolved through duplication and fusion to give rise to modular, often symmetric forms, which later diversified under the influence of evolutionary pressure. we have developed a computational protein design method termed reverse engineer evolution (re volution) to create symmetrically self-assembling protein building blocks. we have used this method to design a perfectly symmetric b-propeller protein called pizza. subsequently, we have engineered a metal binding site into this pizza protein. this new pizza variant carries two nearly identical domains per polypeptide chain, and forms a trimer with three-fold symmetry. the designed single metal ion binding site lies on the symmetry axis, bonding the trimer together. two copies of the trimer associate in the presence of cadmium chloride in solution, and high resolution x-ray crystallographic analysis reveals a nano-crystal of cadmium chloride, sandwiched between two trimers of the protein. this nano-crystal, containing seven cadmium ions lying in a plane and twelve interspersed chloride ions, is the smallest reported to date. our results indicate the feasibility of using rationally-designed symmetrical proteins to biomineralize nano-crystals with applications in bionanotechnology. bacillus licheniformis trehalose- -phosphate hydrolase structures suggest keys to substrate specificity chwan-deng hsiao , min-guan lin , long-liu lin , yuh-ju sun institute of molecular biology, academia sinica, department of applied chemistry, national chiayi university, depaertment of life science, national tsing hua university trehalose- -phosphate hydrolase (trea) of the glycoside hydrolase family (gh ) catalyzes the hydrolysis of trehalose- -phosphate (t p) to yield glucose and glucose- -phosphate. products of this reaction can be further metabolized by the energy-generating glycolytic pathway. here we present the crystal structures of bacillus licheniformis trea (bltrea) and its r q mutant complexed with p-nitrophenyl-a-d-glucopyranoside (r q/ ppng) at . Å and . Å resolution, respectively. the overall structure of bltrea is similar to other gh family enzymes. however, detailed structural comparisons revealed that the catalytic groove of bltrea contains a long loop adopting a different conformation from those of gh family members. unlike the homologous regions of bacillus cereus oligo- , -glucosidase (bcogl) and erwinia rhapontici isomaltulose synthase (nx- ), the active site surface potential of bltrea exhibits a largely positive charge, contributed by the four basic residues his , his , lys and lys . mutations at these residues resulted in significant decreases of bltrea enzymatic activity. strikingly, a hhlk motif and the lys residue played critical roles in bltrea substrate discrimination. crystal structure of engineered lrrtm synaptic adhesion molecule and a model for neurexin binding anja paatero , katja rosti , alexander shkumatov , cecilia brunello , kai kysenius , prosanta singha , henri huttunen , tommi kajander institute of biotechnology, university of helsinki, helsinki, finland, dept of pharmaceutical and pharmacological sciences, ku leuven, leuven, belgium, neuroscience center, university of helsinki synaptic adhesion molecules are key components in the development of the brain, and in the formation of neuronal circuits, as they are central in the assembly and maturation of the chemical synapses. several families of neuronal adhesion molecules have been identified such as ncams, neurexins and neuroligins, and in particular recently several leucine rich repeat protein families, e.g. netrin g-ligands, slitrks and lrrtms. the lrrtms form a family of four proteins. they have been implicated in excitatory glutamatergic synapse function, and were specifically characterized as ligands for neurexins in excitatory synapse formation and maintenance. in addition, lrrtm and lrrtm have been found to be ligands for heparan sulphate proteoglycans. we report here the crystal structure of a stability-engineered mouse lrrtm , with a tm c higher than the wild type protein, while retaining its function. we localized the neurexin binding site to the concave surface based on protein engineering, sequence conservation and prior information on the ligand interaction with neurexins, allowing us to propose a tentative model for lrrtm:neurexin interaction compex. cell culture studies and binding experiments show that the engineered protein is functional and capable of forming synapse-like contacts. small angle x-ray scattering data suggests that the wild type protein forms transient dimers, which may have importance for the function. the structural and functional data presented here provide the first structure of an lrrtm protein, and a model for molecular mechanism of lrrtm function in adhesion. computational design of phenylalanine binder olga khersonsky , gil benezer , sarel fleishman recently, abdesign algorithm was developed in our lab for de novo design of antibodies ( ). it is guided by natural conformations and sequences, and exploits the modular nature of antibodies to abstract generate an immense space of conformations, which can be used as scaffolds for design of stable highaffinity binders. we have used abdesign to design a binder of phenylalanine. , antibody scaffolds were obtained by splicing h and l fragments into a template (pdb id brr), and subsequent optimization of vh and vl orientation. phenylalanine binding site, based on native phenylalanine binders, was introduced into the scaffolds with rosettamatch ( ), and the sequences were subsequently optimized by rosetta enzyme design protocol ( ) . designs were experimentally tested by yeast display for binding of biotinylated phenylalanine ligand. several designs were found to bind the ligand, and we plan to further characterize this affinity and improve it using directed evolution techniques. in collaboration with the group of prof. johnsonn, the resulting phenylalanine binder will be incorporated in a bio-luminescent (lucid) sensor for phenylalanine ( ) . phenylalanine monitoring device would be of primary importance for patients with phenylketonuria, a genetic disease with phenylalanine metabolism problem. cold-adapted enzymes are interesting because of their higher catalytic activity compared to mesophilic and thermophilic homologues. alkaline phosphatase (ap) from a psychrophilic vibrio marine bacteria (vap) has an unusual large surface loop that extends from each of its monomers to stabilize a homodimeric structure ( ). in many cold-adapted enzymes, the loop regions are longer compared to proteins of mesophilic organisms and our aim was to study the functional and structural role of this loop. three substitutions (r l, y f and f y) were introduced within the large surface loop as directed by microsecond molecular dynamics (md) simulations. with the r l mutation, two hydrogen bonds were broken that connect the loop to residues on the adjacent subunit, and further two hydrogen bonds broken with the adjacent q . as a consequence, r l displayed a % higher kcat compared with wild-type and a slight decrease in the km value. overall, the catalytic efficient improved by %. the global heat stability (tm) and the active site sensitivity to heat (t %) were reduced by c and c, respectively. md simulations showed that hydrogen bonds to arg are important for longrange communication to the active site. certain rotamers of two important residues in the catalytic site, ser and arg , were favored, presumably toward states more competent for catalysis upon the replacement of arg with leu. in the y f variant, removal of one hydrogen bond between the loop and the other subunit caused a small drop in stability parameters, whereas both kcat and km were reduced by about half, giving similar kinetic efficiency (kcat/km) to that of wild-type. finally, we changed a residue at the root of the large loop (f y) such that one new intersubunit hydrogen bond could form. this variant maintained the wild-type characteristics. in conclusion, removing hydrogen bonds connecting the major loop of one subunit to the protein surface of the other subunit in vap produced higher catalytic activity and this shows functional connections between loop mobility and the active site. our study also demonstrates that interactions between residues in the large disordered loop and the opposite subunit in the dimeric vap are determinants of its stability. thus, we managed to show that loosening of interface contacts between the two vap subunits by replacement of crucial residues provides a way to orchestrate structural and kinetic dynamics in a productive way. the de novo design of artificial proteins arises as a stringent test of our understanding of the relationship between sequence, structure, and function. examples include the design of a four a-helix bundle, a new protein topology called top , and a series of artificial (ba) -barrels called octarellins. however, de novo design has proven difficult for larger proteins with more than amino acids. here we present two methods to generate the backbone and to perform the de novo design of (ba) -barrel proteins through the use of the software rosetta; both have different advantages and limitations. the first method for generating the backbone is knowledge-based, with a first analysis of a non-redundant database of natural (ba) -barrel proteins in order to obtain statistical analysis on preferred secondary structure element length and amino acidic propensities. with this information we use the rosetta cm software to create more than models which are then ranked in term of rosetta energy. the second method is performed with the parametricdesign package of rosetta, in which only geometrical information are requested (number of strands and helices, radius of the b-and a-barrels, degree of inclination, orientation of the side chains, among others). both methods contain a step of loop refinement and multiple steps of sequence design with the package rosetta design, in order to find low scoring amino acid sequences for each of the starting backbone conformations. thousands of models will be generated by both methods and then analyzed in term of sequence similarity, secondary and tertiary structure prediction, and stability by molecular dynamics simulations. the best candidate sequences will be selected for the experimental verification. in order to identify a putative successfully design, we added a metal binding site during the design step. all the proteins will be expressed in e. coli. the solubility of the designed proteins inside bacteria will be determined thanks to the fusion to green fluorescence protein (gfp). solubility, stability, secondary structure, and cooperativity of folding will be assessed for each protein before determination of their three-dimensional structure. construction of protein capsule possessing drugs controlled release ability shota shimizu , masatoshi nakatsuji , keisuke yamaguchi , yuya sano , yuya miyamoto , takashi inui most compounds that exhibit anti-tumor activities are water-insoluble, thus limiting their clinical use. chemical modification of these compounds and the use of solubilizing agents such as organic solvents, surfactants and ph modifiers improve their solubility. however, chemical modification of compounds decreases their potency, and the use of solubilizing agents causes toxicity in many cases. thus, drug delivery systems (dds) for poorly water-soluble anti-tumor drugs which exploit liposomes, cyclodextrins, and lipid nanoparticles have been studied intensely. in these dds, the controlled release of drugs from the delivery vehicle is one of the most important functions. selective release in target cells leads to adequate therapeutic efficacy with few side effects. in our laboratory, we have already demonstrated that lipocalin-type prostaglandin d synthase (l-pgds), an intravital transporter protein, is a novel and valid drug delivery vehicle for sn- , a poorly water-soluble anti-tumor drug. in this study, we generated l-pgds-based protein capsules with a controlled-release function by introducing a disulfide bond into the upper part of the drug-binding cavity of l-pgds. the intracellular concentration of glutathione ( . mm) is known to be substantially higher than the extracellular concentration ( mm). therefore, it is expected that in the extracellular oxidative environment the disulfide bonds in the protein capsule remain stable, avoiding premature release of the internal drugs during circulation of blood, after reaching the target cells, the disulfide bonds are cleaved in the intracellular redox-environment, and then the internal drugs are released. we generated three kinds of protein capsules which have disulfide bonds in different positions, w c/w c, k c/h c, k c/w c, based on tertiary structure information of human l-pgds (pdb id: o y). firstly, we performed circular dichroism (cd) measurements to confirm the structure of each capsule. the cd spectra of three protein capsules were similar to that of wild-type l-pgds in the far-uv region. therefore, the secondary structures of three protein capsules were not changed from wild-type l-pgds by introducing the mutations. quantitative analysis of the free thiol group in the protein capsule by dtnb assay revealed that the intermolecular disulfide bond was formed by h o -induced oxidation and cleaved by dithiothreitol-induced reduction. in addition, to investigate the solubility of sn- in the presence of protein capsules, we mixed the protein capsule of reduced-form with sn- suspension, and stirred at c for hours. the resulting concentrations of sn- in pbs with mm w c/w c, k c/h c, and k c/w c were mm, mm, and mm, respectively. these values were approximately -fold higher than without protein capsules. sds-page analysis showed that the bond formation decreased in a time-dependent manner, and that new intermolecular disulfide bond was not formed in the protein capsules after hours' incubation. from the above, we succeeded in generating drug delivery vehicles possessing openable and closable lids that are responsive in an oxidation-reduction environment. takaaki miyamoto , mai kuribayashi , satoshi nagao , yasuhito shomura , yoshiki higuchi , , shun hirota graduate school of materials science, nara institutte of science and technology, graduate school of science and engineering, ibaraki university, department of life science, graduate school of life science, university of hyogo, domain swapping has been of interest as a mechanism of protein oligomerization, where a secondary structural region or a domain of one protein molecule is replaced with the corresponding region or domain of another protein molecule. we have previously shown that c-type cytochromes and myoglobin form oligomers by domain swapping. , in this study, we show that a four-helix bundle protein cyt cb , in which the heme of cyt b is attached to the protein moiety by insertion of two cys residues, forms a domain-swapped dimer. dimeric cyt cb was more stable than dimeric cyt b at c, showing that attachment of the heme to the protein moiety stabilizes the domain-swapped structure. absorption and cd spectra of dimeric cyt cb were similar to the corresponding spectra of the monomer, showing that the active site and secondary structures were similar between the dimer and monomer. the redox potential of dimeric cyt cb was also similar to that of its monomer. the dissociation temperature of dimeric cyt cb was c, and its dh on dissociation to monomers was . kcal/mol (per dimer). according to x-ray crystallographic analysis, dimeric cyt cb exhibited a domain-swapped structure, where the two helices in the n-terminal region (helices and ) in a protomer and the other two helices in the c-terminal region (helices and ) of the other protomer interacted between each other. the heme coordination structure of the dimer was similar to that of the monomer. we have previously shown that domain-swapped oligomers of horse cyt c form through intermolecular hydrophobic interaction between the n-and c-terminal a-helices at the early stage of folding. it has been suggested that helices and form first at the initial stage of folding in wild-type apo cyt b . therefore, we propose that cyt cb forms a domain-swapped dimer when helices and interact intermolecularly at the initial stage of folding, whereas the intramolecular interaction of helices and results in formation of a monomer. a highly buried and conserved tryptophan residue close to the dimer interface in a cold-adapted phosphatase is phosphorescent and important for activity. jens g. hj€ orleifsson and bjarni asgeirsson. department of biochemistry, science institute, university of iceland, dunhagi , reykjavik, iceland. alkaline phosphatase (ap) from vibrio g - is a cold-adapted dimeric enzyme with one of the highest catalytic efficiency reported for known aps. it contains five intrinsic tryptophan (trp) residues and one additional trp located on the c-terminal streptag used for expression and purification. in this study, we made several single trp-substitutions to determine the role of each of the trp in the fluorescence emission spectrum. we also determined their solvent exposure by acrylamide fluorescence quenching. the results indicate that trp , trp and trp are mostly responsible for the fluorescence emission. quenching experiments with acrylamide indicated that all the trp residues were about equally accessible for quenching, except trp which was shown to be highly buried in the core of the protein. interestingly, the enzyme was found to be highly phosphorescent at c, having two phosphorescence lifetimes. the longer lifetime is due to trp . trp is located close to the dimer interface and points towards a helix in the active site where his binds an active-site zinc ion. in other aps, an aromatic amino acid is conserved in the location occupied by the trp residue. in most cases for cold-adapted aps it is indeed a trp. interestingly, the mutation of the trp to a phenylalanine affected both stability and activity of the enzyme. kcat/km was -fold lower than for wild-type. overall, this study reveals that trp can be used as a phosphorescent probe of local dynamics and could possibly also serve to study the dimer-monomer equilibrium due to proximity to the dimer interface, an area clearly crucial for enzyme activity and stability. modulating protein-protein interaction with a molecular tether helen farrants , oliver hantschel , kai johnsson ecole polytechnique f ed erale de lausanne (epfl) high-affinity scaffolds for protein-protein interactions, such as monobodies and darpins can be engineered in vitro to bind to protein targets. we speculate that the affinity for the target protein can be modulated by incorporating these evolved scaffolds and a synthetic intramolecular tether into protein switches, in a protein construct of composed of snap-tag, a monobody and a circular permutated dihydrofolate reductase. the tether, attached to the construct via snap-tag, was composed of a linker and trimethoprim, which interacts reversibly with the circular permutated dihydrofolate reductase. we have investigated the affinity between the n-sh domain of the phosphatase shp and an evolved monobody in such a protein construct using a fret assay. when the intramolecular tether was bound the circular permutated dihydrofolate reductase ("closed" conformation), there was an increase in the affinity of the construct to the target n-sh . in the presence of a small molecule competitor ("open" conformation) the affinity of the monobody construct to its target was reverted to the value reported in the literature. the intramolecular tether in these protein constructs combined with engineered scaffolds for protein-protein interactions may be a general approach towards protein switches. because most proteins are long polymers of amino acids with twenty or more chemically-distinct sidechains, there are an enormous number of potential protein sequences. here, we report the construction of biologically active proteins with minimal chemical diversity. transmembrane domains of proteins can specifically interact with other transmembrane domains to modulate the folding, oligomerization, and function of transmembrane proteins. for example, the bovine papillomavirus e protein is a -amino acid transmembrane protein that transforms fibroblasts to tumorigenicity by binding directly to the transmembrane domain of the platelet-derived growth factor b receptor (pdgfbr), resulting in ligandindependent receptor activation and cell transformation. these studies showed that a free-standing transmembrane domain could fold properly in cells and act in trans to modulate the activity of a larger transmembrane protein target. because of the relative chemical simplicity of transmembrane domains and this ability to act even when not linked to more complex soluble protein domains, we reasoned that short transmembrane proteins could be used to define the minimal chemical diversity sufficient to construct biologically active proteins. to accomplish this, we infected cultured mouse cells with a retroviral library expressing -amino acid proteins consisting of an initiating methionine followed by a randomized sequence of leucines and isoleucines, two hydrophobic amino acids that differ only by the position of a single methyl group, and selected rare proteins with transforming activity. we isolated numerous proteins consisting of diverse sequences of leucine and isoleucine that cause morphologic transformation, escape from contact inhibition and focus formation, and growth factor independence. genetic and biochemical analysis of these proteins indicate that like e they interact with the transmembrane domain of the pdgfbr to specifically activate the receptor and transform cells. mutational analysis of individual proteins identified specific leucines and isoleucines required for transforming activity, and insertion of a single isoleucine at a particular position in a stretch of leucines is sufficient for activity. these proteins identify the minimal chemical diversity required to generate a biologically active protein and have important implications for biochemistry, protein evolution, protein engineering and synthetic biology. yusuke azuma , donald hilvert virus-like particles that are precisely loaded with functional cargo are an important tool to study the effect of spatial confinement and create novel entities with application in biotechnology and medicine. by genetic fusion to a positively supercharged green fluorescent protein (gfp( )), an enzyme retroaldolase (ra) was efficiently targeted to the negatively charged lumen of an engineered protein cage, aquifex aeolicus lumazine synthase variant (aals- ). the encapsulation is quantitative under mild aqueous condition up to a mixing ratio of guest enzymes per host cages. the chromophoric tag is used for precisely quantifying the enzyme concentration, which allows detailed characterization of the effect of encapsulation on the enzyme activity. the generality of the encapsulation system was examined with structurally different enzymes. introduction and purpose: in the immune system, high affinity antibodies are generated by selection of b cells activated by antigen-stimulation followed by additional optimization through somatic hyper mutation of antibody genes. in the artificial antibody libraries, such as phage libraries, selection of specific antibody clones from the library is performed by in vitro selection process called biopanning and the subsequent binding screening. however, in spite of high efficiency of enrichment in biopanning, there is a possibility that we overlook the minor antigen-specific clones in the screening because of the limitation of the number of clones employed for screening. in recent years, high-throughput analysis of dna sequences by the next-generation sequencer (ngs) has become available not only for genomic analysis of organisms but also repertoire analysis of antibodies. in this presentation, we report the successful isolation of a variety of antigen specific antibodies from patients-derived antibody phage library by a combination method of high throughput sequence analysis on ngs and biopanning. method: we constructed two kinds of human single chain fv (scfv) antibody libraries from pooled mrna of five cancer patients and of a wheat allergy patient, respectively. after biopanning against a cancer antigen or wheat allergy antigen "gluten", the phagemid vector dna prepared from the pooled phages before or after biopanning was used for pcr amplifications of vh genes, adding the index and adapter sequences for ngs analysis. the high throughput sequencing was performed on miseq (illumina) using miseq reagent kits v . after discarding the short sequences and low quality data, '-and '-reading sequences were unified by a merge program. the frequencies (%) of all vh sequences were evaluated using a program based on usearch . clustering software and the changes of the frequency (%) of each sequence between before and after panning were assigned as amplification rate. results and discussion: vh sequences at each round of pooled phages after biopanning against cancer antigen were analyzed on ngs. after three rounds of biopanning, three clusters of antibody sequences were specifically enriched suggesting these are specific binders. to check this, scfv gene were regenerated by pcr using h-cdr specific primers and scfv-displaying phages reconstructed were subjected to binding analysis. all three phages showed a clear specific binding to cancer antigen in elisa. subsequently, to test the usefulness of this method, we applied it to identify allergen-specific scfv from allergy patient-derived antibody phage library. the phylogenetic tree analysis of vh sequences which showed the amplification rate higher than . by a single round of biopanning elucidated total eleven clusters of vh sequences. the vh sequences in the two clusters with the highest amplification factor were selected and the regenerated scfv-displayed phages were tested for binding analysis. the prepared scfvdisplayed phages and also scfv proteins showed a clear binding ability to allergen. thus, it is suggested that the analytical method of vh sequences on ngs before and after biopanning is very useful to isolate a variety of disease related antigen-specific novel antibodies quickly with high degree of certainty. biochemical analysis of the recognition helix of z-dna binding proteins: roles in conformational specificity yang-gyun kim , xu zheng , so-young park conversion of right-handed b-dna into left-handed z-dna is one of the dramatic structural transitions in biological processes including gene regulation and chromatin remodeling. z-dna binding motif, zalpha (za), was first discovered from human adar . subsequently, with sequence and structure similarity to the hzaadar , families of proteins including viral e l, interferon-induced protein dai (zbp ) and pkz has been identified to have za domain(s). interestingly, the za domain of the e l protein from vaccinia virus (vvzae l) was confirmed to have the ability of z-dna-binding, but it does not have the b-to-z conversion activity. here, we showed that the replacement of the a -helix of vvzae l (vvzae l-a ) with that of hzaadar results in acquiring the ability to converting b-dna to z-dna. the detailed biochemical analysis of the a -helix mutants of vvzae l further suggested that the contribution of positively charged residues in the c-terminal part of the a -helix is crucial during the b-to-z transition. in addition, hydrophobic residues of the n-terminal part of the vvzae l-a also influence on the b-to-z conversion activity, possibly through forming a tightly-packed structure. in conclusion, our results revealed the previously-unknown contribution of amino acid residues existed in the a -helix of the za domains to the b-to-z conversion. moreover, it strongly implies that such residues may play important roles in initiating conformational changes of dna structure during the b-to-z conversion event. the ability of switching the activity of proteins at will is of great interest from an application point of view. one promising approach utilizes a protein modification with an organic photochromic molecule. linking two protein side chains with the photochrome that undergoes a light induced conformational change, protein secondary and tertiary structure can be stabilized or destabilized and thus the structure dependent activity can be switched "on" and "off" by light irradiation. for this the photochrome must fulfil several requirements. foremost, it must possess two states of comparable stability that differ significantly in their geometry. it must further be water soluble and non-toxic, and should not experience fatigue phenomena upon multiple irradiations. there are two classes of molecules that fulfil those requirements: azobenzenes and spiropyrans. we are pursuing two different strategies for the design of photoswitchable proteins. in the first approach we attach an azobenzene compounds to side chains of the alpha-helical antifreeze protein type i. the end to end distance of the photochromic molecule is sterically compatible with the folded helix only in one form, photoisomerization therefore switches the folding state between an active helical state and an inactive unfolded form. in a second, more general approach we use the trp-cage domain as a switching unit. the trp-cage is the smallest known folded protein ( amino acids). its folding is induced by hydrophobic interactions of a tryptophan side chain in a short helical segment. after modification with a photochromic molecule in appropriate positions, its structure is rendered sensitive to the state of the chromophore. by creating protein chimera of such a trp-cage and biologically active peptides with helical propensity, we aim at conferring the light-dependent fold of the cage to the attached peptide moiety. salt-bridges are electrostatic interactions between groups of opposite charges. net interaction energy (ddgnet) of a salt-bridge is partitioned into bridge (ddgbrd), desolvation (ddgdsolv) and protein (ddgprot) energy-terms of which estimation of ddgdsolv and ddgprot are only possible by computational means. thus, general purpose poisson-boltzmann equation solver: "delphi" (in commercial package of insight-ii) and "apbs" (open-source) are popularly used to determine these energy-terms. nevertheless, the computation-method is highly involved one than other uses of these solvers. moreover, protein-specific saltbridges, grid-points, center, hydrophobic-isosteres-mediated mutation-files of original charge-radius file and others are to be worked out prior to the computation. this might answer as to why only limited numbers of structure files ( % of crystal-structure-database) are worked out till date. at this juncture, an efficient fully automated all-in-one-procedure that could analyze large dataset in a single run would be useful. to the best of our knowledge, such procedure is truly lacking in public domain. at this end, our fully automated all-in-one procedure: adsetmeas (available freely at http://sourceforge.net/projects/adsetmeas/along with detailed documentation) uses "apbs" method to compute component as well as net energy-terms of salt-bridges and redirect compact output in excelformat. further, micro-environments of salt-bridges are also been reported based on the presence of polar, dipolar, acidic, basic and hydrophobic side-chains in their proximity. the procedure provides versatility to users in choosing a] model for computation of energy-terms to-date available in the literature and b] method (default or advanced) for parametric optimization in "apbs" calculations. it works in unix like environment including cygwin. it processes all proteins present in the working directory with any number of salt-bridges in them. a pre-released version of the procedure was successfully applied for energy-terms on salt-bridges from halophilic proteins. overall, our adsetmeas provides intricate details on salt-bridge energetic from crystal structures and find application in the field of computational structural biology. these and other results will be discussed in the conference. next generation analgesics -targeting ion channels with antibody-drug conjugates (adcs) anna wojciechowska-bason , clare jones , chris lloyd postdoctoral fellow, adpe, medimmune, cambridge, ria, medimmune, cambridge, adpe ion channels are common targets for chronic pain therapies. small molecule analgesics are widely used therapeutically, but due to poor specificity they often cause a wide range of side effects. as a result, efficacy of existing treatments is very limited. we believe that to achieve the required specificity and efficacy, a novel and innovative approach is required that would combine the potency of the small molecule with the selectivity of an antibody. therefore, we propose to apply antibody-drug conjugates (adcs) to deliver small molecules or peptides to ion channels in order to specifically modulate pain signalling pathways. voltage-gated sodium channel nav . has a well characterised role in the perception of pain. here we present the activity of the peptide huwentoxin-iv (hwtx-iv) and small molecule inhibitors ptc-a, ptc-b and ptc-c on voltage gated sodium channels nav . and nav . . in novel findings, we report that these inhibitors show little selectivity between the voltage-gated sodium channel family members, nav . and nav . , and that the ic values and the impact on channel biophysics (voltage-dependence of activation and fast inactivation) of the inhibitors are largely similar for both channel types. therefore, the use of hwtx-iv and other small molecule inhibitors of nav . for pain therapy could be dose-limited due to side effects mediated by the inhibition of channel nav . . in conclusion, we propose that hwtx-iv and the investigated small molecule inhibitors could be used for the treatment of pain as part of a nav . antibody-drug conjugate (nav . -adc), establishing nav . specificity and minimising side effects. maria antonietta carillo , daniel varon silva malaria is one of the most infectious diseases caused by plasmodium species parasites. the merozoite surface protein (msp ) is the most abundant protein on the surface of the plasmodium species merozoite stage, which plays an important role during the erythrocytes invasion process [ ] . msp is synthesized as a -kda glycosylphosphatidylinositol (gpi) anchored protein precursor which is processed at the end of the schizogony into four different fragments. the primary processing step produces a complex of four fragments that are present on the merozoite surface. the secondary processing step at erythrocytes invasion results in the detaching of the complex from the surface, except for the cterminal -kda domain (msp ), which remains anchored to the parasite surface by the gpi moiety. in human malarial infections, the gpi is considered to be a toxin that causes the expression of various host genes and induces a pro-inflammatory immune response, making it a valuable candidate for the development of anti-malarial drugs. in order to study the function of the gpi and evaluate the effects, msp fragment has been expressed, purified and anchored to the synthetic gpi molecule using protein trans-splicing strategy based on the split intein method [ ] . the role of the gpi moiety will be studied through protein folding experiments and the effect of the anchored protein will be evaluated in vitro in order to understand the function of the gpis. assessment of uch-l substrate selectivity using engineered ubiquitin fusions with varying linker lengths peter suon, mario navarro, john love san diego state university, san diego state university, assessment of uch-l substrate selectivity using engineered ubiquitin fusions with varying linker lengths peter suon, mario navarro, and john j. love san diego state university the ubiquitin proteasome system (ups) is a complex system composed of multiple structural and functional elements that play key roles in cellular processes such as signal transduction, cell cycle regulation, apoptosis, and protein degradation. proteins destined for degradation are first tagged with the protein, ubiquitin, which is covalently attached to internal lysine residues. once the target has be degraded by the proteasome; the enzyme ubiquitin carboxy hydrolase l (uch-l ) is believed to prepare ubiquitin for additional rounds of ubiquitination by cleaving small peptides and chemical adducts from the ubiquitin c-terminus. previously in our laboratory, protein substrates of uch-l were engineered and used to characterize uch-l substrate selectivity. the engineered substrates consisted of n-terminal monoubiquitinated test variants derived from streptococcal protein g (protein gb ) and staphylococcal protein a (spab). the thermal denaturation temperatures (tm) of the fusion proteins were measured using circular dichroism and span a range of over c. more importantly, the rate of hydrolysis for the fusion proteins is demonstrated to be directly correlated to the tm of the test variant fused to the c-terminus of ubiquitin. previously, the engineered substrates were designed to emulate natural ubiquitin fusions and thus did not contain any 'linker' residues between the c-terminus of ubiquitin and the n-terminus of the test protein. to explore the effects of linker length on uch-l hydrolysis we are engineering new uch-l substrates that contain an unstructured amino acid linker between ubiquitin and the test protein. to further explore the catalytic efficiency of uch-l we will revisit diubiquitin (ub-ub), which is not hydrolyzed by uch-l , and will make mutations in the hopes of generating a hydrolysable substrate. using rational design, the new variants will be engineered to destabilize the c-terminal ubiquitin to determine if this results in hydrolysis of the new ub-ub construct. the thermal stability of these new fusion protein substrates will be measured using circular dichroism spectroscopy (cd) and uch-l hydrolysis rates will be characterized using existing assays. our goal is to continue the use of engineered substrates to further explore the catalytic properties of uch-l activity and the potential role in protein trafficking and degradation within living cells. we present a biophysical study of a suite of helical proteins that have been modified to contain and -amino acid additions on their termini that impart increased resistance to degradation in e. coli abstract recombinant expression systems. the b domain of staphylococcal protein a (ab) and the homeobox dna-binding domain from d. melanogaster engrailed (en) are small -helix bundles. these domains do not appreciably accumulate in the e. coli bl (de ) cytoplasm when expression in a pet vector is chemically induced. this is likely due to host protein degradation/recycling factors that function to efficiently degrade these two proteins. addition of sequences encoding either of two amino-terminal beta-hairpins to either the n-or c-terminus of ab and en results in the accumulation of large amounts of these new chimeric proteins. additionally, destabilization of the ab or en sequence does not abolish the expression enhancement effect of the beta-hairpin addition. we have investigated the biophysical origins and effects of the beta-hairpin additions using circular dichroism (cd) spectroscopy, and have determined that the added sequence does not significantly perturb the secondary structure of ab or en, nor does it significantly influence the unfolding temperature (tm). while investigation into the origin of the accumulation effect is ongoing, we hypothesize that the addition of the sequence is disruptive to recognition events in the native protein degradation machinery in e. coli. thus, this approach represents both a biotechnological tool for expressing helical peptides recalcitrant to expression, as well as a system well-suited to probing mechanisms of protein recycling and homeostasis. a special class of these proteins are lipidated proteins containing a glycosylphosphatidylinositol (gpi) glycolipid moiety at the c-terminus. the lipid chains of the gpi anchor molecule are responsible for the membrane association of the attached protein. a unique feature of gpi-anchored proteins is that after isolation they can be reinserted into the membrane of recipient cells with the retention of the biological function. accordingly, the exogenous introduction of fluorescent gpi-anchored protein analogues into cell membranes is a useful method for visualizing the cellular traffic of membrane associated proteins and for engineering cell surfaces. we have recently shown that cholesterol can be applied for anchoring proteins to the plasma membrane of live cells without perturbing the membrane. in order to introduce proteins containing covalent modifications that are not genetically encoded, an enzymatic method was considered and fused with the c-terminal cholesterylation method. the usefulness of the method is demonstrated via the preparation of multimeric model proteins of kda monomers, that is an appropriate representation of the ligation of domain size proteins. transmembrane domain dimerization drives p ntr partitioning to lipid rafts irmina garc ıa carpio , marc¸al vilar sociedad de biof ısica de españa. sbe p neurotrophin receptor (p ntr), is best known for its role in mediating neuron cell death during development or after injury but it also regulates cell proliferation, axon guidance or survival. the key to understand its signaling could rely in its structure and conformational states. it has been described that p forms disulfide-linked dimmers through the cys in the transmembrane domain which are essential for its ngf mediated signaling. previous studies have shown that p is present in lipid rafts, where it interacts with intracellular adaptors to activate different signaling pathways. we design several p mutants in the tm domain that impairs dimerization and study the role of tm domain dimerization in lipid rafts recruitment. our analysis suggests that p tm domain dimerization influences lipid raft partitioning. these results could be a key role to understand its signaling and processing pi- bioluminescent sensor proteins for therapeutic drug monitoring of the monoclonal antibody cetuximab martijn van rosmalen , remco arts , brian janssen , natalie hendrikse , dave wanders , maarten merkx therapeutic drug monitoring (tdm) -adapting the drug dosage scheme to the individual patient's pharmacokinetic and pharmacodynamic characteristics -is still uncommon for therapeutic monoclonal antibodies, despite preliminary studies showing its potential benefits. one of the factors impairing tdm implementation is the lack of equipment and trained personnel to regularly measure drug concentrations in patients receiving treatment. point-of-care diagnostic devices which could be used by patients themselves or by their general practitioners would greatly advance the feasibility of tdm. here we present a biosensor for the therapeutic monoclonal antibody cetuximab. we developed a series of cyclic peptides that specifically recognize cetuximab, covering a fourfold range of affinities, and incorporated these cyclic peptide sequences into a set of luminescent sensor proteins. the sensors translate cetuximab concentrations into a change in emission color that can be read out using a mobile phone camera. together, these sensors can quantify cetuximab levels within the relevant therapeutic concentration range and we propose that they can be used for therapeutic drug monitoring applications. genetically encoded biosensor for cell permeability of inhibitors of the p -hdm interaction silvia scarabelli , thomas vorherr , kai johnsson ecole polytechnique f ed erale de lausanne, the evaluation of the permeability across the cellular membrane is a key step in the development of therapeutics, since it affects the distribution and the efficacy of the latters. reliable and versatile techniques for the determination of structural permeability determinants of molecules and information about the entry kinetics are still missing. we introduced in the past a class of semi-synthetic ratiometric sensor proteins (snifits) that has been shown to be suitable for the measurement of intracellular metabolites concentrations. here we describe a totally genetically encoded sensor based on the snifits modular design for the assessment of the cell permeability of small molecules and peptides inhibitors of the protein-protein interaction between p and hdm . we show that our sensor detects the presence of hdm -binding stapled peptides in vitro, and, when expressed in mammalian cells, it responds to the perfusion of the known small molecule hdm inhibitor nutlin- a. moreover, experiments made with an automated microscope show that the sensor is suitable for measuring and comparing the kinetics of entry of different kinds of inhibitors in the cytosol of living cells. in parallel, we are developing an hcaii-based sensor protein for the sensing of sulfonamides and eventually their peptide derivatives. we show that the sensor responds to the presence of different kinds of hca-inhibitors in vitro and in perfusion experiments. this second sensor would broaden the range of molecules and peptides whose permeability can be studied with our tools beyond the family of the hdm -binders. our sensors overcome the limitations of the already existing techniques for measurements of permeability while offering a simultaneous measurement of the cell permeability and of the binding efficiency of small molecules and peptides of interest. archer: predicting protein function using local structural features. a helpful tool for protein redesign. jaume bonet , javier garcia-garcia , joan planas-iglesias , narcis fernandez-fuentes , baldo oliva structural bioinformatics lab, grib, upf, division of metabolic and vascular health, university of warwick, the advance of high-throughput sequencing methodologies has led to an exponential increase of new protein sequences, a large proportion of which remain unannotated. the gap between the number of known proteins and those with assigned function is increasing. in light of this situation, computational methods to predict the function of proteins have become a valid and necessary strategy. here we present archer, a server that exploits archdb's hierarchy of super-secondary structures to map go and enzyme functions upon protein regions and, thus, infer the function of a protein. the server relies on either the sequence or structure of the protein of interest and returns the mapping of functional subclasses extracted from archdb. moreover, it computes the functional enrichment and significance of each subclass, combines the functional descriptors and predicts the function of the query-protein. combining the functional enrichment analysis of the super-secondary structures with the structural classification of archdb, users can select variants of the target sequence that swap the region of a supersecondary structure by another that putatively fits in the same scaffold minimizing the effect on the global tertiary structure. only variants that modify the predicted function are offered for selection, thus providing a rational, knowledge-based, approach for protein design and functionalization. the archer server is accessible at http://sbi.imim.es/archer. phytochromes are natural photoreceptors known to regulate photosynthesis in plants, fungi and bacteria. phytochromes found in bacteria share common architecture and consist of a pas-gaf-phy photosensory core and a c-terminal output module, responsible for biological function. a bacterial phytochrome, bphp , from rhodopseudomonas palustris undergoes reversible conversion from the farred absorbing state (pfr) to the red-absorbing state (pr) followed by the conformational change upon nm light irradiation. as most of bacterial phytochromes, bphp forms a dimer. it was shown that nm light causes a protomer swapping between the bphp dimers; and likely, the output module is involved in this process. however, the mechanism of the light-induced swapping is poorly studied. we tested an ability of the protomer swapping between bphp dimers using pull-down biochemical assay. for this, strep-tagged bphp was immobilized on strep-tactin sepharose beads in the presence of untagged bphp fused to mruby at different concentrations. after incubation, the proteins were eluted and visualized in sds-gel using a zinc-induced fluorescence assay. an amount of the bound to beads protein was estimated by densitometry. it was found that more than % of heterodimers (streptagged-bphp and bphp -mruby ) form within . h of incubation under nm light at -fold excess of one of the interacting partners. in darkness, the swapping was much slower. in the similar setup we checked the amount of heterodimers after , and min of incubation. no difference was observed for different time points, suggesting that the protomer swapping is relatively fast process. next, a role of the c-terminal effector domain of bphp in the light-induced interaction was studied. for this, kinetics of the pfr-to-pr transition was analysed by measuring of absorbance at nm and nm for full-length bphp and a bphp mutant with the deleted c-terminal domain. while full-length bphp showed the normal pfr-to-pr transition, absorbance of the mutated bphp at nm did not raise. however, nm absorbance changes were similar for both proteins; and surprisingly, the similar dark relaxation kinetics was observed. we propose that the impaired pfr-to-pr transition is caused by restricted pr conformation in the mutant rather than by fast pr-to-pfr relaxation. understanding the mechanisms of the bphp light-induced structural changes and the protomer interaction should advance engineering of bacterial phytochromes into fluorescent probes and optogenetic tools. antibody detection is an integral part of many diagnostic strategies, most crucially so when infectious diseases are involved. currently used assays, such as elisa or spr, enable detection of antibodies in the laboratory with high sensitivity, yet a translation of these technologies to an application outside of the laboratory setting is far from trivial. problematically, the burden of disease for many infectious diseases is carried precisely by those countries where access to laboratory facilities is severely limited. we therefore developed a novel, one-step assay that allows the detection of antibodies directly in solution using a luminescent sensor protein. our strategy is based on the use of a bright luciferase, nanoluc, tethered to a green fluorescent protein (mneongreen) via a semi-flexible linker containing two epitope sequences. crucially, two small helper domains were fused to the protein termini. these domains keep nano-luc and mneongreen in close proximity in the absence of antibody, enabling efficient bioluminescence resonance energy transfer (bret). binding of antibody to the epitopes in the sensor proteins linker domain pulls the bret partners apart, effectively changing the color of emission from green to blue. the assay allowed the detection of picomolar amounts of anti hiv -p antibodies directly in solution, both under optimized buffer conditions and in blood plasma. in principle. the modular sensor architecture should allow detection of any antibody with a well-defined epitope of sufficient affinity. to demonstrate this, the hiv-epitopes were substituted for two ha-tag epitopes, yielding a sensor that enabled the detection of picomolar amounts of anti-ha antibodies. the simple optical readout provided by the sensor system allowed us to record the emitted signal with a conventional mobile phone camera. a simple software application that analyzes the image based on rgb values sufficed to interpret the recorded image vis-a-vis the presence of antibody. bearing in mind the eventually envisioned application in a point-of-care diagnostic setting, this combination of sensor recording and interpretation using nothing more than a mobile phone and a software application holds considerable diagnostic potential. beyond point-of-care diagnosis of infectious diseases, a simple assay to detect and quantify antibodies directly in solution could also have a substantial impact in other fields. antibodies are ubiquitous in biotechnology, and this is reflected by the plethora of potential sensor applications, which range from a role in microfluidic circuits or monitoring the biotechnological production of antibodies, including validation of bispecificity, to veterinary applications, diagnosis of autoimmune diseases and monitoring the success of vaccination campaigns. the continually growing protein data bank (pdb) has been a key resource for general principles of protein structure. for example, parsing structural observations in the pdb into simple geometric descriptors has given rise to statistical energy functions. here we present a novel strategy for mining the pdb on the basis of local tertiary structural motifs (term). we define a term to be the structural fragment that captures all local secondary and tertiary structural environments of a given residue, and query the pdb to obtain quantitative information for each terms. first, we show that by breaking a protein structure into its constituent terms, we can describe its sequence-structure relationship via a new metric we call "structure score." using submissions in recent critical assessment of structure prediction (casp) experiments, we find a strong correlation (r . ) between structure score and model accuracy -a performance that exceeds leading atomistic statistical energy functions. next, we show that querying terms affected by point mutations enables the quantitative prediction of mutational free energies. our simple approach performs on par with state-of-the-art methods fold-x and popmusic on mutations, and provides superior predictions in certain cases where other methods tend to fail. in all, our results suggest that the data available in the pdb are now sufficient to enable the quantification of much more sophisticated structural observations, such as those associated with entire terms, which should present opportunities for advances in computational structural biology techniques, including structure prediction and design. exploiting natural sequence diversity for protein crystallization sergio mart ınez-rodr ıguez , valeria risso , jos e m sanchez-ruiz , jos e a. gavira , departamento de qu ımica-f ısica, universidad de granada, laboratorio de estudios cristalogr aficos, iact-csic-ugr granada during the last decade, different rational and high-throughput approaches have been successfully applied in the protein crystallography field to widen thejjso-called "protein crystallization bottleneck" [ , ] . despite the enormous efforts carried out by our community, the statistics presented by structural biology consortiums [ ] suggest that so far only the easy-to-pick fruit has been attained; thus, new approaches are necessary to further expand the crystallization limiting step to relevant targets. on the basis of previous hypothesis suggesting that the difficulties found in protein crystallization might be a result of evolutionary negative design [ ] , we have used two different protein engineering approaches exploiting natural sequence diversity using beta-lactamase as toolbox: i) ancestral reconstruction and ii) consensus approach [ ] . both approaches resulted in hyperstable and promiscuous ancestral derivatives. furthermore, our initial crystallization results also suggest that both approaches increased the crystallizability of the resulting enzymes when compared to the extant tem- beta-lactamase. the adipocyte-derived hormone adiponectin has become a key player for the understanding of overweight related diseases like obesity, diabetes, atherosclerosis or the metabolic syndrome. one of its abstract major functions are the insulin sensitizing effects, which are mediated by the activation of ampk, p -mapk and ppara ( ). furthermore adiponectin is involved into glucose regulation and fatty acid oxidation. recently, three adiponectin receptors adipor , adipor and t-cadherin have been described while an unknown fourth receptor is hypothesized ( ) . for only two of them (adipor and adipor ) the signaling transduction via adiponectin has been confirmed ( ). in order to find new binding partners or co-receptors, we cloned and expressed full length adiponectin as a fusion protein with a c-terminal intein and a chitin binding domain (cbd) as well as an n-terminal his -tag. by using the impactsystem, the fusion protein was cleaved to form the corresponding thioester. to separate the starting materials as well as the cleaved intein chitin binding domain, the purification was performed with chitin beads. furthermore, the product was concentrated by ni-nta-affinity chromatography. accordingly, the obtained adiponectin thioester was reacted with a tamra-or a biotin labeled peptide, respectively, to receive the corresponding ligation product. finally the functionalized adiponectin was purified by size exclusion chromatography. further studies will allow screening for interacting molecules in cell and tissue derived samples. departamento de quimica fisica, facultad de ciencias university of granada, dpto. de quimica fisica biologica. instituto de quimica fisica rocasolano, departamento de quimica organica, facultad de ciencias university of granada, rational design of non-natural enzyme activities has proved challenging. here, we report the introduction of catalysis of the kemp elimination (a model of proton abstraction from carbon) in scaffolds corresponding to precambrian nodes in the evolution of the antibiotic resistance protein b-lactamase. we used a single-mutation, minimalist approach based on chemical intuition, and obtained catalysis levels similar to those reported in the literature for computational kemp-eliminase designs involving multiple mutations. remarkably, the approach was unsuccessful when performed on modern b-lactamases. we provide experimental evidence that enhanced conformational flexibility contributes to the success of the minimalist design in the ancestral scaffolds. this work has implications for the understanding of function emergence in protein evolution and demonstrates the potential of ancestral protein resurrection in enzyme engineering and design. exploring the importance of dimerization for dj- function through engineered domain fusions sierra hansen , jiusheng lin , mark wilson parkinson's disease is a progressive neurodegenerative disease that affects approximately . million people worldwide and is characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta. dj- (park ) is one of several genes that are mutated in rare forms of familial parkinsonism. dj- is a dimeric cytoprotective protein that defends against oxidative stress and preserves mitochondrial function. dimerization of dj- is thought to be essential for this function, as some diseaseassociated mutations cause poor folding and disrupt the dj- dimer. however, recent reports suggest that dj- may be functional as a monomer. to test this, we have engineered a non-dissociable dj- dimer that is a fusion of two human dj- domains. this construct cannot dissociate into monomers and thus will provide a stringent test of the importance of monomeric dj- . our engineered construct is modeled on plant dj- homologs, which feature naturally occurring duplicate dj- domains separated by a small ( amino acid) linker region. using x-ray crystallography, we confirmed that this engineered non-dissociable human dj- dimer has identical structure to the naturally occurring dimeric protein. we have investigated the influence of enforced dimerization of the pathogenic effects of the parkinsonian l p and l p mutations. cd spectroscopic analysis reveals that single and double l p mutations in the non-dissociable dj- dimer maintain a higher degree of structure than l p mutations in the native protein. additional characterization of the protective capacity and subcellular trafficking of this non-dissociable dj- dimer is underway. the purification, crystallization and preliminary characterization of sdre from s. aureus the purification, crystallization and preliminary characterization of sdre from s. aureus staphylococcus aureus (s.aureus) is an important human opportunistic pathogen which colonizes about % of the human population persistently [ ] . surface proteins of s.aureus can excretion a kind of sortase, which represents a surface organelle responsible during the pathogenesis of bacterial infection the host circulation [ ] . sdr proteins were a component of cell wall anchored family proteins, including sdrc, sdrd and sdre [ ] . sdre could combine with the complement regulatory protein factor h to escape the alternative pathway of complement [ ] . to further investigate the functions of sdre, we have expressed and purified the adhesive domain (residues -' : ), and crystallized the recombinant protein. in addition, we also constructed the mutant s.aureus, and the cell experiments confirmed that sdre gene participate in the bacteria invasion. bacterial microcompartments (bmcs) are proteinaceous organelles that sequester key metabolic reactions to increase enzymatic efficiency or to prevent the loss of volatile or toxic intermediates. there is an increasing desire to engineer bmcs for non-native enzymatic processes. it is thought this will increase multi-enzyme pathway efficiency and allow the expression pathways that may produce toxic or volatile intermediates in bacteria. the mechanisms of small molecule transport and retention of toxic intermediates by bmcs remain poorly understood. better understanding of the bmcs pores critical to engineer bmcs for these non-native pathways. in order to better understand the bmc pore we have undertaken structure-guided modifications of the the hexameric pdua shell protein of the , -propanediol utilization microcompartment (pdu mcp). these modifications include pore mutations in an attempt to alter substrate specificity and permutations of pdua to allow more drastic alterations to the structure of the protein. crystal structures of pdua pore mutants, solved to atomic resolution ( - . Å) provide evidence of the pore residues that confer specificity. further, a pdua permutation (pduap) has resulted in a closed icosahedral cage. this novel pduap cage shows a ph and salt dependent assembly and may serve as a reaction vessel or be utilized for cargo delivery. ( , ) . anm-mc is used to identify targeted transition pathways and intermediates between open and closed states of proteins. at each step of this iterative technique, the protein is deformed along the collective anm mode showing the best overlap with the target direction and its energy is minimized via short mc run. in this work, optimization of simulation parameters (number of mc moves and their perturbation strength, anm deformation factor in each cycle and force constant for backbone bonds) was performed in order to increase the efficiency of this technique. as a result, this technique can now be applied to much larger systems and conformational changes. the transition pathway between apo and dna-bound conformations of the yeast rna polymerase, which is a hetero- -mer with more than residues, will be presented here. moreover, the pathway intermediates for more than diverse proteins were analyzed in terms of changes in local strain energy and backbone torsional angles during apo-to-complex transitions. certain residues interacting with the ligand are detected to exhibit large changes with respect to any of these two parameters for more than half of the proteins in our dataset. department of chemistry and chemical biology, harvard university, howard hughes medical institute, harvard university, transgenic crops have radically reshaped the agricultural landscape. since their introduction in the late s, transgenic crops have affected economic gains greater than us$ billion globally due to reduced production costs and increased yield gains. crops modified to produce biological insecticides derived from the soil bacterium bacillus thuringiensis (bt) are among the most robust methods of pest control. bt toxins offer many advantages over traditional insecticides, chiefly their inability to affect human biology and exquisite selectivity for defined pest species. however, the evolution of resistance to bacillus thuringiensis oendotoxins (bt toxins) in insects has been widely observed in the field, and greatly threatens the use of this mechanism of pest control in the future. we developed a phage-assisted continuous evolution (pace) platform for the rapid generation of high-affinity protein-protein interactions and validated the system by evolving known high affinity antibody mimetics in < days of pace. we applied this system to the evolution of the bt toxin protein cry ac to recognize a non-cognate cadherin-like receptor from trichoplusia ni, a pest for which bt toxin resistance has been observed in both the laboratory and the field. the resulting evolved cry ac variants exhibits high affinity for the target receptor, and kill insect cells more potently than wild-type cry ac. our findings establish that the directed evolution of novel receptor recognition in bt toxins can be used to target resistant pests, and has far-reaching implications for biological reagents and therapeutics. optimization of a designed protein-protein interface brian maniaci , collin lipper , john j. love san diego state university, university of california protein-protein interactions play key roles in practically every biological process. protein-protein interactions vary with composition, affinity, and lifetime of the complex. studying designed protein-protein interactions will provide insight into the underlying principles of complex assembly and formation. computational protein docking and amino acid sequence design were used previously to generate protein dimers from monomeric proteins. the normally monomeric b domain of streptococcal protein-g (gb ) was computational docked to itself, followed by optimization of the interfacial side chains. two variants, monomera and monomerb, were computationally derived as a result of a designed protein-protein interface. these designed proteins were characterized using analytical ultracentrifugation and heteronuclear nmr techniques. this design resulted in a pair of protein monomers that formed a heterodimer of modest binding affinity. a tetrahedral metal-templated interface design strategy was implemented in an attempt to strengthen the monomera-monomerb complex by introducing cross-monomer metal coordination. another advantage of using the metal-templated interface is the ability to control the protein-protein interaction both temporarily and spatially. a number of newly engineered variants of monomer a and monomer b with metal coordination sites were designed, produced, and tested for increased affinity of the protein-protein complex. while the generation of a metal-templated monomera-monomerb complex was unsuccessful, we were able to obtain monomera variants that form a homodimer assembly only in the presence of zinc (ii) ions. the crystal structures of metal-templated monomera variants in the presence of zinc provide an explanation for the observed dimer formation. the crystal structure indicates that the protein-protein interaction is not driven by the designed protein interface, but rather non-specific association via edge-strand interactions. new variants were designed with the goal of engineering a high affinity homodimer in a helix-to-helix orientation as the originally designed protein-protein interface. current evaluation of monomera variants for self-association via metal coordination are being evaluated using size exclusion chromatography with a multi-angle light scattering detector for oligomerization state quantification. the results of this protein design project should lead to a greater understanding of the biophysical parameters that drive natural protein-protein interactions. continuous evolution of site-specific recombinases with highly reprogrammed dna specificities the ability to precisely modify the genome of human cells has enormous potential as a novel therapy and a powerful research tool. in contrast to reprogrammable nucleases, such as talens or a cas / sgrna pair -which specifically cleave dna but then rely on stochastic host cells processes to effect gene insertion -site specific recombinases directly catalyze genomic integration with high efficiency. a major limitation of this approach is that recombinases, such as cre, natively bind with high specificity to long dna target sequences (loxp in the case of cre) that do not exist in the human genome. previous attempts at evolving cre resulted in modest changes to its specificity, or required hundreds of rounds of manual protein evolution. we developed and validated a phage assisted continuous evoluiton (pace) selection for rapidly altering the dna specificity of cre recombinase towards a site present in a human genomic safe harbor locus. the pace experiments resulted in cre variants capable of recombining a substrate with nearly % of the nucleotides altered compared to loxp. we successfully used one of these variants to integrate exogenous dna into the genome of unmodified human cells. we are currently using sequencing methods to determine the specificity of the new recombinase clones. aleardo morelli , burckhard seelig generation of comprehensive deletion libraries mediated by in vitro transposition analysis of protein enzymes and ribozymes from nature, and from in vitro evolution, revealed that deletions of up to dozens of amino acids (or nucleotides) can be structurally tolerated. furthermore, shortened variants can exhibit better stability and increased catalytic activity. in order to investigate the effects of deletions, we developed a new procedure based on in vitro transposition to build libraries of more than , deletion mutants in three to four days. we tested our procedure on dna sequences coding for an artificial rna ligase called ligase c. we used the generated library for an mrna display selection, and isolated two active mutants containing and amino acids n-terminal deletions. structural characterization of ppsc, a multi-domain polyketide synthase from mycobacterium tuberculosis using a fragment-based approach alexandre faille , nawel slama , anna grabowska , david ricard , annaik qu emard , lionel mourey , jean-denis pedelacq polyketide synthases are of great interest in numerous scientific fields. they are composed by multiple domains, each having a different role to play in the catalysis of sequential reactions including condensation, reduction and esterification. their reaction products, named polyketides, represent a large variety of chemical compounds, from antibiotics to immunosuppressors or even anticancer drugs. ppsc is a kda polyketide synthase, organised into six catalytic domains (ks-at-dh-er-kr-acp) with singular functions. along with other type i polyketide synthases, ppsc is responsible for the biosynthesis of an essential polyketide for the virulence of mycobacterium tuberculosis (mtb) and thus is a target of choice for the design of inhibitors. to date, no structural information of any type i polyketide synthase in its entire form has been described. main reasons are the length of these large size enzymes and the flexibility imposed by the linkers between domains, thus making them very difficult to crystallize. numerous questions about domain-domain interactions, spatial arrangement of this complex machinery, substrate specificity and stereochemistry are still unanswered. addressing the structural and functional characterization of ppsc would then help answering these questions and provide valuable information for drug design. to overcome the length-and flexible-dependent problem originating from the presence of multiple domains and linkers, we decided to study domains expressed alone. for this purpose, we used our domain trapping strategy to identify soluble fragments representing a single domain from ppsc [ ] . it has the advantage of not relying on the bioinformatically designed domain boundaries and can even sometimes include parts of linkers to obtain more soluble fragments. using this strategy, we were able to identify relatively small and highly soluble fragments representing each domain of ppsc, thus facilitating the downstream structural and functional characterization. more than fragments have been submitted to crystallization trials. among these, gave crystals and allowed us to determine the x-ray structure of ppsc at, er, in addition to the dh domain in complex with a substrate analog for which activity was confirmed in vitro. the computational design of proteins that bind small molecules remains a difficult challenge in protein engineering. the ability to computationally design native-like interactions with high accuracy and efficiency would be an asset towards therapeutic development, enzyme design, and engineering functional proteins. we have developed a systematic approach to designing interfaces. we first identify ligands with naive binding affinity to our protein scaffold, then use rosettaligand to computationally dock the ligand while designing the interface for a tighter interaction. this way, we are taking a 'shot in dim light' for design as opposed to a 'shot in the dark', allowing us to more thoroughly investigate the successful and not-so-successful designs, and improve the computational methods. of ligands screened, we identified weakly-binding hits in the range of - mm. thus far, rosettaligand has successfully designed one tighter protein-ligand interface, from mm to mm. in progress experiments include designing and experimentally validating more designed interfaces. structural studies of human acidic fibroblast-growth factor (fgf ) mutants with a probable anticancer activity lectins are carbohydrate-binding proteins ubiquitously present in nature. they play a role in biological recognition phenomena involving cells and proteins. the interaction lectin-carbohydrate is highly specific, and can be exploited for the development of nanoparticles containing on their surface lectins specifically directed to carbohydrate residues present only on malignant cells and absent on healthy ones ( ) . lectins have been found to possess anticancer properties and they are proposed as therapeutic agents, binding to cancer cell membranes or their receptors, causing cytotoxicity, apoptosis and inhibition of tumor growth. some lectins are able to prevent the proliferation of malignant tumor cells because they recognize the t-antigen (gal b - galnac) found specifically on the surface of tumor cells ( ) . the main problem is that their use as a detection agent for the t-antigen in clinical studies is not possible because the immune system can recognize them as foreign molecules and develop an immune response. previous studies with x-ray crystallography made in our laboratory have characterized a lectin found in mushrooms called bel b-trefoil which has antiproliferative activity on tumor cell lines, because it contains three binding sites for the t-antigen. unlike other lectins with this property, bel b-trefoil shows structural homology with a human protein, acidic fibroblast growth factor (fgf ) ( ). superposition of their structures suggests that the human protein could be mutated to contain at least one of the binding sites for the t-antigen. such mutations should create in fgf the potential capacity of recognizing tumor cells with less immunogenicity than the fungal protein. fgf is mitogenic and chemotactic, and mediates cellular functions by binding to transmembrane receptors, which are activated by ligand-induced dimerization requiring heparin as co-receptor. to reach our purpose, the fgf cdna was cloned into a bacterial plasmid and then mutated in five different positions to eliminate its mitogenic activity and to engineer in the protein the t-antigen binding capacity. attempts to crystalize the mutants of fgf were made using the hanging drop technique with the final aim to carry out their structural characterization by x-ray diffraction analysis of the crystals. the de novo synthesis of proteins in response to the activation of cellular signaling pathways is a crucial element of many high-level biological processes, including the synaptic plasticity underpinning memory formation in the brain. while of fundamental biological importance, there has been a shortage of tools with which to specifically target pools of newly synthesized proteins of interest for study. thus, we have developed timestamp and smash, methods for drug-dependent tagging, or destruction, respectively, of newly synthesized copies of proteins of interest. both methods rely on protein tags that remove themselves by default via an internal hepatitis c virus (hcv) ns protease, but which are retained in the presence of cell-permeable small molecule protease inhibitors. the timestamp tag contains split yfp halves and epitope tags which are reconstituted and preserved, respectively, on proteins of interest following drug application, whereas the smash tag contains a strong degron which remains attached to proteins of interest following drug application, resulting in their clearance. one limitation of time-stamp and smash is that they can only be used to independently manipulate one protein of interest at a time. furthermore, the application of timestamp and smash to study endogenous protein pools in mammals has not yet been explored. here, we report on efforts to extend these techniques by reengineering ns proteases which can be inhibited by two different drugs orthogonally to one another. by incorporating different drug resistance mutations into two ns protease variants, we engineered ns protease domains that are inhibitable either by asunaprevir only, or by telaprevir only. we found that these tags permit simultaneous and independent control over the newly synthesized pools of two proteins of interest within the same population of cells. we also report the development of transgenic knock-in mouse strains incorporating timestamp and smash tags, which allow the interrogation of newly synthesized pools of specific endogenous synaptic proteins in the context of their endogenous regulatory elements, and without relying on overexpression. infectious diseases are often diagnosed by the presence of specific antibodies that are produced in response to the invading pathogen. one example are antibodies that are present in patient blood after infection with the dengue virus serotype and that are directed against an epitope on the virus' nonstructural protein (ns- ). traditional antibody diagnosis relies on time-consuming multi-step assays that require sophisticated equipment in a laboratory environment. a promising alternative are protein switches that are based on bioluminescence resonance energy transfer (bret). these switches comprise a luciferase (nanoluc) and a green fluorescent protein (mneongreen), which are connected via a semiflexible linker. the linker contains two epitope sequences of ns- to which the antibodies bind specifically. if no antibodies are present nanoluc and mneongreen are held in close proximity via two helper domains and bret can occur; thus green light originating from mneongreen is visible. if antibodies are present, they bind to the specific epitopes in the linker of the switch and cause stretching of the linker and therewith break the interaction of the helper domains. as a result, nanoluc and mneongreen are separated in such a way that bret cannot occur anymore; thus only blue light originating from nano-luc remains visible. using this principle, monoclonal anti-ns- antibodies were detectable in a controlled buffer system and in spiked plasma samples. furthermore, the developed antibody switch was applied to plasma samples of macaques after a primary infection with dengue virus serotype . signal readout was possible using a laboratory-based plate reader as well as the camera of a standard smartphone. we demonstrate that this bret-based protein switch can quickly detect antibodies in solution in a single-step assay format using simple equipment for signal readout, such as a standard smartphone. this simplified antibody detection platform has the potential to be carried out outside of a laboratory, thus in areas with limited laboratory infrastructure and a high number of diverse infectious diseases. proteins expressed from more than two-thirds of the human genome reside within intracellular compartments. of these proteins many are important disease-related targets such as kras and c-myc which cannot be easily addressed by conventional small molecule approaches. some of the weaknesses of small molecules can be addressed by biologic drugs, for example high target specificity and inhibition of protein-protein interactions. the challenge for biologics is how to engineer recombinant proteins to access the intracellular space. one strategy is to use systems evolved by bacteria and viruses to deliver material inside the cells. an example of such pathway is used by pseudomonas exotoxin a (pe). the modularity of pe allows the catalytic domain to be replaced with a biologic payload against desired intracellular target. an additional benefit of pe-based delivery is a possibility of targeting the drugs only to relevant cells in the body by modifying the cell-targeting domain of the pe. the aim of this project is to deliver functional payloads against k-ras and c-myc into the cell using a pseudomonas exotoxin a translocation domain. we used phage and ribosome display to select antibody mimetics that bind k-ras and c-myc. here, we present their activity in biochemical assays and the initial results on generation of pe-based constructs. ( ) . hemagglutinin is synthesized as ha molecule assembled as noncovalently bound homotrimers on the viral surface. this precursor protein is cleaved by trypsin-like proteases to yield two subunits ha and ha linked by a single disulphide bond ( ) . ha is also post-translationally modified by n-glycosylation ( ). it is well established that the virus hemagglutinin is the main antigen, inducing the neutralizing antibodies. in the attempt towards developing influenza vaccine production (the egg-based manufacturing lasts several months) that would be faster and safer the utilization of recombinant antigen alone is currently being observed. recently we demonstrated that yeast produced influenza h protein although cleaved into two subunits induced strong immunological response in mice ( ) . in this report, we describe the biochemical and immunological characterization of the h antigen, based on hydrolytic domain of the h n gene, with deletion of multibasic cleavage site and expressed in yeast system. the ha encoding gene from h n virus with deletion of nucleotides was cloned into ppiczac vector. rha fusion protein with his -tag was secreted into the culture medium and was purified to homogeneity in one step using ni-nta agarose. the efficiency of the antigen purification was mg/l. glycosylation sites of rha were determined using lc-ms-ms/ms. analysis of the n-linked glycans revealed that the rha is glycosylated at the same sites as the native ha in the vaccine strain. next we investigated if the hemagglutinin with deletion of the cleavage site oligomerize into higher molecular forms. to determine the oligomeric forms of the recombinant antigen various approaches were applied e.g. native-page, size exclusion chromatography or dynamic light scattering. as a final experiment to measure the size of oligomers in a protein sample a combined technology sec-mals was conducted, using multi angle light scattering (mals) as a detector. the immunological activity of rha was tested in chicken and mice, where antigen elicited high immune response. the data presented here demonstrate that new influenza antigen produced in p. pastoris is highly immunogenic and might be consider as a candidate for subunit vaccine. structural motifs capture redundant patterns that frequently occur in proteins. motifs associated with contiguous fragments of structure (i.e., secondary structural motifs) are well studied and have been successfully used to capture "rules" describing sequence-structure relationships in protein design and structure prediction. we have extended this concept to motifs that capture tertiary information-(i.e., tertiary structural motifs or terms. we have discovered that a relatively small alphabet of terms describes the known structural universe (all secondary, tertiary and quaternary information in the pdb) at sub-angstrom resolution. this alphabet of universal motifs reveals the remarkable degeneracy of the protein structure space, with just a few hundred terms sufficient to accurately capture half of the known structural universe. we have begun to demonstrate the considerable promise this structural alphabet has for applications such as protein design, structure prediction, and docking. we have developed a novel protein design framework that selects amino acid sequences, given a desired structure, using solely information from the universal terms. we show that given a native backbone, this framework recovers the native sequences to a level on par with state-of-the-art atomistic protein design methods, indicating that the motifs capture the salient structural rules governing native proteins. further, predicted sequence distributions agree closely with observed evolutionary variation. given the apparently high degeneracy among even complex features of protein structure, methods based on mining the pdb for tertiary information should provide ample opportunities for advancement in problems of computational structural biology. sortase-mediated synthesis of protein-dna conjugates for sensitive biosensing bedabrata saha , marieke op de beeck , remco arts , maarten merkx in recent years, semisynthetic protein-dna conjugates have emerged as attractive biomacromolecules for different applications in bio-nanotechnology, biosensing, diagnostics and therapeutics. in protein-dna conjugates, synthetic oligonucleotides allow the construction of desired molecular architecture with high specificity, while maintaining the original functionality of the protein molecules for desired application. however, the synthesis of site-specific and stoichiometric protein-dna conjugates can be challenging. due to the diversity in composition and physico-chemical properties of the proteins, few generic strategies are available for conjugation of protein molecules to a dna scaffold. a common approach is to use thiol-based covalent conjugation, but the introduction of additional cysteines can lead to the formation of intermolecular disulfides or interfere with the formation of native disulfide bonds. as an alternative, here we have developed a site-directed protein-dna conjugation strategy based on sortase mediated trans-peptidation reaction. the sortase recognizes a 'sorting motif' (i.e. lpxtg, x any amino acid), which is recombinantly introduced by site-directed mutagenesis at the cterminal end of the protein molecule. the sortase cleaves the t-g peptide bond and catalyzed the formation of a new amide bond between the lpxt peptide and the n-terminal amine of any molecule bearing an n-terminal oligoglycine motif. for this purpose, a triglycine motif was introduced at the 'end of single-stranded dna (ssdna). on-column synthesis of triglycine modified ssdna, protected on a controlled pore glass beads, simplified the purification process and enhanced the yield of triglycinemodified ssdna (> %). we used this conjugation strategy in several biosensing applications. for example, we used the method to conjugate ssdna linkers at the c-termini of a range of single-chain antibody fragments (scfv) and applied these constructs to allow oriented display of capture molecules on biosensor surfaces. ssdna-scfv were using an excess of triglycine modified ssdna, we achieved % conversion scfv-ssdna conjugate, which can be further purified by in two step purification process consisting of ni-nta affinity column and ion-exchange chromatography. we also extended this sortase-based conjugation strategy to develop a bioluminescence based assay for sensitive target oligonucleotide detection. in this regard, the ' and ' end triglycine-modified ssdna molecules were successfully conjugated with a bret protein pairs, nanoluc luciferase and mneongreen fluorescent protein. the introduction of a c-terminal sortase-his tag and and n-terminal strep-tag allowed efficient purification of theseprotein-ssdna conjugates from excess oligonucleotides and unreacted protein. mass spectrometry based proteomics to identify the protein differences in human breast milk from breast cancer patients and controls devika channaveerappa , roshanak aslebagh , kathleen f. arcaro , costel c. darie breast cancer is the second leading cause of cancer death in women. about % women in the us develop breast cancer. death rates due to breast cancer have been declined over the years due to advancements in mammography and treatment. although, mammography helps in the early detection of breast cancer, it has few limitations. dense breast tissue makes mammogram less accurate. breast milk can be assessed to evaluate the risk of one getting breast cancer by comparing the proteomes of breast milk from healthy and breast cancer suffering individual. this study makes use of mass spectrometry based proteomics to identify the differences between the control and cancerous samples which would further help in identifying potential biomarkers for breast cancer. firstly, sds-page was used to separate the proteins from the whole milk sample. the gel bands for each sample was then excised and cut into small pieces. the gel pieces were washed and trypsin digested in order to extract the peptides. peptide mixtures in the solution were cleaned using c zip-tipp and then analyzed by liquid chromatography-tandem mass spectrometry (lc-ms/ms). minutes and minutes gradient were used for lc-ms/ms analysis. raw data obtained were converted to pkl files using proteinlynx global served (plgs version . ). raw data were then submitted to mascot database search for protein identification. the mascot results were then exported as .dat files and further analyzed using scaffold version . software. three breast cancer milk samples were investigated against healthy control milk samples. in the sds-page gel, after coomassie staining, the protein patterns did show minor differences. after lc-ms/ms analysis, the proteins identified by mascot database search were imported into the scaffold software and compared for the relative ratio between the proteins from the milk sampled from control donors and the donors with breast cancer. there were significant differences identified in the proteomes of the two sets of samples. some of the proteins were upregulated in the breast cancer samples and some were down regulated when compared with the controls. additional investigation of more breast milk samples is ongoing. this study focuses on identifying biomarkers directly in the milk of donors with breast cancer. leukolike vectors: leukocyte-inspired nanoparticles claudia corbo , , alessandro parodi , , roberto palomba , , roberto molinaro , michael evangelopoulos , francesco salvatore , , ennio tasciotti the houston methodist research institute, fondazione irccs sdn, nanomedicine aims to improve drug efficiency by enhancing targeting and biocompatibility, and reducing side effects. multiple surface modifications have been proposed to provide nanocarriers with these features, based on complex synthesis processes and very often inefficient in contemporary providing biological tolerance and targeting properties [ ] . bio-inspired approaches based on surface coatings developed from the purified cell membrane of immune cells represents a new paradigm shift for the development of carrier enable of prolong circulation and proper tumoritropic capabilities. we showed that nanoporous silicon (nps) particles coated with leukocyte cellular membranes -leukolike vectors (llvs) -possess cell-like properties [ ] . llvs can escape macrophage uptake, delay sequestration by the reticulo-endothelial system, target tumor inflamed vasculature and accumulate within the cancer parenchyma [ ] . llvs were fully characterized for their shape, size, surface charge and coating through dynamic light scattering and scanning electron microscopy. in addition we characterized the content and function of the leukocyte's proteins transferred onto the llvs coating through high-throughput proteomic analysis and the results revealed the presence and the correct orientation of several important markers of leukocytes: cd , cd and mhc-i were identified as key players in determining llvs biocompatibility, while leukocyte associated function- (lfa- ) and mac- contributed to the llvs targeting ability and bioactivity towards inflamed endothelium [ ] . recent investigation showed that the coating induced the formation of a singular protein corona (i.e. the protein adsorption layer) on the surface of the nanoparticles compared to negative control following in vivo injection. in addition, the proteolipid coating favored active extravasation of the llvs in the tumor vasculature by molecular mechanisms similar to those used by tumor infiltrating leukocytes. this work shows that is possible to transfer biologically active leukocyte membrane proteins onto synthetic nanoparticles, thus creating biomimetic carriers retaining cell-like functions that are not affected by the protein corona effect that occurs in vivo. the targeting of the inflamed endothelium can be applied to a broad range of diseases and the approach used to formulate the system could open new avenues for the fabrication of the next generation of personalized treatments by using as cell membrane source the immune cells of patients. references: [ ] alessandro parodi, claudia corbo, armando cevenini, roberto molinaro, roberto palomba, laura pandolfi, marco agostini, francesco salvatore, ennio tasciotti. enabling cytoplasmic delivery and organelle targeting by surface modification of nanocarriers. nanomedicine uk. accepted. steroid hormone receptors are intracellular receptors that initiate signal transduction in response to steroid hormones, including oestrogen and androgens. generally, the binding of the steroid to the nuclear receptor induces the protein to form a dimer and relocate onto the chromatin, although the order of these events may vary. the location of receptor binding on the chromatin is defined by specific hormone response elements (hre). once located, the receptor promotes gene activation by the recruitment of other co-factors. it is this process that makes the complex of receptor protein and co-factors play a pivotal role in the regulation and activation of genes. the failure to regulate this process correctly is a key step in the development of several endocrine-driven cancers. for example: oestrogen receptor positive (er ) breast cancer is one of the most common forms of cancer and accounts for % of all breast cancer cases. in er tumours, the oestrogen receptor (er) drives the tumour growth and cell proliferation. understanding the interactions of the er with other proteins, either directly or indirectly, can provide vital insight to the regulation of the system that drives this cancer. the progesterone receptor (pr) has also been implicated in breast cancer, and the androgen receptor (ar) is a known driver in the majority of prostate cancers. to meet the challenges of elucidating these systems, we have developed methods to purify and analyse cross-linked regulatory complexes bound to dna by mass spectrometry (chip-ms). this allows for the enrichment of proteins involved in gene regulation. chip-ms, combined with tandem mass tags (tmt), makes it possible to realise a quantitative method to investigate the dynamic network of interactions between proteins within complexes that undertake the regulation of biological systems. chip-seq is a well-established method for identifying where these protein complexes are bound to the genome. this work focuses on how to combine these technologies with my previous development of cross-linking coupled mass spectrometry techniques (xcms) to provide a strategy for visualising the dynamic organisation of the proteins on the chromatin. global kinetic analysis of caspase protein substrates in cell lysate reveals selective roles and target specificity olivier julien , min zhuang , arun wiita , james wells caspases are cysteine proteases that play important roles in development, cell differentiation and cell death. however, the limited number of known caspase substrates hinders our understanding of caspase function. here we performed a non-biased identification and kinetic analysis of caspase- and caspase- proteolytic substrates in cell lysate, using an enzymatic n-termini enrichment approach followed by mass spectrometry. we identified and potential substrates for the initiator caspase- and putative executioner caspase- , respectively. our results not only confirm known substrates but also identify many more new substrates with the precise location of proteolysis. given the emerging roles of caspases- and in inflammation and neurodegeneration, these new substrates may provide molecular insight into the progression of related diseases. the sequence consensus logo of caspase- targets was very similar to a classical executioner caspase motif (devd), while caspase- revealed a vevd motif. using selected reaction monitoring (srm), we quantified the kinetics of proteolysis of a large subset of these substrates by measuring the appearance of the caspase cleavage product over time. in the end, we measured and kcat/km values for individual substrates cut by caspase- and caspase- , respectively. by comparing these data with our previous analysis of caspase- , , and , we found that substrates that are shared between caspases are often cleaved at rates that differ by orders of magnitude. thus, despite having nearly identical primary sequence motifs, the caspases exhibit remarkable substrate specificity that may reflect their specialized roles within the cell. the rockefeller university, new york university school of medicine, johns hopkins university school of medicine line- (l ) retrotransposons are catalysts of evolution and disease whose sequences comprise a significant proportion of the human genome. despite tremendous influence on genome composition, l rnas only encode two proteins. consequently, l particles include a combination of permissive host factors that are essential to their lifecycle as well as repressive factors that constitute defenses against l 's mutagenic activity. we previously characterized host proteins associated with synthetic and natural human l retrotransposons, as expressed in cell culture, using a combination of techniques including metabolic labeling and affinity proteomics. to build on these analyses, we have implemented a series of d separations and post-purification treatments to produce a multi-dimensional interactomic characterization of affinity isolated l s. these studies have revealed the presence of at least two populations of putative transposition intermediates that may exhibit distinctive intracellular localizations. we report a comprehensive, quantitative survey of the proteins partitioning within these distinct l populations and their associated in vitro activity. our observations provide a basis for the classification of l interactors with respect to their physical and functional links, facilitating hypotheses to direct in vivo experimentation. polyubiquitin recognition by continuous ubiquitin binding domains of rad probed by modeling, small-angle x-ray scattering and mutagenesis sangho lee , trung thanh thach , namsoo lee , donghyuk shin , seungsu han , gyuhee kim , hongtae kim rad is a key protein in double-strand break dna damage response (ddr) pathways by recognizing k -linked polyubiquitylated chromatin proteins through its bipartite ubiquitin binding domains ubz and lrm with extra residues in between. rad binds k -linked polyubiquitin chains as well as k linked ones and mono-ubiquitin. however, the detailed molecular basis of polyubiquitin recognition by ubz and lrm remains unclear. here, we examined the interaction of rad ( - ), including ubz and lrm, with linear polyubiquitin chains that are structurally similar to the k -linked ones. rad ( - ) binds linear polyubiquitin chains (ub , ub , ub ) with similar affinity to a k -linked one for diubiquitin. ab initio modeling suggests that lrm and the extra residues at the c-terminus of ubz (residues - ) likely form a continuous helix, termed 'extended lr motif' (elrm). we obtained a molecular envelope for rad ubz-elrm:linear ub by small-angle x-ray scattering and derived a structural model for the complex. the rad :linear ub model indicates that elrm enhances the binding of rad with linear polyubiquitin by contacting the proximal ubiquitin moiety. consistent with the structural analysis, mutational studies showed that residues in elrm affect binding with linear ub , not monoubiquitin. in cell data support that elrm is crucial in rad localization to dna damage sites. specifically e seems to be the most critical in polyubiquitin binding and localization to nuclear foci. finally, we reveal that the ubiquitin-binding domains of rad bind linear ub more tightly than those of rap , providing a quantitative basis for blockage of rap at dsb sites. taken together, our data demonstrate that rad ( - ) forms continuous ubiquitin binding domains, comprising ubz and elrm, and provides a structural framework for polyubiquitin recognition by rad in the ddr pathway at a molecular level. optimization of a protein extraction method for the proteomic study of pozol cynthia teresa leyva-arguelles , carmen wacher , rosario vera , romina rodr ıguez-sanoja instituto de investigaciones biom edicas, unam., facultad de qu ımica, unam., instituto de biotecnolog ıa, unam key words: proteomics, fermentation, pozol pozol is a mexican traditional no alcoholic beverage elaborated by various ethnic groups in the southeastern of mexico. pozol is obtained from the natural fermentation of nixtamal (heat-and alkali-treated maize) dough. the main carbohydrate in maize dough is starch ( - %), because others such as sucrose, glucose and fructose are mostly lost during nixtamalization; so, the starch remains as the major carbohydrate available for fermentation [ ] . a wide variety of microorganisms have already been isolated from the fermentation of pozol; these microorganisms include fungi, yeasts, lactic acid bacteria, and non-lactic acid bacteria [ ] . however, only few bacteria are amylolytic in this fermentation and all of them are weakly amylolytic [ ] . in an attempt to explain how a very low content of soluble sugars can support a diverse and abundant microbiota, a proteomic approach was designed to understand the fermentation of pozol [ ] . nevertheless, the extraction of proteins from pozol remains a limiting step in proteomic analysis mainly due to the complexity of the sample. on the basis of the aforementioned reasons, the aim of this work was to obtain a suitable extraction method of proteins for proteomic analysis. therefore, the fermentation of pozol was continued for h and samples were taken at , , and h. for each sample, the total sugar content was determined by the dubois et al. method [ ] and protein extraction was performed by two methods: a) direct extraction from the dough [ ] and b) initial extraction of microorganisms and soluble proteins (this work). comparison between the two protein methods was performed on two-dimensional gels with silver stain. then, gels underwent to image analysis by the image master d platinum software. comparing the d-gels, more proteins spots were obtained with method b than that with method a, indicating a more efficient protein extraction with method b. although, using method a higher concentration of total proteins was observed, they were mostly maize proteins, that in turn overlap and reduce the efficiently extraction of the microbial low abundant proteins. then, method b allows a better extraction of those low abundant proteins and removes sample components that may interfere with the determination. these results could help us to find the proteins involved in carbohydrate metabolism of the microbiota and finally elucidate the dynamics of pozol fermentation. proteomics has been applied to the enology field for numerous purposes including fermentation control, improvement of fermentation processes, ensuring wine quality, etc. according to rodriguez et al., ( ), the information provided by wine proteomics is not only useful for these intentions, but also offers excellent prospects for innovation and diversification of winemaking processes in the near future. in this context, our group has focused research on the identification of proteins that might be important for yeast survival under typical wine elaboration conditions (standard fermentation, sherry wine biological aging and sparkling wine second fermentation) as well as proteins that configure the content of metabolites which are ultimately responsible for wine quality. by using novel proteomic (offgel fractionator and ltq orbitrap xl ms) and metabolomic techniques (sbse-td-gc-ms) we have identified a high amount of up-regulated proteins involved in processes like oxidative stress response (in biological aging) or protein biosynthesis (in second fermentation) as well as thirty-three proteins directly involved in the metabolism of glycerol, ethanol and seventeen aroma compounds excreted by the yeast under biological aging conditions. further, in order to validate proteome data; null mutants of genes codifying proteins up-regulated in the biological aging condition were constructed. analyses of correlated phenotypes are in progress. this technique and its combination with metabolomics within the enology context will provide enough knowledge to design or choose yeasts or conditions that satisfy wine production and/or wine characteristics such as color/aroma/texture/flavour profile demands of winemakers and consumers. additional binding sites for cytochrome c on its redox membrane partners facilitate its turnover and sliding mechanisms within respiratory supercomplexes blas moreno-beltr an , antonio d ıaz-quintana , katiuska gonz alez-arzola , alejandra guerra-castellano , adri an vel azquez-campoy , miguel a. de la rosa , irene d ıaz-moreno ibvf, ciccartuja, universidad de sevilla -csic, bifi -iqfr (csic), universidad de zaragoza, departamento de bioqu ımica y biolog ıa molecular celular, universidad de zaragoza, gliding mechanisms of cytochrome c (cc) molecules have been proposed to shuttle electrons between respiratory complexes iii and iv within plant and mammalian mitochondrial supercomplexes, instead of carrying electrons by random diffusion across the intermembrane bulk phase [ ] [ ] . in this work, the binding molecular mechanisms of the plant and human cc with mitochondrial complexes iii and iv have been analyzed by nuclear magnetic resonance and isothermal titration calorimetry. our data reveal that both cc-involving adducts possess a : stoichiometry -that is, two cc molecules per adduct -. the presence of extra binding sites for cc at the surfaces of complexes iii and iv opens new perspectives on the mitochondrial electron transport chain, where membrane respiratory complexes can be either in independent, free diffusional motion or forming macromolecular assemblies. in the latter context, such new binding sites for cc facilitate the turnover and sliding mechanisms of cc molecules within supercomplexes. indeed, the accommodation of several cc molecules between complexes iii and iv in supercomplexes provide a path for cc diffusion from complex iii to iv. such path could have physiological significance in the electron flow, which is controlled in supercomplexes to optimize the use of available substrates [ ] [ ] [ ] . can bio-functionalities be deciphered from protein sequence information using computational approaches? background: the processes of uncovering bio-functionalities such as pharmacological activities, disease processes, physiological and structural properties by means of clinical approaches are irrational. this is because they are resource and time consuming. sometimes, they involve sophisticated and expensive equipments, reagents and animal tissues. contrarily, sequence information-based computerized approaches are rational and have become relevant in assessing bio-functionalities. they include geno pheno [coreceptor] [ ] , position-specific scoring matrix (pssmsi/nsi and pssmcxcr /ccr ) [ ] , and informational spectrum method (ism)-based phylogenetic analysis (istree) [ ] . aim: this presentation demonstrates how bio-functionalities could be deciphered from sequence information using computational approaches. method: ism procedure and peptides, vipmfsals and capagfail are engaged. results: protein sequences of the peptides are converted into bio-functionality (affinity). affinity between the two peptides is demonstrated as significant amplitudes at the point of common interaction also referred to as consensus frequency, signifying remarkable affinity. discussions: bio-functionalities of bio-molecules are known to be expressed in one or two genes, which have been found to provide as much biological information as the bio-molecules. this indicates that biological characteristics, represented in these genes and proteins can now be extracted from their sequence information. for example, multi-drug resistances arising from a variety anti-microbial agent from several classes including alkaloids, flavonoids, etc can be retrieved from the sequence information of their encoding genes (mdr and mdr ). similarly, translation of hiv infection to aids disease can be extracted from the protein sequence alterations in the hiv gp . similarly, effectiveness of anti-retroviral agent, maraviroc on the hiv isolate h bx and ndk can be deciphered from the sequence information of their v observed at the predicted sequences. these positions are important as they surround the cleavage site in the three-dimensional structure, and are probably less tolerant to change. moreover in previous studies, cys at p position has been shown to be the dominant determinant for cleavage efficiency, while cys, pro and glu at p position have also been shown to be correlated with increased cleavage efficiency of ns / a protease. for adv cysteine protease, on the other hand, bsst produces similar significant results for both type (xgx-g) and type (xgg-x) consensus cleavage sites, where p and p ' positions have gly with highest percentage in type (xgx-g) while p and p positions have gly in type (xgg-x). these indicate that the bsst seems to provide a powerful methodology for predicting the substrate specificity for the hcv ns / a serine protease and adv cysteine protease, which are targets in drug discovery studies. protein plasticity improves protein-protein binding description chiara pallara , juan fern andez-recio an accurate description of protein-protein interactions at atomic level is fundamental to understand cellular processes. however the current structural coverage of protein-protein interactions (i.e. available experimental structures plus potential models based on homologous complex structures) is below % of the estimated number of possible complexes formed between human proteins. , for these reasons, computational docking methods aim to become a complementary approach not only to solve the structural interactome but also to elucidate the basis of the protein-protein association mechanism. in spite of the advances in protein-protein binding description by docking, dealing with molecular flexibility is a major bottle-neck, as shown by the recent outcomes of the capri (critical assessment of prediction of interactions) experiment. this data clearly confirms that the protein dynamics plays a key role in protein-protein association. the use of conformational ensembles generated from unbound protein structures in combination with computational docking simulations might represent a more realistic description of protein-protein association. here, we present the first systematic study about the use of precomputed unbound ensembles in docking, as performed on a set of cases of the protein-protein docking benchmark . . the primary aim of our work is to understand the role of the protein conformational heterogeneity in protein-protein recognition. to do this, small conformational ensembles were automatically generated starting from the unbound docking partners, and then an extensive analysis of their binding properties was performed in the context of pydock docking scheme. the results show that considering conformational heterogeneity of interacting proteins can improve docking description in cases that involve intermediate conformational changes in the unbound-to-bound transition. more interestingly, we found that protein plasticity increases chances of finding conformations with better binding energy, not necessarily related to bound geometries. the relevance for future docking methodology development and for understanding protein association mechanism will be discussed. purpose of the research: there is increasing interest in the development of protein scaffolds that can be used to develop affinity reagents that are alternatives to antibodies. the affimer scaffold is based on the cystatin protein fold. the affimer scaffold is biologically inert, biophysically stable and capable of presenting a range of designed or random binding surfaces defined by peptides inserted at different loops. the result is highly specific, high affinity interactions with a wide range of targets including ones that are inaccessible to antibodies. affimers are designed to work in the same way as the very best antibodies, but with a number of key advantages. affimers are quick to develop (typically weeks) without using animals. they contain no disulphide bonds, are expressed easily in e. coli and have no batch to batch variability. affimers are small molecules ( aa, kda), robust and stable (resistant to ph range, thermally stable and not sensitive to edta). affimers can be a direct replacement for antibodies -no process or workflow change required -and perform identically to antibodies in assays such as elisa, facs, ihc, western blots, affinity purification, microarray and potentially therapeutics. we describe some applications of the technology in regards of affimer development for custom targets on one hand and for the biomarker discovery workflow using affimer microarrays on the other. main results: by screening of our very large ( x ) library against yeast sumo protein we identified affimers with high affinity allowing their use for elisa. moreover, no cross-reactivity was observed when affimers were used on western blots leading to a unique band specific to yeast sumo when compared to human proteins. a library of , random affimers, expressed in e. coli, was printed on glass microscope slides and challenged with plasma from children (n ) with sepsis and from healthy children (n ). unsupervised hierarchical clustering based on the , affimers allowed differentiation between the control and patient samples. affimers were found to differentially bind proteins between the groups with a > fold change. the affimer arrays identified a strong signature of sepsis and roc curve analysis allowed confident prediction of disease (auroc of . ). affinity purification and preliminary mass spectrometry analysis identified known biomarkers of sepsis and also potentially novel biomarkers not previously associated with this disease. major conclusions: this work demonstrates the scope of affimer affinity reagents to develop alternative binders to antibodies, where affimers perform identically in most assays without the disadvantages associated with antibodies. moreover, affimers enable a new protein microarray-based biomarker-discovery workflow and we predict that array-based validation of signatures identified using discovery arrays prior to affinity purification and mass spectrometry will offer a cost-and time-effective methodology compared to purely mass spec-driven workflows. tau pathologies, called 'tauopathies', are related to several neurodegenerative diseases including alzheimer disease (ad). in ad, tau protein is observed hyper-phosphorylated and aggregated as paired helical filament (phf). the neuronal tau protein is an intrinsically disordered proteins (idps). nuclear magnetic resonance spectroscopy (nmr) is here used to study the tau protein phosphorylations and protein-protein interactions (ppis). in in vitro assays, tau phosphorylation by rat brain extract is considered as an hyperphosphorylation model that was furthermore pointed out to enable tau aggregation [ ] . in a first step, we have identified all the phosphorylation sites of rat brain extract phosphorylated-tau, using the analytical capacity of nmr. we showed that the protein is modified at ser/thr sites. among the kinases that we have characterized so far using tau as substrate, only the extracellular signal-regulated kinase (erk ) shows an ability to modify in vitro tau protein on so many sites. we have indeed identified phosphorylated ser/thr-pro motifs out of potential phosphorylation sites in the sequence of full length -residue tau. in addition, we showed using transmission electron microscope (tem) a similar in vitro aggregation capacity of erk-phosphorylated tau protein compared to that of rat brain extract phosphorylated-tau. this shows that phosphorylation by the erk kinase generates an hyperphosphorylated tau. given the high efficiency of erk towards tau, we have next looked into the mechanism of tau recognition. erk kinase possesses two well-characterized docking domains: d recruitment sites (drs) and f recruitment sites (frs), which recruit complementary docking sites and increase the specificity and efficiency of the interaction with both its upstream regulators and downstream substrates [ ] . as the interaction between tau protein and erk kinase is analyzed by nmr spectroscopy, multiple sites of interaction are observed along the tau sequence, similar to drs docking sites, all located in the so-called microtubule binding domain of tau. these sites are short sequences loosely matching the reported consensus for d sites w - uxu (w, u, and x refer to positively charged, hydrophobic, or any intervening residues, respectively) [ ] , and also the reverse sequence uxuw - .to confirm the mapping of the interaction, two tau recognition sites were produced as recombinant peptides of about amino-acid in fusion with an n-terminal his-tag sumo. interaction assays using d [ h, n] hsqc spectra of the peptides confirm their binding to erk kinase. the potential of these peptides to inhibit erk activity with tau as substrate is now being investigated. while rigid-body docking has become quite successful for predicting the correct conformations of binary protein complexes, determining whether two given proteins interact remains a difficult problem. successful docking procedures often give equally good scores for pairs of proteins for which there is no evidence of interaction. studies investigating what we define as the 'pre-docking' problem via in silico approaches have only recently become feasible with the help of supercomputers and gridcomputing systems. in a previous work, on a restricted set of protein complexes, we showed how predictions of interacting partners could be greatly improved if the location of the correct binding interface on each protein was known. experimentally identified complexes are found to be much more likely to bring these two interfaces into contact, at the same time as yielding good interaction energies. we present data from a complete cross-docking (cc-d) study of a database of proteins, including the treatment of more than , potential binary interactions. the performance of the interaction index we developed to predict binding probability compares well with other methods. by studying the interaction of all potential protein pairs within a dataset, cc-d calculations can also help to identify correct protein interaction interfaces. the present large-scale study also reveals the influence of various protein families (enzyme-inhibitor, antibody-antigen, antigen-bound antibody, etc.) on binding specificity, showing, in particular, the distinctive behavior of antigenic interfaces compared to enzymes, inhibitors or antibodies. the performance of our approach is encouraging. although identifying interaction interfaces significantly helps in the identification of interacting proteins, further refinements will be necessary to make in silico cross-docking a viable alternative to high-throughput experimental methods. whole-protein mass spectrometry reveals global changes to histone modification patterns in hypoxia sarah wilkins , kuo-feng hsu , christopher schofield chemistry research laboratory, oxford university cells respond to limiting oxygen availability (hypoxia) by altering the gene expression profile. this primarily involves changes at the level of transcription via the activity of hypoxia-responsive transcription factors, although increasing evidence suggests that changes in chromatin structure (i.e. from a condensed 'silent' state to a more open or 'active' state) are required in order for transcription to take place. in particular, post-translational modifications (ptms) to histones have an important regulatory function in gene expression under hypoxic conditions. the n-terminal tails of histone proteins are accessible to a set of enzymes capable of 'writing' and 'erasing' ptms including acetylation, methylation, ubiquitylation, sumoylation and phosphorylation. to date, studies in hypoxia have employed antibody-based methods to investigate changes in histone modifications, and so have focused on individual marks in isolation. the interplay between coexisting ptms is thought to be much more important than the effect of any single mark. therefore, a global view of the histone modification profile is essential to gain a complete understanding of the function of histone ptms and their roles in gene regulation. in this study, we apply whole protein mass spectrometry to investigate hypoxia-induced changes in histone marks. this 'top-down' approach provides insight into combinational modification patterns that are difficult to establish by antibody-based methods or peptide ms analysis. we investigated changes in the global ptm profiles of histones from a range of human cell-lines and tissues under severe hypoxia (< . % o ). we find that hypoxia causes a shift in the overall profile towards a more highly modified state, with significant changes in methylation and phosphorylation. marked changes in histone ptms were also observed following treatment of cells with epigenetic inhibitors and commonly used hypoxia mimetics, including several iron chelators currently in clinical trials for the treatment of anaemia. finally, we show that this method can be used to identify the histone variant h ax, whose phosphorylation at serine is an indicator of double-stranded dna breaks in cancer. overall, these data provide important insights into the epigenetic changes associated with hypoxia in normal and disease contexts. we hope to further develop this method in combination with different labelling strategies to enable quantitative analysis of histonemodifications in cells. mass spectrometry-based protein biomarker discovery in neurodevelopmental disorders interactions. there is currently no biological diagnosis or known cause of asd. slos is characterized by a cholesterol deficiency due to a mutation on the dhcr gene. approximately / , babies are born with slos. diagnosis is achieved by measuring cholesterol and -dehydrocholesterol ( dhc) levels in the blood, however, there is currently no proven treatment for slos. because of this, research is increasing to determine biomarkers for these disorders. here, samples from people with asd (sera and saliva) and slos (saliva), and matched controls were analyzed using a combination of gel electrophoresis (tricine-page, sds-page and blue native page), in gel digestion or insolution digestion and nanoliquid chromatography-tandem mass spectrometry (nanolc-ms/ms) to investigate differences between the proteomes of people with these neurodevelopmental disorders and matched controls. several alterations in protein expression were identified. these differences may lead to potential biomarkers for diagnosis, possible therapeutic targets and an altogether better understanding of the disorders. understanding protein recognition using structural features protein-protein interactions (ppis) play a crucial role in virtually all cell processes. thus, understanding the molecular mechanism of protein recognition is a critical challenge in molecular biology. previous works in this field show that not only the binding region but also the rest of the protein is involved in the interaction, suggesting a funnel-like recognition model as responsible of facilitating the interacting process. further more, we have previously shown that three-dimensional local structural features (groups of protein loops) define characteristic patterns (interaction signatures) that can be used to predict whether two proteins will interact or not. a notable trait of this prediction system is that interaction signatures can be denoted as favouring or disfavouring depending on their role on the promotion of the molecular binding. here, we use such features in order to determine differences between the binding interface and the rest of the protein surface in known ppis. particularly, we study computationally three different groups of protein-protein interfaces: i) native interfaces (the actual binding patches of the interacting pairs), ii) partial interfaces (the docking between a binding patch and a non-interacting patch), and iii) back-to-back interfaces (the docking between non-interacting patches for both of the interacting proteins). our results show that the interaction signatures in partial interfaces are much less favoured than the ones observed in native and back-to-back interfaces. we hypothesise that this phenomenon is related to the dynamics of the molecular association process. back-to-back interfaces preserve the exposure of the real interacting patches (thus, allowing the formation of a native interface), while in a partial interface one interacting patch is sequestered and becomes unavailable to form a native interaction. structural characterization of the cytoplasmic mrna export platform laboratory of cellular and structural biology, the rockefeller university., laboratory of mass spectrometry and gaseous ion chemistry, the rockefeller univ., university of california, san francisco, the new york structural biology center, department of biochemistry, faculty of medicine, university of montreal mrna biogenesis is an intricate process that begins within the nucleus and culminates with the remodeling and nuclear export of the mrnp particles through the nuclear pore complex (npc). defects in this conserved mechanism have been shown to cause serious human diseases. the protein assembly that performs the last steps in mrnp biogenesis and export is located at the cytoplasmic face of the npc and is formed by different proteins, organized into several subcomplexes whose arrangement and molecular architecture are poorly understood. in this study we applied an integrative approach, combining cross-linking and mass spectrometry (cx-ms), electron microscopy and available high-resolution structures, to describe the molecular architecture of the endogenous npc cytoplasmic mrnp export machinery. we generate a hybrid, close-to-atomic structure of the yeast native nup complex, the core of the assembly. our map also reveals how the nup complex organizes the entire cytoplasmic mrnp export machinery, and how this in turn docks into the architectural core of the npc. mapping of phenotypic profiles into our structures allows us to generate a first functional map of the ensemble. we expect that our map will serve as a framework to understand the molecular mechanisms underlying this key step of mrnp biogenesis. study of candidate proteins to pore associated with p x receptor in different cell types carla oliveira , anael alberto , mônica freitas , luiz alves laborat orio de comunicac¸ão celular -fiocruz, centro nacional de ressonância magn etica nuclear -ufrj aim: the p x r is a purinergic receptor, which differs from others subtypes due to its structural and pharmacological characteristics. when exposed for extended time or to high concentrations of its agonist (atp), promotes an increase in membrane permeability, allowing the passage of molecules up to da. there is a controversy among several authors that leave in doubt if this receptor needs a second protein for the pore formation and which protein could be. we select five pore-forming proteins: trpv , trpa , connexins- (cx- ), pannexin- (panx- ) and vdac. we believe that different mechanisms and proteins could be associated with p x r, depending on the cell type and their microenvironment stimuli. in this context, our main goal is identify possible proteins that could be associated with the p x r pore in different cells and species. methods and results: we started with rt-pcr technique of cell lines: j .g , n a, u , u , hek- and primary cells from wistar mouse and swiss mice. we used different primers and pcr cycle for each target at different species. we observed that the p x , panx- and cx- are the most abundant and are present in all cell types except the absence of p x in u cells and panx- in mice macrophages and u cells (n> ). however, trpv was seen at n a and u cells and trpa in and primary cells from mouse and mice and in j .g cells (n> ). regarding to the vdac, it is present in mouse macrophages, j .g and hek- cells (n> ). the further steps, we verified if those proteins could be physically associated with the p x r. we coimmunoprecipitated the p x r of j .g (with or without atp), mice macrophages, hek- and u cells. the samples were applied in two separated . % bis/acrylamide gels: one destined to mass spectrometry (ms) and the other to western blot. at this point, we confirmed the presence of p x r, and observed several others proteins associated to p x r at different cell conditions, mainly when we exposed, j .g cell, to mm atp (n ). at this condition, we found by ms, hsp , , and ; alpha and b tubulin; myosin va; alpha, b and g actin; malate and lactate dehydrogenase (n ). although u and hek- had not received atp treatment, we found several proteins associated to p x . the next step was to immunoprecipitated those proteins in j .g (treated or not with atp) and use it to verify if p x are physically associated to them. as result we saw the p x associated to panx- in j .g cells. conclusion: we conclude that the p x r activated by extracellular atp triggers the recruitment of variety different proteins. at this condition, we can suggest that maybe there is a conformational change, regardless of the numerous recruitment structural proteins. in addition, apparently, the pore-forming protein pannexin- is associated with p x r, and the others pore forming proteins (vdac, cx- , trpv , trpa ) seems not be linked to p x r at j . recently, we developed a series of molecular modeling tools for structure-based studies of protein functions and interactions. these tools are publicly available as web servers that are easily operated even by non-specialists: cabs-fold server for protein structure prediction [ ] ; cabs-flex server for modeling of protein structure flexibility [ ] ; aggrescan d server for prediction of protein aggregation propensities and rational design of protein solubility [ ] ; and cabs-dock server for prediction of peptide binding sites and peptide docking [ ] . the web servers are freely available from the laboratory website: http://biocomp.chem.uw.edu.pl/tools sandy on , pinghui feng university of southern california, keck school of medicine, developing a technique to detect deamidated proteins and peptides using rig-i sandy on, pinghui feng university of southern california, norris comprehensive cancer center, department of microbiology, and molecular biology, los angeles ca perhaps the most notable type of post-translational modification of proteins and peptides into a higher order structure is deamidation of asparagine and glutamine. deamidation occurs when an amine group is removed, degrading the molecule for purpose of regulating intracellular levels. previous studies have demonstrated that this notable post translational modification has been uncovered over time for use in dna recombinant technology as well as use as a biological clock to facilitate the rapid turnover of biologically important components of the cell. while the effects of this non-enzymatic chemical reaction have been widely studied, the method to uncover modification sites over a large quantity of proteins remains an issue. one of the most common types of deamidation is of asparagine and glutamine residues. at this time, most researchers will depend on mass spectrometric based proteomic techniques for identification of these post-translational sites. the issue is that mass spectral analysis of deamidated proteins and peptides is complication and can lead to misassigned identification attributed by an overlapping of c peak of the amidated form with the deamidated monoisotopic peak; these two peaks are only separated by . mda. while these issues can be mediated by using a mass spectrometer with a high mass measurement accuracy, and high resolving power, it is essential to establish simpler methods for identifying substrates that have undergone deamidation. if deamidation is present, different protein bands will be exhibited in the western blot, which will be compared to a triple mutant rig-i, which resists deamidation, to observe the location of this modification on the protein. with enough testing, i will determine specific sites of digestion and use this information to make conclusions of unknown proteins. i will make results regarding whether the protein has been modified based on the digestion sites. i will use mass spectrophotometry analysis to compare the proteins on a wider scale and double check my results. i have narrowed it down to a couple of different digestion sites that indicate deamidation. though the analysis work can be tedious, it is crucial to ensure the sites we isolate are accurate in order to establish this technique. from my research, we can apply this method for wider scale use such as in clinical settings. in areas of inflammation of parkinson's' patients, we can review specifically the infected cells versus uninfected and isolate the proteins, usually deamidated, responsible often smaller in size and more specific. in addition, research articles have already shown that suppressing modification of certain cells such as bcl-xl playing a major in leading the regulation of cancer cell death by apoptosis. by leading the discovery of a simpler methods to uncovering deamidation in cells, researchers will more easily and quickly be able to scan through various proteins, some of which discovered eventually may play pivotal roles in cancer research. influenza virus (iv) hemagglutinin (ha) is a homotrimeric integral membrane glycoprotein that mediates receptor-binding and membrane fusion. it constitutes the prominent viral surface antigen and a main target for neutralizing antibodies. bacterial, recombinant ha-based vaccines indicate high potential to confer protection against highly pathogenic (hp) avian iv (aiv) h n and arise as alternative for the traditional egg-or cell culture-based manufacturing. relatively short time of bacterial has production can be of great importance in case of a pandemic. escherichia coli produced protein, based on the ha sequence of a/swan/poland/ - v / (h n ) hpaiv*, has been successfully expressed in the form of inclusion bodies at institute of biotechnology and antibiotics. refolded and purified antigen was obtained in a soluble form, isolated by reversed phase hplc and identified with peptide mass fingerprinting using matrix-assisted laser desorption/ionization time of flight mass spectrometry (maldi tof/tof ms). the performed research in a great extent allowed to confirm the amino acid sequence of the recombinant ha (rha) assumed based on the cdna and allowed to establish the location of a total of six disulfide bridges. however, during purification and storage of the rha, apart from desired higher order rosette-like structures of the protein, other non-native species resulting from posttranslational modifications, misfolding, aggregation and degradation may occur what results in reduced vaccine potency. here, besides the properly folded monomers, we indicate non-native aggregates induced by disulfide crosslinking. moreover, several free cysteine residues and unexpected intrachain s-s were identified in rha tryptic peptide maps. cys was found most susceptible to formation of disulfide bridges between the distinct chains of rha. the above findings allow to assume that not all rha particles fold to form the native structure. reduced cys residues exhibit tendency to undergo oxidation and uncon- new strategies and approaches to understand how antibodies recognize and neutralize snake toxins represent a challenge to improve the antivenoms. the neurotoxic activity of micrurus venom is carried majority by two distinct proteins families, ftx and pla . the conserved structural folding of these toxins can be appreciated as model to generate inhibitors against them. in this regard, monoclonal antibodies (mabs) can be used as tool to find hot spots for inhibit the toxins and represent the first step in order to develop recombinant neutralizing molecules. in this work our goals were analyse a set of monoclonal antibodies against the most toxic components of m. altirostris venom by proteomics approaches. the venom was fractionated; its major toxic proteins identified by in vivo tests based on murine lethal toxicity analyses (approved by the ethical committee for animal experimentation from center of health and science of the federal university of rio de janeiro -no. . / - ). the toxic components were used to generate a panel of five monoclonal antibodies. elisa and antivenomics results allowed us identify the specificity of all mab and their neutralizing efficacy was measured by in vitro tests. three mabs showed reactivity towards ftx and two against pla . all monoclonal antibodies against ftx lack a broad recognition. however, we identified a pair of monoclonal antibodies able to recognize all pla molecules of m. altirostris venom and showed a synergism to inhibit the catalytic activity of them. moreover, we challenge monoclonal antibodies against to micrurus venom for inhibit the pla activity of naja naja, specie taxonomically out of micrurus cluster. our results showed that pla of m. altirostris venom share a pair of conserved antigenic regions and draw attention to use these epitopes to miming antigen to generate antibodies for antivenom production. moreover, face to the cross reactivity and the pla activity inhibition capability by mabs towards the naja naja venom, our results highlight the conservation of neutralizing epitopes across the elapidae family. protein-protein interactions are known to play key roles in the most important cellular and biological processes such as signaling, metabolism, and trafficking. one major goal of structural biology is the structural characterization of all protein complexes in human and other organisms. these efforts can be complemented by computational approaches. in this context, computational docking attempts to predict the structure of complexes from their monomeric constituents. the docking problem presents two main challenges: the generation of structural poses or sampling, and the identification of the correct structures with a scoring function (sf). docking methods can be successful if the interacting partners undergo small conformational changes. however, in a general situation, these algorithms generate a large number of incorrect predictions, and therefore the predictive success strongly depends on the accuracy of the sf used to evaluate the docked conformations. a variety of strategies have been developed to score putative protein-protein docked complexes. they are usually based on atomic level potentials, residue level potentials, or a combination of both. in current work, we have evaluated different sf, taken from cchappi server, on the results of different rigid body docking methods, ftdock, zdock, and sdock, using the docking benchmark . and a docking set built from capri scorers experiment. our results show sf that showed better or similar success rate than the in-built sf. some of these sf increase the docking success rates especially for flexible or weak-binding cases, which are the most challenging for docking. of them are residue level sf robust enough to detected solutions in cases with large conformation change. in particular we found two sf that shows outstanding robustness, one designed for protein modeling and shared among docking methods, and the other is for protein docking which is also the best success rate in the top ranking in the capri scorers set. the other atomic level sf display high success rate to find a solution within weak binding proteins. the most successful sf are shared between the docking methods and display high success rate in the hard cases of the benchmark . and in the capri scorers set. the difference between them in the resolution level at which they work, one being atomistic the other residue-based. we found that they success rate vary according to the docking method chosen, allowing them to explode different properties of the sampling used. this way to characterize a protein complex can help to develop new combined scoring functions in protein docking or a new ranking strategy to enhance the success rate. multi-ptk antibody: a powerful tool to detect a wide variety of protein tyrosine kinases (ptks) isamu kameshita , noriyuki sueyoshi , yasunori sugiyama the eukaryotic protein kinases consist of large families of homologous proteins and play pivotal roles in various cellular functions. these enzymes are classified into two major groups; protein serine/threonine kinases and protein tyrosine kinases (ptks). ptks are believed to be involved in various cellular events such as cell cycle, proliferation, differentiation, apoptosis, and cell adhesion in multicellular eukaryotes. as many as ptk genes have been identified in the human genome and many of these ptks are known to be closely correlated with various diseases such as cancer. therefore, it is important to elucidate the expression profiles of the entire ptk family in cells and tissues. to investigate the expression profiles of the cellular ptks, we produced an antibody that detects a wide variety of ptks. for production of the antibody, antigenic peptides corresponding to amino acid sequences of a highly conserved region (subdomain vib) of ptks were synthesized and immunized to balb/c mice. among various antigens, a peptide with amino acids, cyvhrdlraan, efficiently produced a polyclonal antibody with a broad reactivity to ptks. we established a hybridoma cell line producing a monoclonal antibody, yk , which appeared to cross-react with various ptks. at least ptks could be detected by yk antibody, as evidenced by its reactivity with the recombinant src tyrosine kinases whose subdomain vib had been replaced by those of the other ptks. when differentiated hl- cells were analyzed by western blotting after two-dimensional electrophoresis with yk antibody, we observed significant changes in the immunoreactive spots in hl- cell extracts along with the changes in the morphology of the cells. these results suggest that the multi-ptk antibody, yk , will be a powerful tool for the analysis of a variety of cellular ptks. analysis of the siglec- and hvap- interactions leonor carvalho , vimal parkash , heli elovaara , sirpa jalkanen , xiang-guo li , tiina salminen structural bionformatics laboratory, department of biosciences, medicity research laboratory, department of pharmacology, drug development and therapeutics, sialic acid-binding immunoglobulin (ig)-like lectins (siglec) are type i transmembrane proteins. siglec- has an n-terminal v-set domain followed by two c -set domains in the extracellular region. it contains an immunoreceptor tyrosinebased inhibitory motif (itims) in its cytoplasmic tail and can function as an inhibitory receptor by dampening the tyrosine kinase-driven signaling pathways. these proteins are expressed primarily on leukocyte subsets and, thus, are thought to be involved in regulation of leukocyte functions during inflammatory and immune responses. recently, phage display screening experiments identified siglec- as leukocyte surface ligand for human vascular adhesion protein (hvap- ; aoc gene product) and their interaction was confirmed by cell adhesion and enzymatic assays (kivi et al., ; aalto et al., ) . based on our preliminary data, hvap- sugar units with sialic acid (sa) might mediate interactions with the v-set domain in siglec- . furthermore, it is known that the siglec peptides binding to hvap- are located in the ce loop of the second c -set of domain (siglec- _c ). based on current hypothesis an arginine in siglec- _c interacts with the tpq residue in the active site of hvap- . the ce loop of siglec- _c has two arginines (r and r ) and, therefore, the interacting arginine is unclear. we will now study the interaction mode of hvap- and siglec- in silico to predict the role of the arginines in the c domain and the role of sa-binding using the d model of the full-length ectodomain of siglec- and the hvap- crystal structure. the in silico analysis will be conducted in parallel with experimental site-specific mutational studies and the result will be combined to elucidate the mechanism of hvap- -siglec- interaction. adam middleton , catherine day attachment of ubiquitin to substrate proteins regulates almost all cellular processes, including protein degradation and cell division. ubiquitylation involves a cascade of three families of proteins: ubiquitin activating (e ), ubiquitin conjugating (e ) and ubiquitin ligase (e ) enzymes. the . kda protein can be attached as a monomeric moiety or as a polyubiquitin chain, and the type of modification spells out the 'ubiquitin code' that directs the fate of the substrate. polyubiquitin chains can be formed via eight different linkage types, and the arrangement of chain formation is typically directed by the e enzymes. forming a polyubiquitin chain involves binding of two molecules to the e : the donor (ubd) and acceptor (uba) ubiquitin. ubd is linked to the e via a thioester bond between its c-terminal gly and the active site cys of the e , and when primed for catalysis it interacts with a particular face of the e . in contrast, coordination of uba by e s is transient and cannot be easily measured; however, uba binding defines the linkage type of polyubiquitin chains. the e , ube k, directs lys chain synthesis, which results in modified proteins being degraded by the proteasome. we generated a stable form of the ube kub conjugate and crystallized it, and showed that both ube k and its ubiquitin conjugate are monomeric. using molecular docking, we modelled the position of both ubd and uba and investigated the interfaces with site-directed mutagenesis. these experiments led to a molecular model that revealed how ube k can synthesise lys -linked ubiquitin chains. this molecular explanation provides a foundation for understanding how other e s generate lys -linked polyubiquitin chains. the two chromophorylated linkers of r-phycoerythrin in gracilaria chilensis marta bunster, francisco lobos-gonz alez, jos e aleikar v asquez, carola bruna, jos e mart ınez-oyanedl fac de cs biol., universidad de concepci on the two chromophorylated linkers of r-phycoerythrin in gracilaria chilensis. francisco lobos-gonz alez, jos e aleikar v asquez, carola bruna, jos e mart ınez-oyanedel, marta bunster. departamento de bioqu ımica y biolog ıa molecular, facultad de ciencias biol ogicas, universidad de concepci on. phycoerythrin is a phycobiliprotein present in phycobilisomes in gracilaria chilensis as a complex with chromophorylated linker proteins. our interest is to discover the role of these linkers in the function of phycobilisomes. phycobilisomes(pbp) are auxiliary light harvesting protein complexes in charge of channeling energy towards photosystem ii in alga, cyanobacteria and cryptophyta. this is possible thanks to fluorescent proteins called phycobiliproteins (pbp) and the chromophores (phycobilins, open-chain tetrapyrrols) attached to specific cysteines. phycobiliproteins share a common general structure; they are organized as (alfab) heterodimers which themselves assemble as trimers(alfab) or hexamers (alfab) ; this complexes are organized in high order structures to form the core and the rods. besides pbps, pbs have linker proteins in charge of the assembly and stabilization of the complex, and also it has been proposed that they collaborate in the fine tuning of the energy transfer steps between chromophores. these linkers are located within the rods, the rod-core interface, the core and the core-membrane interface. although most linker proteins are colorless, chromophore bearing linkers have been described, which suggest its participation in the energy transfer process. two of them, g and g are associated to r-phycoerythrin in gracilaria chilensis, nevertheless the information available on these linkers in eukaryots is still limited. to understand how these linkers collaborate with the function of the phycobilisome, we need structural information, especially the coordinates of all the chromophores present in the complex; we have sequenced both linkers from the genomic dna, performed sequence analysis and also we have purified the linkers by anion exchange, molecular sieve and hpl chromatography. the characterization was performed by denaturant electrophoresis, absorption and emission spectroscopy and by mass spectrometry. the results show that they have molecular masses as predicted, with a peptide signal for chloroplasts, an internal sequence repeat; residues - with residues - for g and residues - with residues - for g , and the presence of conserved cysteine residues putative sites of chromophorylation. the spectroscopy shows that they have different composition of phycobilins and a very short t / . a preliminary model for both linkers shows that they belong to aa structural class and that they share a common fold (heat like motifs) frequently involved in protein-protein interactions. dept. of phys., chuo univ., grad. sch. of inform. sci. and eng., tokyo tech, rigid-body docking algorithms are useful for predicting tertiary structures of near-native protein complexes. however, this algorithms generate many protein complex poses including false positives. then, near-native poses are searched in a post-docking process. there are many computational softwares with rigid-body docking algorithms, for example, zdock. we developed a high-performance protein-protein interaction prediction software, megadock, which is basically used on supercomputing environments for a large scale and network level in this work, we then tried to use these docking softwares and the profile method for understanding mechanisms of protein-protein interactions. we focused on some physicochemical properties, electrostatic and hydrophobicity, of a set of protein complex poses generated by a rigid-body docking process. from these poses, we obtained sets of possible interacting amino acid pairs. a set of interaction profiles has some information of docking spaces. from the view of a network prediction, the docking spaces of a set of protein complex poses are one of the properties for discriminating native protein-protein pairs from non-native pairs. in this work, ensemble docking process is performed by megadock ver. . and zdock ver. . . . cluster analysis is used with profiles of physicochemical properties. we used a dataset composed of typical monomer-monomer protein pairs and will discuss mainly differences between native and non-native protein pairs. the structural studies of the two thermostable laccases from the white-rot fungi pycnoporus sanguineus marta orlikowska , grzegorz bujacz institute of technical biochemistry, lodz university of technology, poland laccases (ec . . . , benzenodiol oxygen oxidoreductases) are enzymes that have the ability to catalyze the oxidation a wide spectrum of phenolic compounds with the four-electron reduction of molecular oxygen to water [ ] . it has been found that the active site is well conserved in between laccases from different organisms. it contains four copper atoms: one paramagnetic type cooper (t ) that is responsible for their characteristic blue color and where the oxidation of the reducing substrate occurs, one type cooper (t ) and two type coopers (t ) that conform a trinuclear cluster in which molecular oxygen is reduced to two molecules of water [ ] . laccases are present in many different species and they have been isolated from plants, fungi, prokaryotes, and arthropods in most cases laccases are monomeric glycoproteins of around amino acids with molecular weights in the range of - kda. the various functions carried out by those enzymes include the antagonistic ones such as their involvement in lignin biosynthesis (in plants), lignin degradation, pigment production, fruiting body formation, pathogenesis (in fungi) and spore protection against uv light (in bacteria) [ , ] . the diversified functions of laccases make them an interesting enzyme for study from the point of view of their structure, function and application. laccases of white-rot fungi (wrf) are of special interest because one of its role is to degrade lignin and most of them are extracellular enzymes helping purification procedures [ ] . during the last two decades, there has been an increasing interest in the genus pycnoporus for its ability to overproduce high redox potential laccases as the ligninolytic enzymes. we present the crystal structures of two thermostable lacasses produced by strain pycnoporus sanguineus cs (laci and lacii). the molecular weights of laci and lacii, determined by sds-electrophoresis, is and kda, respectively [ ] . both isoforms shows high amino acids sequence similarity ( %) between them and high thermal stability, at c and c. they remained active at high concentration of organic solvent (acetonitrile, ethanol or acetone). the unique properties make them promising candidates for industrial applications in wasterwater treatment. laci exerted a higher thermal and ph stability, tolerance against inhibitors and was a more efficient catalyst for abts and dmp (laccases substrate) then lacii [ ] . based on the structures we would like to understand the isoforms differences that confers laci a markedly better performance than lacii in ph and thermal stability as well as better resistance to inhibitors. analysis of liver proteome in cystathionine ß-synthase deficient mice using d ief/sds-page gel electrophoresis, maldi-tof mass spectrometry, and label-free based relative quantitative proteomics izabela bieli nska , Łukasz marczak , hieronim jakubowski , institute of bioorganic chemistry, polish academy of sciences, rutgers university, new jersey medical school homocysteine (hcy) arises from the metabolism of the essential dietary protein amino acid methionine. levels of hcy are regulated by remethylation to met and transsulfuration to cys. cystathionine bsynthase (cbs) catalyzes the conversion of homocysteine to cystathionine (first step of transsulfuration reaction). human cbs deficiency is a recessive inborn error of homocysteine metabolism that casues severe hyperhomocysteinemia (hhcy) and diverse clinical manifestations, including fatty liver disease [ ] . although the causes of fatty liver disease in cbs deficiency have been studied the underlying mechanism is not understood. we hypothesize that cbs deficiency induces changes in gene expression that could impair liver homeostasis. to test this hypothesis and gain insight into hepatic functions of cbs we analyzed the liver proteome of cbs -/-and cbs / mice [ , ] using d ief/sds-page gel electrophoresis and maldi-tof mass spectrometry (n ) we identified twelve liver proteins whose expression was significantly altered as a result of the cbs gene inactivation. expression of three proteins was upregulated and of nine down-regulated by the cbs-/-genotype. two up-regulated liver proteins are involved in iron metabolism (ftl and fth). those proteins are associated with oxidation stress and inflammation. third up-regulated liver protein (cbr ) is related to oxidation-reduction process. the downregulated protein are involved in the hydrolysis of n-acylated or n-acetylated amino acids (acy ), regulation of endopeptidase activity (a at ), cholesterol biosynthetic process (fpps), amino acid degradation (huth), cellular calcium ion homeostasis and l-ascorbic acid biosynthetic process (rgn). using label-free based relative quantitative proteomics (n ) we identified fourteen liver proteins whose expression was significantly altered as a result of the cbs gene inactivation. expression of four proteins was up-regulated and of ten proteins was down-regulated. the down-regulated liver proteins are linked with regulation of bone mineralization and inflammatory response (ahsg) or regulation of mrna splicing (roa ). the up-regulated liver proteins are involved in tricarboxylic acid cycle (suca), oxidation-reduction process (cy ), cholesterol metabolic process, iron ion homeostasis (fech), fatty acid metabolic process (ssdh; eci ) and response to oxidative stress (lonm). our findings suggests that cbs interacts with diverse cellular processes, including lipid metabolism, that are essential for normal liver homeostasis. deregulation of genes involved in lipid metabolism provides a possible explanation for fatty liver disease associated with cbs deficiency. transcription factors play central roles in coordinating developmental processes, as evidenced by the increasing number of transcription factor-related developmental disorders being uncovered by nextgeneration sequencing and genome-wide studies of copy number variation. the action of a transcription factor in regulating gene expression depends on interactions with other transcription factors, coactivators/co-repressors and chromatin modifying and remodeling complexes. transcription factors are commonly regulated by post-translational modifications. however the study of protein-protein interactions and post-translational modifications of transcription factors by common techniques such as coimmunoprecipitation and mass spectrometry is hampered by the difficulty in preserving interactions and modifications through cell lysis. to circumvent this issue, we developed a bioluminescence resonance energy transfer (bret) assay, which allows protein-protein interactions to be observed in live cells. in this assay, a protein of interest is expressed as a fusion with luciferase from renilla reniformis, and its putative interaction partner as a fusion with yellow fluorescent protein (yfp). upon addition of a cell-permeable substrate, the distance-dependent non-radiative transfer of energy from luciferase to yfp is quantified by measurement of light emission at two wavelengths to assess the interaction between the two fusion proteins. to validate the utility of this assay for investigating transcription factor interactions, we confirmed homodimerization of the foxp transcription factor, haploinsufficiency of which causes a rare and severe speech and language disorder, as well as interaction of foxp with other members of the foxp family. we also confirmed the interaction between foxp and multiple candidate interactors identified through yeast two-hybrid assays, including the autism-related transcription factor tbr , the co-repressors ctbp and ctbp , and post-translational modification enzymes of the pias family. the role of pias enzymes in sumoylation -the covalent modification of proteins with small ubiquitin-like modifier (sumo) proteins -led us to further explore this process, which is notably difficult to investigate because of the dynamic and labile nature of the modification, which is also typically present on only a minor fraction of molecules of a given protein. combining the bret assay with gel-shift techniques we demonstrated that foxp is sumoylated. finally, we used the bret assay to examine the effects of etiological foxp variants in speech and language disorder on protein-protein interactions and post-translational modification. in summary, the bret assay is a sensitive, reliable and potentially high-throughput technique for exploring protein biology in the context of live cells. we have demonstrated applications of the assay in validating putative protein-protein interactions, assessing posttranslational modifications, and investigating functional effects of protein variants identified in patient cohorts. these investigations have provided novel insights into the function of the foxp transcription factor in neurodevelopment and into the etiology of foxp -related speech and language disorder. the directly interaction between pres of human virus b and human heat shock protein (hsp ) deqiang wang , chen ke , jun zhang key laboratory of molecular biology on infectious disease, the department of cell biology and genetics the directly interaction between pres of human virus b and human heat shock protein (hsp ). hepatitis b virus (hbv) has infected billion people worldwide, and million of them are chronically infected. the chronic virus infection, a major public health problem worldwide, leads to bout two-thirds of hepatocellular carcinoma (hcc). the hbv envelope consists of the large (l), middle (m) and small (s) envelope proteins, which contain pres -pres -s, pres -s, and s domain alone, respectively [ ] . the pres domain is believed to mediate virus attachment to the high-affinity receptor. yan et al employed a novel technique to propose sodium taurocholate co-transporting polypeptide (ntcp) as the candidate hbv receptor, and consequently, ntcp is a target for a new family of anti-hbv agents [ ] . whereas, it remains a query to clarify that ntcp is the only or major hbv receptor in vivo. to illuminate if other host proteins cooperatively participate the hbv infection, we detect the interaction between pres and many candidate host proteins. fortunately, we have found that the human heat shocking protein (hsp ) could directly interact with the pres domain of the hbv virus protein. both the pull down and the size exclusion chromatography experiments verify that the grp have the ability binding to pres . whereas, whether the interaction between hsp and pres relates to the hbv infection need further experiments to clarify. the member sponsorship in the vast world of naturally occurring peptides, where more than peptides are known and approximately peptide therapeutics are currently being evaluated in clinical trials (fosgerau & hoffmann, ), the rapid and accurate determination of their physicochemical properties is key in peptide drug discovery. among these properties, hydrophobicity is crucial for understanding molecular recognition and biomolecular aggregation. hence, there is a great interest in determining hydrophobicity scales for amino acid structures. in this work, octanol/water partition (log p) and octanol/water distribution (log dph, fig. ) of n-acetyl-l-amino-acid methyl amides were determined by means of quantum mechanical ief-mst solvation calculations taking into account the intrinsic conformational preferences of each amino acid according to dunbrack's libraries (dunbrack & karplus, ; ) . the results reveal log d . differences for a-helical and b-sheet conformations in arg, lys, hid, asn, gln, met, cys, leu and ile. furthermore, by decomposing the octanol/water transfer free energy into electrostatic and non-electrostatic components, we estimated that the non-electrostatic cost of transferring the amino acid side chain amounts to . . cal/mol.Å , in agreement with previous estimates reported in the literature. comparison of our scale with other theoretical and experimental hydrophobicity scales yields satisfactory results, leading to correlation coefficients ranging from . to . . additionally, the mstderived hydrophobicity scale led to significant correlations with the rp-hplc retention factors measured for eight decapeptides (r . ) and for influenza virus hemagglutinin -mer (ac-ypydvp-dyaslrs-amide) peptides (r . ). finally, the hydrophobicity scale was able to reproduce the experimental log p for random neutral peptides (r . ) and log d . for ' : random charged peptides (r . ), fig. . future studies will address the application of this methodology to nonproteogenic amino acids, the prediction of peptide hydrophobicity at global and atomic level in peptides, and the scoring of peptide-protein interactions. docking-based tools for discovery of protein-protein modulators docking-based tools for discovery of protein-protein modulators. protein-protein interactions (ppis) play an essential role in many biological processes, including disease conditions. strategies to modulate ppis with small molecules have therefore attracted increasing interest over the last few years. although protein-protein interfaces (ppifs) are considered difficult to target with small molecules given its lack of well defined cavities. successful ppi inhibitors have been reported into transient cavities from previously flat ppifs. recent studies emphasize on hotspots (those residues contribute for most of the energy of binding) as promising targets for the modulation of ppi. pydock algorithm is one of the few computational methods that use energy of solvation to predict protein-protein interfaces and hotspots residues. we present an approach aimed at identifying hotspots and transient pockets from predicted proteinprotein interfaces in order to find potential small molecules capable of modulating ppis. the method uses pydock to identify ppifs and hotspots and molecular dynamics (md) techniques to propose putative transient cavities. we benchmarked the protocol in a small set of protein-protein complexes for which both structural data and ppi inhibitors are known. the method applies to the unbound proteins of the complexes the fast fourier transform algorithm, followed by the energy-based scoring from pydock to calculate the normalized interface propensity (nip) values derived from rigid-body protein docking simulations to predict the ppifs and hotspots residues without any prior structural knowledge of the complex. then we used md to describe the possible fluctuations of the interacting proteins in order to suggest transient pockets that could be useful as targets of small molecules for the modulation of ppis. finally, we evaluated by ligand docking, the validity of predicted hotspots and pockets for in silico drug design. we found that the nip-based method from pydock protein-protein docking identifies hotspots residues that are located within the binding site of known inhibitors of ppis. predicting ppifs from a three dimensional structure is a key task for the modulation of ppis. the use of the nip-based hotspots prediction method improve the identification of transient cavities from md simulation when compared to known binding cavities. this approach can be extremely useful in a realistic scenario of drug discovery targeting ppifs, when there is no information at all about the protein-protein complex structure. protein complexes are the fundamental molecular organizations that assemble multiple proteins to achieve various biological processes. identification of protein complex membership should provide a genotype-phenotype map to elucidate human gene-disease associations. it has been routinely assumed that network clusters with dense connections inside and sparse connections outside would form functional protein complexes. therefore, searching highly modular subgraphs in protein-protein interaction networks was explicitly or implicitly implemented in the algorithms to find protein complexes. however, to our surprise, we found a large portion of complexes with a medium-to-low modularity from the analysis of experimentally confirmed protein complexes. we also discovered that these complexes have cellular functions enriched in highly time-and space-dependent expression, such as signal transduction or subcellular localization. we further developed an algorithm to find such complexes by weighing network connections to capture transient interactions with intrinsically disordered regions. we confirmed that our method improved the identification of biologically relevant members of protein complexes and covered more complexes with a medium-to-low modularity. furthermore, newly discovered subunits in protein complexes could explain more disease-gene associations, indicating its utility to expand current genotype-phenotype map of human diseases. expanding template-based protein-protein complex prediction using ab-initio docking sergio mares-s amano , luis angel rodr ıguez-lumbreras , juan fern andez-recio structural characterization of protein-protein interaction (ppi) networks is crucial for understanding the underlying molecular mechanisms whereby life processes and disease arise. however, due to inherent limitations of experimental techniques, such characterization only covers an extremely reduced fraction of the human ppi network (interactome). recent studies have shown that although available structural templates may suffice to model a significant proportion of the interactome, model accuracy and binding specificity remain unsolved problems. consequently, improving the ability to predict ppis structurally will help to provide a better d profile of the known interactome, which may ultimately lead to the development of new therapeutic applications. here we show a novel approach that combines templatebased modeling with protein-protein computational docking to the structure-based prediction of ppis. our approach samples different protein-protein structural models derived from docking simulations. models are subsequently ranked using a function that incorporates an energy-based scoring term and a structural template similarity score. the energy-based scoring function includes electrostatics, van de waals and desolvation calculations, whilst the template similarity score accounts for the degree of structural similarity of models against a high-resolution and diverse dataset of structural templates. our approach highly improved the predictive success rate over individual ab-initio docking and templatebased techniques across a large benchmark dataset, including protein-protein complexes. when compared to the performance of the ab-initio docking algorithm, we found that the approach increased consistently the success rate, by approximately %, for the top , top and top solutions. the success rate improvement was even more notorious when the comparison was performed against the predictions from the traditional template-based docking. though incorporating ab-initio docking expands considerably the scope of the template-based docking method, challenges remain for interacting proteins in which high conformational changes occur upon binding and also the size and diversity of the repertoire of structural templates needs to be increased. is essential for the development of multicellular organisms. in mammalian cells, early events in pcd involve the release of cytochrome c (cc) from mitochondria to the cytoplasm, so letting cc play a key role in assembling the apoptosome and triggering apoptosis. in plants, pcd is part of a general process -the so-called hypersensitive response -in which mitochondrial cc is likewise released into the cytosol but its further role and cytoplasmic partners remain veiled. such a coincidence in cc release made us think of a common link for pcd in such evolutionarily distant species along evolution. to go deeper in understanding the pcd-dependent role of cc, a proteomic approach based on affinity chromatography with cc as bait was run using human and plant cell extracts. upon combining this approach with bimolecular fluorescence complementation (bifc), a total of eight and nine unknown proteins interacting with cc under pcd conditions were identified in human and plant cells, respectively [ , ] . such novel cc-partners -which are located in the cytoplasm and even in the nucleus -are involved in protein folding, translational regulation, oxidative stress, dna damage, energetic and mrna metabolism [ ] . strikingly, some of the novel human cc-partners are closely related to those for plant cc, so indicating that the evolutionarily well-conserved event of cc release from mitochondria could involve a common signalosome consisting of a wide range of common targets [ ] . to also understand such a promiscuity of cc from a structural point of view, the cc surface residues involved in complex formation with each one of its counterparts were mapped by using nmr spectroscopy. the resulting data shows that the heme crevice of cc is at the cc-partner interface in most of the complexes, which is in agreement with the vast majority of known redox adducts of cc. in contrast, however, to the high turnover number of the redox cc adducts inside the mitochondria, the complexes formed by cc under pcd conditions lead to the formation of rather stable nucleo-cytoplasmic ensembles. altogether, these findings suggest that extra-mitochondrial cc interacts with nuclear and/or cytoplasmic pro-survival, anti-apoptotic proteins in both humans and plants so as to lead living cells to dye. keywords: cytochrome c, programmed cell death, signalosome. post-translational phosphorylation often modulates the function of proteins. in particular, they affect the role that cytochrome c (cc) plays in cell life and death [ ] . cc is phosphorylated in vivo in tyr and tyr residues [ , ] , but recently, two new phosphorylation sites have been described at positions and [ ] . hence, we aim at understanding the structural and functional changes induced by thr and ser phosphorylation cc. for this purpose, we designed two phosphomimetic mutants of cc by replacing either thr or ser by the canonical amino acid aspartic acid (t d and s d). as control, two other mutants at the same two positions (t a and s a) were analyzed so as to differentiate the effects due to the presence of a negatively charged residue. remarkably, the s a mutant is significantly less stable than the wild-type species. we found that phosphorylation at position thr diminishes the redox potential and oxygen consumption. in addition, t d mutation affects the ability of cc to bind the distal site pcc , thereby suggesting that phosphorylation at this position affects the electron carrier capacity of cc. mass spectrometry (ms) is widely used techniques to gain knowledge about biomolecules [ , ] . it produces a high amount of data which is often presented as a list containing thousands of proteins. that list usually contains few hits interesting for our research. the pocess to select those proteins may include integrating experimental with annotation data. it requires spending some time in both, performing calculus and searching in databases. in this poster we present msbiodata analysis tool, a web service thought to deal with this tedious work. with this tool, researchers can set rules to select the most interesting hits in his lists using both, experimental data and gene ontology [ ] annotation. the data can be upload to the web using an excel spreadsheet or a flat files in a mztab format, and rules are easily constructed by means logical sentences. those sentences are composed by one or more terms linked by logic operators (and and or). each term in the logical sentence indicates to our program the conditions that selected hits must meet. once the alysis is finished, the results are delivered by email. msbiodat analysis tool do not requires any programming knowledge to be used and is freely available at: http://msbiodata.innomol.eu keywords bioinformatics/data analysis/proteomics/data mining/ mass spectrometry. beside the rate of protein synthesis, the regulation of protein degradation plays a crucial role in the white muscle protein accumulation and overall fish growth. intracellular proteolysis in salmonid species, such as atlantic salmon, salmo salar l. and rainbow trout, oncorhynchus mykiss walb., was studied to evaluate the basic mechanisms of protein degradation that could possess a potential target to regulate the body mass accumulation in farmed fish. a number of white muscle proteases such as cathepsins b, l, and d, proteasomes, and calcium-dependent proteases (m-and m-calpains), was studied in the juvenile specimens of different size-and age-groups both wild and farmed salmonids. the correlations between the protease activity and expression levels and morphometric characteristics of fish were found. the size-and age-related differences in intracellular protease activity revealed in fish muscles indicate both general role of proteolysis regulation in salmonid growth and the specific role of the individual proteolytic enzymes as well. the data on negative correlation of cathepsin d and calpain activity in muscles and the rate of weight increase in juvenile salmonids were obtained. a revealed positive correlation of cathepsin b activity and morphometric parameters in fish young presumably indicates its primary contribution to non-myofibrillar protein turnover. ubiquitin-proteasome system seems to contribute to background protein turnover as the proteasome activity was not corresponded with growth rate. summarizing the data obtained the autophagy-lysosomal and calpain-related protein degradation pathways were recognized to be directly involved in body growth and muscle protein retention in salmonid fish. the work was carried out using technical facilities of ib karrc ras equipment centre and financially supported by the russian science foundation, grant no. - - "salmonids of the north-west russia: ecological and biochemical mechanisms of early development". solving the proteomic organization of fitness-related genes in uropathogenic escherichia coli in life threatening sepsis. nowadays, complete genomes for almost all major bacterial pathogens are available, helping researchers to identify virulence factors. however we still ignore how these genes are organized at the proteome level and how this association influences bacteria pathogenicity. we integrated available databases on upec e. coli (strain cft ) to investigate the genomic and proteomic organization of genes related to upec fitness in the host. intriguingly, we found that most fitnessrelated genes have orthologs not only in other pathogenic strains but also in non-pathogenic bacteria such as e. coli k- . these genes are organized in clusters and operons with similar structure. by integrating protein-protein interaction data we observed that genes with high impact on fitness also display a highly clustered organization when compared to other genes. overall, our results show that proteinprotein interaction clusters associated to upec fitness in the host represent a promising target for the design of new antibiotics. elucidating the molecular mechanisms by which the hnh endonuclease gp activates the terminases in bacteriophage hk ( ) . hnh endonucleases are characterized by two highly conserved his residues and an asn residue( ). gp is essential for phage head morphogenesis, likely because gp enhances the activity of the hk terminase enzymes toward the cos site ( ) . notably, enhancement of the terminase-mediated cleavage of the phage cos site requires the presence of an intact hnh motif in gp . mutation of the canonical metal binding his in the hnh motif abrogates gp mediated-terminase activity. although phages are widely studied, there is no definitive structural or mechanistic evidence as to how the hnh endonuclease within gp functionally interacts with the adjacent terminase enzymes to facilitate phage morphogenesis. previous work on hnhcontaining bacteriophage proteins does not address explicitly how the requirement for divalent metal binding at the hnh endonuclease site induces interaction with the terminase enzymes that are so crucial for phage dna packaging during morphogenesis ( , ) . in addition, gp possesses no sequence similarity to hnh proteins for which the structure has been determined ( ), making structural studies of gp necessary. toward these ends, we use nuclear magnetic resonance (nmr) spectroscopy to probe metal and terminase binding of gp in the wild type state and bearing metal binding mutations. we also report backbone resonance assignment of gp . our nmr studies have elucidated residues within gp required for metal binding and terminase activity. these data are being used to assess the role of specific gp residues in phage morphogenesis. together, this work will identify the enigmatic role describing how metal binding in hnh endonucleases is crucial in the replication and morphogenesis of phages. meat production from pigs for human consumption is a resource heavy process, indeed every part of the animal that is not used constitutes a protein food-chain loss, which is neither economically nor environmentally viable. the goal of this project is to better harness slaughterhouse waste such as the keratin rich pig bristles and nails through microbial conversion. instead of using identified single microorganisms, it is the goal to define microbial consortia where microorganisms synergistically show the ability of efficient keratin degradation/conversion. candidate consortia have been obtained by selecting for microorganisms growing on enriched media that contains milled pig bristles as sole carbon and nitrogen source. by using mass spectrometry and various biochemical analyses to investigate keratinolytic enzymes, methods will be established for identifying and characterizing suitable consortia. protein families likely to be involved are keratinases, which are specialized proteases including serine, cysteine and metallo proteases, as well as systems capable of reducing or otherwise breaking disulfide bonds which are highly abundant in hair and nails. furthermore, interactions and symbiosis of microorganisms in a consortium will be investigated at the meta-proteomics level. the project will lead to development of biotechnological degradation of keratin rich fibers, and provide new insights into functional dynamics and efficacy of microbial consortia. a comprehensive protein domain analysis to map cancer-type-specific somatic mutations interpretation of the genome-wide association studies (gwas) of cancer patients to find cancer-typespecific biomarker is challenging due to the mutational heterogeneity of cancer types. network approaches to find cancer-type-specific variants and biological pathways are increasing since genes tend to act together to display phenotypic or disease outcomes. phenotype similarity has proven to reflect the relationship of functionally related genes. we applied phenotype similarities between various diseases for expanding molecular connections of cancer-type-specific variants to discover cancer-type-specific modules. specifically, cancer-type-specific variants of cancer types from the cancer genome atlas (tcga) were analyzed to find phenotype-inferred relationships among the variants. we find that cancer variants that cause the similar disease phenotypes tend to be linked as a cluster of biological pathways or functions. moreover, cancer-type-specific modules could explain the underlying pathogenicity of specific symptoms which manifest in particular cancer types. cancer-type-specific modules and pathways found from phenotype similarity/dissimilarity based on cancer symptoms improved the discrimination performance to sort cancer-type-specific variants to accurately predict patient groups. our method will be further developed to find genetic biomarkers for the diagnosis or prognosis of specific cancer types pk- engineering a stable, symmetric membrane protein scaffold amanda duran , jens meiler computational protein engineering has the potential to contribute to various fields including drug design, protein therapeutics, and materials science. protein-ligand interface design and the construction of large, stable proteins rely on stable scaffolds. symmetry is a great tool for protein stability both in protein engineering and nature. several membrane protein structures exhibit pseudo-symmetry and are proposed to be the result of gene duplication, fusion and diversification events originating from a monomeric gene. aquaporins (aqp) are a class of membrane proteins that exhibits a two-fold inverted pseudo-symmetry. the escherichia coli aqp glycerol facilitator protein (glpf) was originally computationally engineered to be perfectly symmetric in sequence and presumably in structure. the symmetric gene was assembled, cloned, and expressed. however, after facing many challenges experimentally, the computational study has been expanded to aqps of known structure for a more extensive symmetric backbone search. mammoth structural alignment was used to align the structures to their inverted counterparts. cutpoints were calculated based on a-carbon distance. finally, the rosetta protein modeling software suite was used to refine and energetically minimize the symmetric backbones. from over generated symmetric backbones, candidates were chosen for experimental verification. these studies are ongoing.currently, the symmetric backbone models have scored to be more stable than the wild-type proteins. experimental verification of these symmetric backbones will provide valuable information for the current state of membrane protein modeling and design using computational methods. intrinsically disordered proteins drive heritable transformations of biological traits daniel jarosz , james byers , sohini chakrabortee , sandra jones , amelia chang , david garcia stanford university, whitehead institute for biomedical research, rockefeller university the transmission of information from one generation to the next generally occurs via nucleic acids. the only known protein-based molecular memories are prions, which drive heritable biological traits based upon self-templating changes in protein conformation. these protein-based genetic elements have previously been identified systematically, but at least three do not share the sequence biases or structural characteristics that have informed such studies. here we employed a comprehensive library of yeast proteins to examine the breadth of protein-based inheritance. transient overexpression of more than forty proteins created new traits that were heritable and beneficial. some shared properties of known prions, but most employed distinct genetic and biochemical mechanisms to act as elements of inheritance. traits with these characteristics were common in wild yeast strains and could also be elicited using orthologous mammalian proteins. the inducing proteins were strikingly enriched in intrinsically disordered sequences that have been widely conserved across evolution. intrinsically disordered proteins are associated with human disease and with dosage sensitivity in yeast, flies and worms. our results suggest another widespread role for such intrinsically disordered sequences: induction of heritable epigenetic switches that transform phenotypic landscapes and drive adaptation to stressful environments. prediction of binding affinity in protein complexes: contacts do matters almost all critical functions in cells rely on specific protein-protein interactions. understanding these is therefore crucial in the investigation of biological systems. despite all past efforts, we still lack a thorough understanding of the energetics of association of proteins. here, we introduce a new and simple approach to predict binding affinity based on functional and structural features of the biological system, namely the network of interfacial contacts. we assess its performance against a protein-protein binding affinity benchmark and show that both experimental methods used for affinity measurements and conformational changes have a strong impact on prediction accuracy. using a subset of complexes with reliable experimental binding affinities and combining our contacts-and contact types-based model with recent observations on the role of the non-interacting surface in protein-protein interactions, we reach a high prediction accuracy for such a diverse dataset outperforming all other tested methods. free radical oxidation -a new method for obtaining stable protein coatings on magnetic nanoparticles magnetically targeted nanosystems (mtnss) are now considered to be applicable in different areas of biology and medicine such as hyperthermia, magnetic resonance imaging, immunoassay, cell and molecular separation, a smart delivery of drugs to target cells. proteins are promising materials for creation of coatings on magnetic nanoparticles (mnps) due to their biocompatibility, an ability to protect magnetic cores from influence of biological liquids and prevent agglomeration of mtnss in dispersion, their possible functional activity as therapeutic products and biovectors. the creation of stable protein coatings with retention of native properties of molecules is still an important biomedical problem because of disadvantages of the commonly used methods such as formation of a polydisperse ensemble of particles, nonselective linking of proteins leading to cross-linking of macromolecules in solution, and desorption of coatings. a novel method in obtaining stable single-layer coatings assembled from protein molecules on the surface of magnetite nanoparticles has been developed. it is based on protein liability to free radical modification, leading to the formation of intermolecular covalent cross links. free radicals are locally generated on the surface of nanoparticles via the fenton reaction thereby proteins adsorbed on the surface are subjected to the cross-linking. o-phenylenediamine was used for detection of free radical generation initiated by nanoparticles. the proteins drastically differing in their structure and properties, namely, serum albumin, thrombin and immunoglobulin g were selected for creating the protein coatings. the properties of the obtained coatings and their stability have been studied with the help of dynamic light scattering (dls), uv/vis spectrophotometry, antibody-antigen test and the method of spectral-fluorescent probes. albumin molecules in mnps coatings have been shown to retain their capability of binding with a dye and be conformationally stable. the dye , '-di-(g-sulfopropyl) , 'diphenyl- -ethiloxacarbocyanine-betaine interacting with albumin with a growth of fluorescence and with partial cis-trans conversion of the dye has been used. it has been proven that coatings composed of protein macromolecules are ) stable, ) formed around individual nanoparticles and ) have several nanometers in thickness. the free radical linking of thrombin and immunoglobulin g on the surface of nanoparticles has been shown to almost completely keep native properties of the protein molecules. the free radical linking method reveals new possibilities for design of single-layer multiprotein polyfunctional coatings on the surfaces of all the nano-, micro-and macroobjects containing metals of variable valence (for example, fe, cu, cr). the spectral-fluorescent investigation was supported by the russian foundation for basic research, project nos. - - and - - mol_a. regulation of neuronal snares by accessory proteins shrutee jakhanwal , reinhard jahn regulation of neuronal snares by accessory proteins shrutee jakhanwal and reinhard jahn department of neurobiology, max planck institute of biophysical chemistry, fassberg, goettingen, germany- . synaptic vesicle exocytosis lies at the heart of the process of neurotransmitter release. and, the family of proteins that is central to the process of synaptic vesicle exocytosis is the family of snare proteins. there are three kind of neuronal snare proteins namely syntaxin, snap and synaptobrevin. these three snare proteins interact through their snare-motifs to form a highly stable four-helix bundle, which in turn, pulls two membranes together to mediate fusion. years of work in this field have established that the four-helix bundle is critical for the membrane fusion to occur. however, the process of regulation of snare-mediated fusion remains very poorly understood. the major regulatory proteins involved in the process are munc , munc , synaptotagmin and complexin. the major aim of my project is to obtain a closer look at the regulation process of snare-mediated fusion by focusing on the interaction between the snare proteins and the regulatory proteins. to achieve this objective, i express and purify the different proteins involved in the process of snare-mediated fusion and thereafter subject them to appropriate biochemical characterization. in order to assess the role of the purified proteins in the process of fusion, i reconstitute them into liposomes and perform in-vitro lipidmixing assays. these assays are based on f orster resonance energy transfer (fret). based on the discretion of assessing the protein-protein or protein-lipid interactions, either the proteins or the lipids can be fluorescently labeled. also, the lipid compositions can be varied in order to assess the effect of lipid on the function of the respective protein. fluorescence-based anisotropy measurements can also provide information about the degree of freedom of a protein, indirectly providing information about the kinetics of a reaction. employing these techniques, i observe that munc - leads to displacement of syntaxin from a complex of syntaxin and snap . also, a complex of syntaxin and munc is resistant to the action of the aaa-atpase, nsf and its co-factor asnap, implicating this complex as a strong candidate for acting as the starting point for the process of neurotransmitter release. munc also appears to enhance lipid-mixing by interacting with the snare-complex. further investigations on the same lines can provide very useful insights into the process and can help us unravel the secrets that underlie the beauty of the exquisitely regulated process of neurotransmitter release. binding of thymidine nucleotides to a viral thymidine monophosphate kinase aldo a. centro de investigaci on en alimentaci on y desarrollo theme: biochemistry there is great interest in the evolution and activities of fish trypsins, since they appear to have evolved into different families. the cdna for trypsin iii from the monterey sardine (sardinops sagax caerula) was obtained and its deduced amino acid sequence matched its identity with a purified protease from the fish by mass spectrometry analysis. molecular modeling of sardine trypsin iii compared to other homologs showed a typical trypsin fold with all the cognate components for catalysis, and specific amino acid distribution that are possible factors that explain the cold adaptation. from phylogenetic analysis, sardine trypsin iii belongs to the novel y family, which is proposed to have evolved for cold adaptation. the obtained recombinant trypsin iii showed a low catalytic efficiency, but it remained active at cold temperatures, similar to other cold-adapted trypsins. the cold-adaptation of sardine trypsin iii opens a wide range of biotechnological applications for this protease and is also interesting from the serine protease structure-function relationship point of view. fungicidal mechanism of scolopendin , a cationic antimicrobial peptide from centipede heejeong lee , dong gun lee drastically (from . x - to x - colonies) upon deletion of this residues domain from the full length trai. we are investigating the structure and function of this very c-terminal end of trai using nmr spectroscopy. for the backbone assignment we used slice-selectively homonuclear broadband decoupled spectra along with standard experiments. three-bond scalar coupling constants were obtained through real-time j-upscaling experiments. with the backbone assignments, we have the first hand evidence which shows that his domain is for the most part intrinsically disordered, but contains short a-helical regions. structural development, interaction studies to find the binding partner and transition of disorder to order orientation of this domain will be further investigated in this project. here we investigated a model system where mab aggregation is induced by increasing the ionic strength (nacl) at low ph. the aggregation depends both on protein and sodium chloride concentration. with nanoparticle tracking analysis (nta) and micro flow imaging (mfi) the aggregation formation was further characterized. aggregation can be partially reverted by lowering the ionic strength as determined by soluble monomer concentration measurement using se-hplc: parts of insoluble aggregates could be solubilized as soluble aggregates, dimers or even monomers. a quasi equilibrium is formed in between the subtypes. the whole aggregation process was examined by ftir and cd-spectroscopy to identify structural changes of the mab. screen of protective additives: the effect of osmolyte additives on aggregation kinetics and final aggregate concentration is investigated, revealing protective effects in both cases. in a screen with more than compounds not only the aggregation propensity was studied but also structural changes. the aggregation index (quantity for colloidal stability) and the melting point (quantity for conformational stability) measured by differential scanning fluorimetry were determined. the used mtp format screen has potential for buffer optimization and formulation development. structural biology and protein dynamics tetraspanin cd has a broad range of cellular functions, such as integrin association forming tetraspanin-enriched domains, synapse formation between b and t cells, cell adhesion, motility, invasion and signalling. furthermore, cd is one of the four receptors involved in the cell entry of hepatitis c virus (hcv) and therefore infection onset, one of the major causes for chronic liver disease resulting in cirrhosis and hepatocarcinoma. human cd large-extracellular-loop (hcd lel) is composed of a "stalk" and a "head" subdomain; with the latter interacting with hcv-e glycoprotein. we present four novel hcd lel crystal forms. analysis of the fourteen independent observed hcd lel high-resolution x-ray structures suggests that the dynamism of the hcd lel head-subdomain is an inherent molecular property, an observation supported also by molecular dynamics (md) studies. we classify the conformations in three distinct clusters (closed, intermediate and open) , which are seen both in the crystal structures and in the molecular dynamics simulations. the md simulations also show that conformational variability is modulated by ph changes, with distinct probability for each cluster at acidic and neutral ph. furthermore, in silico docking of the recent e core structure with three of the major types of hcd lel head-subdomain clusters highlights hydrophobic interactions as the major forces in the e core: hcd lel recognition mechanism. we propose that the flexibility of the hcd lel is exploited by hcv at different stages of cell entry from virus attachment to internalization and fusion with the endosomal membrane. our results provide important insights on the basic mechanism governing hcv binding to hcd , and can help structure-based drug design of entryinhibitors of hcv. allophycocyanin of gracilaria chilensis: from gene to function jorge dagnino-leone , jos e martinez-oyanedel , marta bunster-balocchi universidad de concepci on theme: structure-function relationship of proteins the phycobilisomes (pbs) are auxiliary photosynthetic complexes that allow cyanobacteria and red algae to enhance the energy uptake in the range of - nm. in gracilaria chilensis, an eukaryotic red algae, pbs is composed of phycoerythrin (pe), phycocyanin (pc) and allophycocyanin (apc); these proteins possess chromophores which capture energy and then transfers it to photosytems. pbps are oligomers of a ab heterodimer; it oligomerizes into a trimer (ab) , this trimer has discoidal shape and it is associated in hexamers (ab) , several of this hexamers forms cylinder-like structures. pbs has components: antennas and core. the antennas are composed of pe and pc, whose function is to capture energy between - and - nm respectively and transfer it to the core. the core is formed by apc, which can absorb energy in the - nm range. apc emission allows transferring energy to the photosystems with high efficiency. pbs is also composed by linker proteins which allow the correct assembly of pbs and possibly regulate the energy transfer. the main goal in our group is to build an atomic model of the gracilaria chilensis phycobilisome. we have solved the crystal structure of pe and pc and created an antenna model. at present we are working in apc and the chromophorilated linker proteins. the objective of the present work is to create a model of the core of gracilaria chilensis; to achieve these we have used molecular biology, biochemistry and bioinformatics techniques. we designed oligonucleotides primers for the four allophycocyanin subunits genes and for the globular domain of the apce linker. these primers were used in pcr experiments to obtain the genes sequences. the sequences were translated to a aminoacid sequences and used to build a d model for apc subunits and trimers using the software modeller. on the other hand we purified and analyzed the spectroscopic properties of apc from gracilaria chilensis using absorption and fluorescence spectroscopy. we also determined apc oligomerization state using gel filtration. molecular docking using the cluspro server was performed to obtain a hexamer and apc cylinder models. based on electron micrographs obtained by our lab a tri-cylindric core model was built. all the models were submitted to a molecular dynamics using gromacs software. finally we determine possible energy transfer pathways in the core model applying the extended forster equation, spectroscopic data from literature and the transition dipole moments of each of the chromophores present in the core. as conclusion of this work we built the first atomic model of gracilaria chilensis phycobilisome core and propose energy transfers pathways inside the core in the context of a phycobilisome. novel practical strategies to access artificial metalloenzymes marco filice , jose miguel palomo departamento de biocat alisis, instituto de cat alisis, csic protein chemistry and engineering since the first report, the design of artificial metalloenzymes has rapidly been converted into an important topic in biological and inorganic chemistry due to their potential applications in synthetic chemistry, nanoscience and biotechnology. the combination of a catalytically active organometallic moiety with a macromolecular host has permitted the creation of biohybrids, a new kind of heterogeneous catalytic entities combining the attractive features of both homogeneous and enzymatic systems. presenting our most recent achievements in this research area, here we describe two novel powerful and promising approaches focusing the practical synthesis and large scale production of heterogeneous artificial metalloenzymes showing chimeric activity. the first strategy is based on the in situ synthesis of noble metal nanoparticles and their supramolecular assembly with a microbial lipase from candida antarctica (fraction b) finally creating an ultra-active organometallic-enzyme heterogeneous nanobiohybrid. in the second approach, combining different protein engineering protocols (molecular biology, orienting immobilization, solid-phase bioorganic modification and bioinformatic tools), an orthogonal solid-phase strategy creating novel unnatural catalytic sites was designed and optimized. the application of such a strategy onto the structure of the lipase from geobacillus thermocatelunatus permitted the generation of a heterogeneous artificial metallolipase with chimeric activity. as proof-of-concept, the combinatorial library of generated artificial metalloenzymes obtained by both strategies was successfully assessed in a set of different synthetic reactions (selective c-c bond formation as suzuki, heck or diels-alder reactions) and also combining both activities (metallic and enzymatic) in cascade processes such as dynamic kinetic resolution of amines or production of arylamines. the obtained results were excellent in all cases. extending this strategy to other enzymes, proteins and catalytic metals, we envisage the creation of a combinatorial library of programmable artificial enzymes useful for a wide set of applications (i.e. fine organic and medicinal chemistry, bioremediation or biomedicine). proteomic examination of the yeast nuclear pore complex dynamics protein turnover and exchange nuclear pore complexes (npcs) are proteinaceous assemblies situated in nuclear envelopes of eukaryotic cells. the main function of the npc is the selective transport of macromolecules. npcs also partake in other functions, such as nuclear organization and gene regulation. the core scaffold of the npc is thought to be a stable structure, while the peripheral components exchange at various rates. however, these phenomena have not been elucidated in detail. the recent findings that yeast daughter cells get a higher proportion of the old npcs and the core scaffold hardly turns over raise the possibility that the exchange of the peripheral nucleoporins can be a repair mechanism. yeast provides a useful organism for the interrogation of nucleoporin exchange, as it performs closed mitosis; hence the only mixing of npc constituents is due to exchange. we have developed a panel of genetic tools providing for conditional induction and repression of nucleoporins. by combining these switches with stable isotope metabolic labeling and affinity capture, cross linking coupled to mass spectrometry, we are able to distinguish between pre-existing and newly synthesized proteins and quantify their relative amounts in the npc. our preliminary findings are in agreement with results obtained in other organisms: the core scaffold of the npc (inner ring, outer ring) appears to be stable, however does exchange slowly over time, while peripheral components exchange faster. by looking at the exchange rates of yeast nucleoporins we hope to gain insight into the npc biology of actively dividing eukaryotic cells. active site clustering identifies functional families of the peroxiredoxin superfamily angela harper , janelle leuthaeuser , patricia babbitt , jacquelyn fetrow department of physics, wake forest university, department of molecular genetics and genomics,-wake forest university, departments of physics and computer science, wake forest university bioinformatics understanding the relationships between proteins is vital to increasing our knowledge of the protein universe. while there are large databases of sequence information, the massive data influx over the past decade has prevented adequate classification of proteins at the molecular function level. however, it has been previously suggested that a protein's active site information may correlate with these known molecular functional differences; thus, active site profiling was developed to use residues around the active site of a protein to relate proteins. subsequently the deacon active site profiler (dasp) was developed to create these active site profiles and search them in a database, such as gen-bank, in order to find proteins with similar active site environments. by using dasp to computationally cluster proteins based on the similarity of their active site profiles, the peroxiredoxin (prx) superfamily was analyzed through active site similarity methods. the residues from the active site of each prx structure were extracted and clustered, and these profiles were iteratively searched in genbank through a multi-level iterative sequence searching technique (misst). the prx superfamily has been studied by experts, allowing the results of these searches to be compared to a well-annotated group of proteins. while previous sequence based evolutionary methods have been unable to identify functional differences between some subgroups of the prxs, notably the ahpc-prx and prx subgroups, misst discretely separates these subgroups. classifying prx proteins into functionally relevant groups using computational active site similarity methods lays the foundation for an automated process for identifying protein functional groups beyond the prx superfamily. synthesis and conformational studies of glycoprotein n homolog of bovine herpesvirus (bhv- ) by using cd, nmr and molecular modelling it serves as a chaperone for viral glycoprotein m and, in its gm-unbound form, acts as an inhibitor constraining the transporter associated with antigen processing (tap). the ul . /gm complex formation is required for the maturation and proper trafficking of both viral proteins. in the absence of gm, ul . blocks transport of antigenic peptides by tap and their mhc i-restricted presentation. the molecular mechanism of ul . activity still remains elusive. in order to investigate the structural requirements for biological function ul . study was conducted using cd, nmr and molecular dynamics methods. the data obtained with the use of high purity synthetic peptides encompassing ul . confirmed the presence of an alpha-helix structure, formed preferentially in the presence of dodecylphosphocholine (dpc) micelles as a membrane-like environment. in order to determine the three-dimensional structure of ul . protein in the present work its nmr solution structure in the presence of membrane-like environment was performed. the nmr data were used as a set of restraints for a simulated annealing protocol that generated dstructures of the colin johnson , sara codding membrane proteins resealing of tears in the sarcolemma of myofibers is a necessary step in the repair of muscle tissue. defects in this repair process are responsible for muscular dystrophy and cardiomyopathy. the repair pathway is triggered by the influx of calcium through lesions in the membrane, which result in membrane fusion and patching of the wound. recently dysferlin has been identified as a calcium binding protein essential for sarcolemma repair, as well as other snare mediated exocytotic events including cytokine and acid sphingomyelinase secretion. in this presentation we demonstrate a direct interaction between dysferlin and the snare proteins syntaxin and snap- . in addition, fret and in vitro reconstituted lipid mixing assays indicate that dysferlin accelerates snare heterodimer formation and snare mediated lipid mixing in a calcium sensitive manner. our results suggest a model whereby dysferlin acts as a calcium sensing snare effector for exocytosis and membrane fusion. exploring the therapeutic potential of a peptide derived from a poxviral immune evasion protein: nmr determination of the solution structure of viper and its inactive mutant toll-like receptors (tlrs) have a role in viral detection leading to cytokine and ifn induction, and as such they are targeted by viruses for immune evasion. the poxviral protein a has been identified to inhibit tlr signaling by interacting with tir domain-containing proteins of the receptor complex to collectively inhibit all tlr adaptor proteins that positively regulate transcription-factor activation ( ). one aa peptide (kysf-klilaey) termed viper (viral inhibitory peptide of tlr ) was reported to retain the inhibitory properties of full length a against tlr signaling. a r homopolymer delivery sequence at the c-terminus provided delivery of the peptide into cells. structural comparisons are presented between r-viper, which is active in preventing tlr -dependent cytokine induction in cell culture, and a mutant that exhibited loss of function ( r-viper l a,e a), through solution nmr spectroscopy. we find that despite a relatively minor sequence difference, the loss of hydrophobicity as well as negative electrostatic interactions result in subtle but potentially significant differences in the region of the peptide proposed to interface with tlr . reference: wake forest university, wake forest university, university of california san francisco protein function prediction the elucidation of protein molecular function lags far behind the rate of highthroughput sequencing technology; thus, it is essential to develop accurate and efficient computational methods to define functional relationships. protein clustering based on sequence similarity has emerged as a simple, high-throughput method for defining protein relationships, but sequence-based techniques often inaccurately define molecular function details. active site profiling (asp) was previously developed to identify and compare molecular details of protein functional sites. protein similarity networks were created using both active site similarity and sequence similarity for four manually curated superfamilies, and results demonstrate that asp-based clustering identifies detailed functional relationships more accurately than sequence-based clustering. building on this, two iterative pipelines were developed using active site profiling and profile-based searches to cluster protein superfamilies into functional groups. first, the two level iterative clustering process (tulip) utilizes active site profiling and iterative pdb searches to divisively cluster protein structures into groups that share functional site features. across eight superfamilies, tulip clusters exhibit high correlation with expert functional annotations. subsequently, the multi-level iterative sequence searching technique (misst) utilizes iterative profile-based genbank searches to identify protein sequences that belong in each tulip group. the results indicate that these asp-based methods accurately and efficiently identify functionally relevant groups through a process that can be applied systematically and on a large-scale. moreover, the approach can be applied more quickly than detailed manual curation, suggesting its value in guiding annotation efforts. dept. biochemistry and molecular biology. university of valencia, lab of peptide and protein chemistry. centro de investigaci on pr ıncipe felipe membrane proteins changes in the equilibrium between pro-survival and pro-apoptotic members of the b-cell lymphoma- (bcl- ) protein family at the mitochondrial outer membrane (mom) induce structural changes that committed cells to apoptosis. bcl- homology- (bh )-only proteins participate in this process activating pro-apoptotic effectors and promoting permeabilization of the mom. the membrane association of bh -only proteins is a controversial issue due to the lack of a canonical carboxyl-terminal (c-terminal) transmembrane (tm) domain. we used an in vitro transcription/translation system to study the insertion capacity of these hydrophobic c-terminal regions of the bh -members bik, bim, noxa, puma and bmf into microsomal membranes, and an escherichia coli complementation assay to validate our results in bacterial cells. furthermore, we have fused these hydrophobic regions to gfp to investigate the subcellular sorting. these results will allow further refinement in the elaboration of the bcl- protein-protein and protein-membrane interactome network. alexis peña , flaviyan jerome irudayanathan , shikha nangia syracuse university, dept. of biomedical and chemical engineering computational modeling, biostatistics, biomedical and chemical engineering tight junctions (tj) are vital intracellular barriers that are responsible for regulating paracellular transport. claudins, a family of abstract small transmembrane proteins with approximately members, are an integral part of the tj strands. tight junctions provide molecular-level protection and prevent infection and toxins from entering the body; in the same sense tjs allow nutrients and vital solutes to pass through. claudins are associated with various diseases including metastatic cancer as well as an entry point for many viruses. despite their importance and abundance in all cell membranes and their ubiquitous nature, the exact -d structure of claudins has remained elusive to traditional x-ray crystallographic and nmr studies. in this investigation, a computational approach was used to determine the claudin structure of claudin - . homology modeling, molecular dynamic simulations, and reverse mapping were employed to predict the protein structures with relative accuracy. understanding structure of claudin proteins and its interaction at the molecular level can lead to effective drug delivery technology. determination of optimal conditions for an isothermal titration calorimetry essay to obtain kinetic parameters of trypsin i from pyloric caeca of monterey sardine (sardinops sagax caerulea) idania emedith quintero reyes , francisco javier castillo y añez , enrique fernando vel azquez contreras , roc ıo sugich miranda , david octavio corona mart ınez , aldo alejandro arvizu flores , ivet cervantes dom ınguez protein kinetics determination of optimal conditions for an isothermal titration calorimetry essay to obtain kinetic parameters of trypsin i from pyloric caeca of monterey sardine (sardinops sagax caerulea) trypsin is the most studied alkaline protease and it s very common to found isoforms from this protein as the case for monterey sardine (sardinops sagax caerulea); as it shows an expression of trypsin i and trypsin iii according to the cdna characterization. trypsin i was determine to be a cold adapted enzyme as it shows a higher catalytic efficiency (kcat/km) than the mesophilic counterparts. the kinetic parameters were obtained by spectrophotometric essays, which are not fallible for all the enzymes because native, recombinant or mutant enzyme activity could be below the detection limit of the assay, opaque or turbid solutions interfere with spectrophotometric detection, etc. alternative tools as the isothermal titration calorimetry (itc) can measure enzyme kinetics using thermal power generated by the enzymatic conversion of substrate to product; were the rate of reaction is directly proportional to thermal power. the objective of this study was to stablish the optimum conditions to obtain kinetic parameters of trypsin i from pyloric caeca of monterey sardine using itc. to reach the objective trypsin i was purified from viscera of monterey sardine using molecular exclusion and affinity chromatography obtaining a yield of . mg/ml. at c kcat and km of tryipsin i form monterey sardine were . s- and . mm respectively. at c were . s- and mm (kcat and km) and at c kcat was . s- and km . mm. the kinetic parameters obtained by spectrophotometric assay at c were kcat and km s- and . mm respectively. at c the kcat was . s- and km . mm and at c kcat s- and km mm. comparing the values obtained for kcat with the spectrophotometric essay were higher fold than those obtained by itc and the values in km were similar by both methods. even though the differences in kcat, we can reassert the psychrophilic behavior of trypsin i as the catalytic efficiency is higher by both methodologies. in the understanding that the kinetic behavior of enzymes is important to not only understanding biochemical pathways and catalytic mechanisms but is again a fruitful area for drug discovery and development; so the itc provides a universal approach to determining the kinetic behavior of enzymes and can yield in a single experiment a complete set of kinetic parameters for an enzyme-catalyzed reaction that can be applied for the different alkaline proteases from pyloric caeca of monterey sardine (sardinops sagax caerulea). mysterious world of stress-responding sigma factors in bacillus subtilis olga ramaniuk protein-dna interaction bacterial transcription is mediated by the rna polymerase holoenzyme containing sigma factors -essential proteins for the initial step of transcription that recognize and bind to promoter dna. the primary sigma factor is essential in exponential phase of growth while alternative sigma factors are active during transcription under stress conditions. this project has three main aims. the first aim is to explore the binding properties of b. subtilis alternative sigma factors; specifically, whether sigma factors lacking the autoinhibitory domain . can bind to promoter dna in the absence of rnap. the second aim explores whether rnap associated with alternative sigma factors is regulated by the concentration of the initiation nucleoside triphosphate. the third aim is to define the regulon of sigma i. in order to achieve our aims, out of alternative sigma factors were successfully purified using affinity chromatography and ion exchange chromatography. we set up in vitro transcription system with selected sigma factors and initiated experiments with sigma i regulon determination. results named above and our future findings will help to better understand gene expression regulation on the level of transcription initiation. this work was supported by grant no. p - -g from the czech science foundation. assessing the costs and benefits of protein aggregation protein aggregation and cell fitness protein aggregation has been associated with numerous diseases but also with important cellular functions such as epigenetic inheritance. here we present a population genetics approach to infer the costs and benefits of protein aggregation on cell fitness. this information is crucial to understand how cellular systems tolerate the formation of protein deposits and which factors modulate this event. using our experimental system, we measured different protein aggregation effects (deleterious, neutral or beneficial) within the same genomic background. single cell analyses, within the same population, showed stochastic variability in the aggregate's size and in its effect on cell fitness. our data indicates that, in certain conditions, protein aggregation can enhance population variability and survival expectancy. overall, these results suggest that the presence and formation of protein aggregates could be almost harmless whereas the associated gain and loss of function are critical for the cell. revealing the key role of negatively charged residues of heme sensor proteins involved in geobacter sulfurreducens' signal transduction pathways marta a. silva , telma c. santos , teresa catarino , carlos a. salgueiro ucibio-requimte, departamento de qu ımica, fct-unl., instituto de tecnologia qu ımica e biol ogica, unl signal transduction proteins bacterial chemotaxis systems sense and regulate the microbe mobility in response to environmental conditions. such mechanisms constitute a striking example of cell motility to gain advantages for cell survival and permit the bacteria to fill important niches in a diversity of anaerobic environments [ ] . geobacter sulfurreducens (gs) is an anaerobic bacterium with a considerable respiratory versatility whose genome encodes for an unusual family of methyl-accepting chemotaxis proteins (mcp), each containing at least one heme c-binding motif [ ] . these sensor proteins, gsu and gsu , are involved in signal transduction pathways mediated by chemotaxis-like systems [ ] . the thermodynamic and kinetic characterization of the sensors gsu and gsu by visible spectroscopy and stopped-flow techniques, at several ph and ionic strength values revealed that sensor gsu midpoint reduction potentials are lower than those of gsu at all ph and ionic strength values and the same were observed for the reduction rate constants [ ] . the origin of the different functional properties of these closely related sensor domains are rationalized in the structural terms showing that gsu has two extra negatively charged residues in the vicinity of the heme group, which have no counterpart in gsu : glu and asp . residue asp is less exposed compared to glu and it was suggested that its carboxylic group might have a role in the modulation of the heme reduction potential of gsu . to investigate this, both residues were replaced by a positively charged amino acid (lysine) and by a neutral one (asparagine or glutamine). for the mutants with enough expression, a functional characterization was carry out, using several spectroscopic techniques, including uv-visible and cd, together with kinetics and potentiometric measurements. significant changes on the reduction potential values are observed when a negative charge is replaced by a positive one at position or . therefore, the decrease of the reduction potential in asp and glu mutants reinforces the hypothesis that the higher reduction potential observed for heme sensor domain gsu is related with the less negative electrostatic surface around the heme. this work provides, for the first time, evidence for the co-existence of two similar methyl-accepting chemotaxis proteins functioning in different working potential ranges. these proteins are responsible to allow geobacter sulfurreducens triggering an adequate cellular response in different anoxic subsurface environments. national autonomous university of mexico, faculty of medicine, national autonomous university of mexico, faculty of chemistry, national autonomous university of mexico, institute of chemistry molecular evolution the glycolytic enzyme triosephosphate isomerase (tim) is an oligomeric (b/alpha) barrel that catalyses the interconversion of d-glyceraldehyde -phosphate and dihydroxyacetone phosphate in a diffusion-limited reaction. although each subunit has its own active site, naturally occurring monomeric tims have not been reported; in fact, monomer association is very tight. tim topology is well conserved among the three domains of life. nevertheless, their folding mechanism and inhibition properties vary across species. comparative studies of proteins have proved to be very useful in understanding the relationship between sequence and physicochemical properties, however, they lack the capacity to give a more integrative and evolutive correlation. in order to elucidate how the catalytic properties, the oligomerization state and the stability of extant tims arose, in this work we examined the molecular history of eukaryotic tim through ancestral protein reconstruction methods (maximum likelihood) and the subsequent physicochemical characterization of the resurrected enzymes. we first characterized in detail the protein corresponding to the last common ancestor of animals and fungi (tim ). the cd and fluorescence spectra of tim are similar to those of extant tims. secondary structure is lost in a cooperative transition with tm . c. the enzyme loses activity upon dilution suggesting that only the dimer is active. dilution experiments followed by isothermal titration calorimetry indicate that dissociation enthalpy is small; moreover the heat capacity change observed is three times higher than the one predicted for a rigid body dissociation process, suggesting partial unfolding of the monomers. when compared with extant tims, the catalytic efficiency of tim is reduced -fold, whereas binding of pgh, a transition-state analogue, shows a similar thermodynamic signature. these data indicate that although monomer association may have been less tight in ancestral tims, catalysis has been always linked to oligomerization. analysis of the crystal structure of tim , obtained at . Å resolution, suggests that the lack of four salt bridges observed in the interface of extant tims is responsible for the low dimer stability. in order to test this hypothesis we also studied the stability of four younger reconstructed ancestors that acquired the salt bridges in two different phylogenetic lineages. we found a correlation between the appearance of stabilizing interactions in the interface, dimer stability and catalysis; suggesting that these salt bridges are partially responsible for extant dimer stability and shed light on the dimeric nature of extant tims. receptor protein-tyrosine phosphatases: dimerization, receptor kinase interaction and allosteric modulation elizabeth dembicer , damien thevenin department of chemistry, lehigh university theme: receptor tyrosine kinase and receptor protein phosphatase signaling many cell-signaling events are regulated through reversible tyrosine phosphorylation of proteins, which is controlled by the counterbalanced actions of two key enzyme families: protein tyrosine kinases and protein tyrosine phosphatases. interestingly, both families include transmembrane receptor-like enzymes, namely the receptor tyrosine kinases (rtks) and the receptor-like ptps (rptps). while the regulation and actions of many rtks are well characterized, the mechanisms controlling the enzymatic activity of rptps and how they interact with their substrates remain to be fully explained. thus, understanding how these receptors function and interact will give fundamental insights into how tyrosine phosphorylation is finely tuned in cells, and how it can be modulated. increasing evidence indicates that rptps, like rtks, are regulated by homodimerization. however, it appears that homodimerization inhibits the activity of most rptps. even though the transmembrane (tm) and the juxtamembrane domains have been proposed to be involved in this process, there is no clear structure-based proposal for the role of these regions. moreover, several rptps have been identified as candidate regulators of rtks. in particular, the receptor-type tyrosine-protein phosphatase eta (ptprj; also known as dep or cd ) is capable of attenuating egfr tyrosine phosphorylation. physical interactions of egfr with ptprj at the cell surface have been documented, but the basis for these interactions is unknown. here, using a dominant-negative transcriptional activator-based assay (dn-aratm), and mutagenesis analysis, we show that: ( ) ptprj has a strong tendency to homodimerize, ( ) ptprj heterodimerizes with egfr through tm-tm interactions, ( ) these interactions are mediated by specific residues, and can be modulated by the delivery of peptide binders. this work represents the first structure-function study of rptp-rtk interaction, and may not only result in significant progress towards a better understanding of the basic biology of rptps in cancer cells, but also offer new possibilities for targeting protein tyrosine phosphatases for therapeutic modulation of egfr in oncology. inhibiting egfr dimerization and signaling through targeted delivery of juxtamembrane domain peptide mimics using phlip anastasia thevenin , kelly burns , janessa guerre-chaley , damien thevenin regulating receptor tyrosine kinase signaling the elevated phosphorylation of key regulatory tyrosines on oncogenic signaling proteins that result from aberrant protein tyrosine kinases activity plays well-abstract established roles in promoting tumorigenesis and in the high frequency with which resistance arises to existing therapeutic treatment. for instance, this is the case for the epidermal growth factor receptor (egfr). thus, there is a clear need for novel specific targeting methods to inhibit the activity of receptor protein tyrosine kinases, such as egfr, in cancer. egfr becomes activated upon ligand binding to the extracellular domain, leading to receptor dimerization. the juxtamembrane (jm) domain of egfr is critical for intrinsic tyrosine kinase activity and receptor dimerization by stabilizing the active conformation of egrr through the formation of a antiparallel helical dimer. therefore, peptides mimicking the jm domain -if specifically delivered to cancer cells -have the potential to prevent egfr dimerization, receptor activation, downstream signaling, and thus to attenuate aberrant egfr activity in cancer cells. here, phlip (ph low insertion peptide), a peptide that can selectively target cancer cells and tumors based solely on their extracellular acidity, is used to selectively translocate the jm domain of egfr in cancer cells to prevent egfr dimerization. at ph above , phlip is soluble and unstructured, however, when exposed to lower ph such as observed in tumors, phlip inserts as a transmembrane (tm) alphahelix, allowing the direct translocation of cargo molecules into the cytoplasm. using the dominant negative arac-based transcriptional reported assay (dn-aratm), which assesses jm and tm domain interactions in cells membranes of e. coli, we show that phlip-jm is able to disrupt egfr dimer by %. current work is focused on testing the ability of such phlip-jm peptide conjugate to perturb egfr homodimerization and decrease downstream signaling through soluble kinases, such as akt and erk, in cancer cells. the thumb subdomain of yeast mitochondrial rna polymerase is involved in processivity, transcript fidelity and mitochondrial transcription factor binding gilberto velazquez , luis brieba , rui sousa universidad de guadalajara, langebio cinvestav, university of texas healthsscience center at san antonio dna protein interaction abstract single subunit rna polymerases have evolved two mechanisms to synthesize long transcripts without falling off a dna template: binding of nascent rna and interactions with an rna:dna hybrid. mitochondrial rna polymerases share a common ancestor with t-odd bacteriophage single subunit rna polymerases. herein we characterized the role of the thumb subdomain of the yeast mtrna polymerase gene (rpo ) in complex stability, processivity, and fidelity. we found that deletion and point mutants of the thumb subdomain of yeast mtrna polymerase increase the synthesis of abortive transcripts and the probability that the polymerase will disengage from the template during the formation of the late initial transcription and elongation complexes. mutations in the thumb subdomain increase the amount of slippage products from a homopolymeric template and, unexpectedly, thumb subdomain deletions decrease the binding affinity for mitochondrial transcription factor (mtf ). the latter suggests that the thumb subdomain is part of an extended bindingsurface area involved in binding mtf . design principles of membrane protein structures vladimir yarov-yarovoy , diane nguyen membrane protein structure membrane proteins play key role in cellular signaling and ion transport. statistical analysis of expanding database of high-resolution membrane protein structures in protein data bank (pdb) provides useful information about membrane protein structure and function. we used rosettamembrane software (yarov-yarovoy v et al ( ) proteins) to analyze unique alpha helical membrane protein structures in pdb and derive knowledge based energy function for membrane protein structure prediction, membrane protein-protein docking, and membrane protein design. the rosettamembrane residue environment energy term is based on amino acid propensities in hydrophobic, interface, and water layers of the membrane and depends on the residue burial state -from being completely buried within a protein environment to being completely exposed either to the lipid or water environments. residue buried state is determined from the number of residue neighbors within and Å spheres. the rosettamembrane residue-residue interaction term is based on the propensities of amino acid pairs to be in close proximity to each other within hydrophobic, interface, and water layers. results of our statistical analysis reveal fine details of favorable and unfavorable environments for all amino acids types in all membrane layers and residue burial states. we find that large hydrophobic amino acids are favorable facing the hydrophobic core of the lipid bilayer. small amino acids are favorable facing the protein core within the hydrophobic layer of the membrane. aromatic or positively charged amino acids and favorable facing the lipid head groups. residue-residue interactions are often favored between polar and charged amino acids and also between some of small and large hydrophobic amino acids inside of the protein core within the hydrophobic layer of the membrane. these data will be useful for rational design of novel membrane protein structures and functions. coordinated gripping of substrate by subunits of a aaa proteolytic machine ohad yosefson , andrew nager , tania baker , robert sauer protein quality control' or 'protein degradation' hexameric aaa protein-remodeling machines use conserved loops that line the axial pore to apply force to substrates during the mechanical processes of protein unfolding and translocation. an open question in the aaa field is whether pore loops from different subunits of the hexameric ring grip the substrate coordinately (all six subunits involved), independently (one subunit at a time involved), or partially coordinated (two or three subunits at a time). to answer this question, we studied covalently linked hexamers of the e. coli clpx unfoldase bearing different numbers and configurations of wild-type and mutant pore loops and challenged these variants with protein substrates with a broad range of stabilities. we find that successful unfolding of increasingly resistant substrates requires the coordinated action of a greater number of wild-type pore loops. our results support a mechanism in which a power stroke initiated in one subunit of the clpx hexamer results in the simultaneous movement of all six pore loops, which coordinately grip and apply force to the substrate. structure and function of the toc m-domain, and its role in targeting the preprotein receptor to the chloroplast outer envelope membrane matthew smith , shiu-cheung lung , prem nichani , nicholas grimberg , j. kyle weston , shane szalai , simon chuong deartment of biology, wilfrid laurier university, department of biology, university of waterloo chloroplast biogenesis and function rely on the import of thousands of nucleus-encoded preproteins from the cytosol. preprotein import is supported by the toc and tic (translocon at the outer and inner envelope membranes of chloroplasts) complexes, which work cooperatively to translocate preproteins across the double-membrane envelope to the chloroplast interior. toc is one of the preprotein receptors of the toc complex, is also encoded in the nucleus and post-translationally targeted to the chloroplast, and is comprised of distinct domains: ) the intrinsically disordered n-terminal acidic (a-) domain; ) the central gtpase (g-) domain; and ) the c-terminal membrane (m-) domain that anchors the protein to the chloroplast outer membrane (com) through an unknown mechanism. the m-domain has no known homologues and does not contain a predicted trans-membrane domain, but does contain intrinsic chloroplast targeting information at the extreme c-terminus. the m-domain also contains a predicted b-helix motif, which may be important for anchoring the protein to the com. we are interested in characterizing the structure of the m-domain and determining the nature of its association with the com, as part of our larger goal of understanding the role toc plays in protein import into chloroplasts. we are also interested in defining the precise nature of the targeting information contained within the extreme c-terminus of toc , elucidating the targeting pathway that is used, and whether other com proteins use this pathway. we will present our most recent data on the structure, function and targeting of the toc m-domain. structural investigation of nlpc/p protein acquired by trichomonas vaginalis through a lateral gene transfer event jully pinheiro , , augusto simoes-barbosa , david goldstone microbiology, school of biological sciences, university of auckland, structural biology, school of biological sciences, university of auckland trichomonas vaginalis is an extracellular flagellated protozoan parasite that causes the most common non-viral sexually transmitted disease, with approximately million cases worldwide annually. nevertheless, the biochemical processes behind t. vaginalis infection and its interaction with the vaginal microbiota are still not well defined. in the draft genome sequence of trichomonas vaginalis strain g was described, identifying , protein-coding genes. of these, nine genes encode nlpc/p -like members. this superfamily is widely represented in the different kingdoms of life and has diverse enzymatic functions, such as amidases, endopeptidases and acetyltransferases. previous studies have shown that members of this superfamily hydrolyze specific peptide linkages in bacterial cell walls affecting germination, vegetative growth, sporulation and division or cell lysis/invasion. as a typical eukaryote, the protozoan parasite t. vaginalis does not have a cell wall itself. previous studies suggest that the t. vaginalis nlpc/p genes were acquired via lateral gene transfer from bacteria and must have an important function, possibly controlling the vaginal microbiota and aiding parasite invasion and infection. to investigate the function of the nlpc/p family of proteins in t. vaginalis we have expressed, purified and crystallized a member tvag_ and report its three-dimensional structure, determined at . Å resolution, by x-ray diffraction. the structure of the protein reveals a typical papain-like fold resembling peptidoglycan hydrolases from the nlpc/p family with a conserved cysteine and histidine; forming the catalytic residues. the protein contains two bacterial sh domains at the n-terminus. this domain acts as a general binding domain and is likely to aid the interaction of the nlpc/p domain with substrate components. combined with biochemical and enzymatic characterization, the structure of this nlpc/p protein will help to elucidate the molecular origin of its hydrolase activity and to decipher their putative role in the parasite infection. novel dna polymerases from red sea brine-pools: new potential polymerases for pcr application masateru takahashi , etsuko kimura , mohamed salem , ulrich stingl , samir hamdan protein biotechnology the polymerase chain reaction (pcr) is a key tool in medical and biological research. the most common pcr reaction relies on the thermal cycling method that consists of repeated cycles of heating and cooling steps for dna melting and extension by the dna polymerase, respectively. the introduction of new dna polymerases to the market is a major area of development that tremendously helped in improving the performance and quality of pcr. nonetheless, pcr still requires optimization of salt and metal ion concentrations leaving a room in the market for introducing new dna polymerases that are robuster in their salt and metal ion concentration dependence. in this study, we will present the characterization of a novel archaeal dna polymerase from the red sea brine-pool (termed br ) and demonstrate how its enzymatic activity reflects on every aspects of the environment of the brine-pool -high tolerance to concentrations and types of salts and metal ions including utilization of zn ions in its active site. these results suggest that the brine-pool microorganisms are likely to contain novel chemical pathways to deal with its exterior harsh conditions. we will further show the mechanism of br polymerase how it was adjusted to be active in harsh condition. structural basis for the identification of the n-terminal domain of coronavirus nucleocapsid protein as an antiviral target ming-hon hou , shing-yen lin , chia-ling liu , yu-ming chang , jincun zhao , stanley perlman institute of genomics and bioinformatics, national chung hsing university., institute of biological chemistry, academia sinica., department of microbiology, the university of iowa drug discovery coronaviruses (covs) cause numerous diseases, including middle east respiratory syndrome and severe acute respiratory syndrome, generating significant health-related and economic consequences. covs encode the nucleocapsid (n) protein, a major structural protein that plays multiple roles in the virus replication cycle and forms a ribonucleoprotein complex with the viral rna through the n protein's nterminal domain (n-ntd). using human cov-oc (hcov-oc ) as a model for cov, we present the d structure of hcov-oc n-ntd complexed with ribonucleoside '-monophosphates to identify a distinct ribonucleotide-binding pocket. by targeting this pocket, we identified and developed a new coronavirus n protein inhibitor, n-( -oxo- , -dihydrophenanthridin- -yl)(n,n-dimethylamino)acetamide hydrochloride (pj ), using virtual screening; this inhibitor reduced the n protein's rna-binding affinity and hindered viral replication. we also determined the crystal structure of the n-ntd-pj complex. on the basis of these findings, we propose guidelines for developing new n protein-based antiviral agents that target covs. thermal and structural stability of ß-glucosidases gh maira artischeff frutuoso departamento de bioqu ımica do instituto de qu ımica da universidade de são paulo enzymology we compared the stability of thermophilic b-glucosidases gh to mesophilic ones in the presence of denaturants as urea and high temperature by following the transitions between the native and unfolded states by tryptophan fluorescence, enzymatic activity and differential scanning fluorimetry (dsf). the bacterial b-glucosidases (bgla) and (bglb) of the mesophile paenibacillus polimyxa and bglucosidase (bglthm) of the thermophile thermotoga maritima were expressed as recombinant proteins in novablue (de ) and purified by affinity chromatography (ni-nta resin). these recombinant enzymes have very similar folding type structure (b/a) barrel, as shown in crystal structures and exhibited a characteristic peak between and nm in the tryptophan fluorescence spectra, indicating that those proteins are folded. circular dichroism analysis in the far-uv region ( nm to nm) also showed typical spectra of folded proteins with secondary structure composition of % of a-helix and % of b-sheets for bgla, % of a-helix and . % of b-sheets for bglb and % of a-helix and % of b-sheets for bglthm. the average degree of accessibility to the exposed tryptophan residues in the native enzyme to increasing concentrations of the acrylamide suppressor (stern-volmer constant -ksv) is greater to bgla ( . ), but similar to bglb ( . ) and bglthm ( . ). the thermal stability determined by dsf was higher for bglb (tm . c) than for bgla (tm . c) . the bglthm was stable at c and remained stable for up to h at c. in addition the thermal inactivation kinetics at c evaluated by the relative remaining activity showed that bgla denaturation (kinactivation of . s- ) is faster than bglb (kinactivation of . s- ). on the other site, bglthm inactivation at c was a two-step process, which exhibited an initial fast step (kinactivation of . s ) followed by a slow step (kinactivation of . s- ). the chemical denaturation by urea followed using tryptophan fluorescence showed a transition pl- covalent structure of single-stranded fibrinogen and fibrin oligomers cross-linked by fxiiia. the influence of free radical oxidation anna bychkova , vera leonova , alexander shchegolikhin , marina biryukova , elizaveta kostanova , mark rosenfeld n. m. emanuel institute of biochemical physics, russian academy of sciences protein structure and function native fibrinogen is a key blood plasma protein whose main function is to maintain hemostasis by virtue of producing the cross-linked fibrin clots under the effect of thrombin and fibrin-stabilizing factor (fxiiia). fxiiia-mediated isopeptide g-g bonds are known to be produced between g polypeptide chains of adjacent fibrinogen or fibrin molecules. but there are apparently conflicting ideas regarding the orientation of g-g bonds. in this study several peculiarities of self-assembly of fibrin(ogen) and induced oxidation of the proteins have been studied with the aid of elastic and dynamic light scattering, uv-, ftir-and raman spectroscopy methods. in the presence of fxiiia both the non-oxidized and oxidized fibrinogen molecules has been shown to bind to each other in the "endto-end" fashion to form the flexible covalently cross-linked fibrinogen homopolymers. to identify the orientation of g-g bonds in fibrin protofibrils a novel approach based on self-assembly of soluble cross-linked fibrin protofibrils and their dissociation in the urea solution of moderate concentrations has been applied. the results of elastic and dynamic light scattering coupled with analytical ultracentrifugation indicated the protofibrils to exhibit an ability to dissociate under increasing urea concentration to yield single-stranded structures entirely brought about by g-g bonds. the results of this study provide an evidence to support the model of the longitudinal g-g bonds that form between the g chains end-to-end within the same strand of a protofibril. since fibrinogen is known to be sensitive to ros the mechanisms of fibrinogen and fibrin self-assembly under induced oxidation have been investigated. in both cases the polypeptide chains of the oxidized fibrin(ogen) proved to be involved in the enzymatic cross-linking more readily than those of unaffected molecules. the enhancing role of the d:d interaction under oxidation could be considered as an compensatory mechanism in the assembly of fibrin when the d:e interaction is impaired. the experimental data on fibrinogen and fibrin oxidation acquired in the present study, being combined with our earlier findings, make it reasonable to suppose that the spatial structure of fibrinogen could be evolutionarily adapted to some ros actions detrimental to the protein function. the study was supported by the rfbr, research projects - - mol_a and - - a. structural and thermodynamic analysis of co-stimulation receptor cd phosphopeptide interactions with grb , gads, and pi -kinese sh domains in addition to the signaling produced by the binding of antigen-major histocompatibility complex to tcell receptors, co-stimulatory signals from other receptor-ligand interactions are required for full activation of t-cells. the cd receptor on the t-cell surface has been well characterized, and the binding of ligand to cd is critical for producing co-stimulatory signals. cd has no enzymatic activity and its cytoplasmic region consists of amino acids that contain the sequence ymnm, in which the tyrosine residue is phosphorylated by kinase. the phosphorylated sequence, pymnm, is recognized by src homology (sh ) adaptor proteins, such as growth factor receptor binding protein (grb ), grb related adaptor downstream (gads), and the phosphatidylinositol -kinase (pi -kinase) regulatory subunit, p . the consensus sequence for the binding of grb sh and gads sh is pyxnx, and that of p n-terminus sh (nsh ) and c-terminus sh (csh ) is pyxxm. we reported the high-resolution crystal structure of grb sh in complex with the cd phosphopeptide [higo et al., plos one , e , ] , and recently determined those of gads sh , p nsh , and p csh . these data along with the results of binding thermodynamics analyzed using isothermal titration calorimetry, helped to elucidate the molecular recognition mechanisms of cd by adaptor proteins. the sh proteins were overexpressed in escherichia coli, and were purified using affinity and gel-filtration chromatography. the cd phosphopeptides, -residue (octp) and -residue (ddcp ), were synthesized using the solidphase supported technique, and were purified using reversed-phase chromatography. the crystals were obtained by the hanging-drop vapor diffusion method. x-ray diffraction data were collected at synchrotron radiation facilities, and the structures were determined by the molecular replacement method. the models of grb sh , gads sh , p nsh , and p csh in complex with octp were refined at . , . , . , and . Å resolutions, respectively. the crystal structures showed that the phosphotyrosine phosphate moiety directly interacted with the side-chain of arginine in sh , which is common in all complex structures. in the grb sh and gads sh complexes, the side-chain of asparagine at the py position forms a pair of hydrogen bonds with the main-chain amide and carbonyl groups of lysine in sh . alternatively, in the p nsh and csh complexes, the side-chain of methionine at the py position is located in hydrophobic pockets of nsh and csh , in which the hydrophobic interactions of csh would be stronger than those of nsh . this idea is supported by the observed binding thermodynamics. the binding affinity of csh to ddcp , because of a favorable enthalpy change, is about -fold higher than that of nsh . the binding affinity of grb sh to ddcp is similar to that of gads sh to ddcp , and is about -fold lower than that of nsh to ddcp . these results indicate that the contribution of hydrophobic interactions of nsh and csh at the py position are stronger than those of hydrogen bonds of grb sh and gads sh at the py position. novel kinetochore protein complex from silkworm holocentric chromosomes takahiro kusakabe , hiroaki mon , jaeman lee the kinetochore, which consists of centromere dna and a multilayered protein complex, plays important roles in chromosome organization and segregation. interactions between chromosomes and spindle microtubules allow chromosomes to congress to the middle of the cell, and to segregate the sister chromatids into daughter cells in mitosis, which is followed cytokinesis. in contrast to monocentric chromosomes, in which the centromere is normally present at a single region on each chromosome, the holocentric chromosomes have centromeric activity along the entire length of the chromosome. it has been known that the silkworm, bombyx mori, has holocentric chromosomes since s, none of silkworm kinetochore proteins, however, have been identified so far. here we report the identification of a novel set of genes for outer kinetochore proteins in silkworm by using bioinformatics and rna interference-based screening. under the hypothesis that depletion of essential kinetochore genes causes cell cycle arrest in mitosis, we performed rnai in the silkworm cell line, bmn -sid , targeting a set of candidate genes. knockdown of five genes caused significant cell cycle arrest at the g /m phase. we also found that these five proteins make a complex, and that all of them are localized along the chromosome arms, indicating that the silkworm kinetochore extends along the chromosome. inactivation of bine aldehyde dehydrogenase from spinach by its physiological substrate bine aldehyde to contend with osmotic stress caused by drought, salinity, or low temperatures some plants synthesize the osmoprotectant glycine bine (gb) from bine aldehyde (bal). the last step-the irreversible nad dependent oxidation of bal-is catalyzed by aldh enzymes that exhibit bine aldehyde dehydrogenase (badh) activity. we here report that the spinacia oleracea badh (sobadh) is reversibly inactivated by bal in the absence of nad in a time-and concentration-dependent mode to approximately % of the original activity. inactivation kinetics are consistent with a partial reversible, two-steps mechanism that involves the formation of an active non-covalent enzyme•bal complex before formation the inactive enzyme-bal complex. crystallographic evidence indicates that in the enzyme previously inactivated by bal the aldehyde forms a thiohemiacetal with the nonessential cys (sobadh numbering) located at the aldehyde-entrance tunnel, thus totally blocking the access to the catalytic cysteine. accordingly, bal does not inactivate the c s sobadh mutant. two crystal structures of the inactivating enzyme-bal complex showed that the trimethylammonium group of bal is inside the active-site aromatic box, as in the productive way of binding. this explains why the inactivation of the a i mutant-where the binding of the trimethylammonium group is hindered-requires non-physiologically high bal concentrations, while the a c mutant-where the binding is allowed-is inactivated similarly to the wildtype enzyme. cys- is conserved in most plant aldh enzymes of known sequence, and in all of them with proven or predicted badh activity. inactivation by bal appears therefore to be a common feature of plants badhs. this short-term regulation may be of great physiological importance since the irreversibility of the badh-catalyzed reaction would unbalance the nad /nadh ratio if the aldehyde concentrations are high, the nad concentrations low and the reaction is not slowed down. plants badhs are prone to this situation since they work under osmotic stress conditions, when high bal concentrations are required for the synthesis of high levels of the osmoprotectant gb. the partial nature of the decamer possesses a donut shaped structure with calcium ions on the surface available for interactions with carbohydrate molecules. binding specificity was evaluated for carbohydrates using differential scanning fluorimetry (dsf) that showed bjcul interacts with galactose and lactose but less with glucose and sacarose. surprisingly, high levels of thermostabilization of bjcul was achieved with the antibiotic aminoglycosides geneticin (g ) and gentamicin in a calcium concentration dependent manner, but not kanamycin. intriguingly, while lactose and galactose inhibited erythrocyte agglutination by bjcul, g and gentamicin did not affect hemagglutination implying a second site of binding. dsf analysis also suggested the presence of a second binding site for the antibiotics and crystallization of the complexes are in progress in order to understand fully this new binding mechanism of c-type lectin with antibiotics. ab initio modelling of structurally uncharacterised antimicrobial peptides mara kozic institute of integrative biology, university of liverpool ab initio modelling of structurally uncharacterised antimicrobial peptides mara kozic * institute of integrative biology, biosciences building, university of liverpool, crown street, liverpool l zb, united kingdom * mara.kozic@liverpool.ac.uk antimicrobial resistance within a wide range of infectious agents is a severe and growing public health threat. antimicrobial peptides (amps) are among the leading alternatives to current antibiotics, exhibiting broad spectrum activity. an understanding of the structure of a protein can lead us to a much improved picture of its molecular function. furthermore, an improved understanding of structure-function relationships facilitates protein design efforts to enhance their activity. currently, the d structures of many known amps are unknown. to improve our understanding of the amp structural universe we have carried out large scale ab initio d modelling of structurally uncharacterised amps. such ab initio modelling is facilitated by the typical small size of amps as well as their tendency to contain disulphide bonds, these providing valuable additional information to simulations. preliminary results reveal unexpected similarities between the predicted folds of the modelled sequences and structures of well-characterised amps. for example, lacticin q was revealed to contain a helical bundle fold that bears a striking resemblance to enterocin a. we also found a remarkable similarity between the predicted structure of silkworm peptide and b-hairpin amps such as tachyplesin i. our results improve the understanding of the structure-function relationship of amps. surface aggregation-propensity as a constraint on globular proteins evolution susanna navarro , marta diaz , pablo gallego , david reverter , salvador ventura institut de biotecnologia i biomedicina and departament de bioquimica i biologia, institut de biotecnologia i biomedicina, universitat aut onoma de barcelona in living cells, functional protein-protein interactions compete with a much larger number of nonfunctional interactions. theoretical studies suggest that the three-dimensional structures of present proteins have evolved under selective pressure to avoid the presence of aggregation-prone patches at the surface that may drive the establishment of anomalous protein contacts. however, no experimental evidence for this hypothesis exists so far. the a-spectrin sh domain (spc-sh ) has been used as a protein model to decipher the sequential aggregation determinants of proteins. here we use it to address the structural determinants of protein aggregation and their link to protein evolution. to this aim we exploit aggrescan d (a d), a novel algorithm developed by our group, which takes into account both protein structure and experimental data to project aggregation propensities on protein surfaces. we used a d to design a series of spc-sh variants with progressively stronger aggregationprone surfaces and characterized their thermodynamic, structural and functional properties. our data support evolution acting to constraint the aggregation propensities of globular protein surfaces in order to decrease their potential cytotoxicity and the protein quality control machinery acting to buffer this negative selective pressure. utilizing d structure for the annotation of structural motifs in the conserved domain database narmada thanki-cunningham , noreen gonzales , gabriele marchler , myra derbyshire , james song , roxanne yamashita , christina zheng , stephen bryant , aron marchler-bauer , farideh chitsaz conserved domain database, structure group cbb/ncbi/nlm/nih the conserved domain database (cdd) is a protein classification and annotation resource comprised of multiple sequence alignments representing ancient conserved domains. cdd protein domain models are curated by ncbi and use d protein structure explicitly to define domain extent and the location of conserved core structures, and to provide accurate alignments between diverse family members via structure superposition. cdd also imports external collections such as pfam and tigrfam. recently, a novel class of annotation labeled as "structural motifs" has been introduced to supplement current capabilities. these annotations define compositionally-biased and/or short repetitive regions in proteins, which are difficult to model as functional domains conserved in molecular evolution. structural motifs include transmembrane regions, coiled coils, and short repeats with variable copy numbers. for many types of short tandem repeats, a few position-specific score matrices (pssms) suffice to annotate more than % of the known instances of that structural motif. unfortunately, a lack of sequence similarity within coiled-coil regions prohibits the development of only a few generic models; therefore, models for coiled-coil regions in the context of specific families have been developed using the spiricoil database as a reference. increased coverage of coiled-coil regions in cdd, specific site annotations of these structural motifs as well as their representation on the webpages will be discussed. specific in vivo ultrasound imaging of e-selectin expression in tumors using a microbubble contrast agent covalently attached to the peptide ligand iellqar, known to bind to e-selectin [ ] . however, it was observed that this probe has a limitation in the imaging of cardiovascular diseases where higher shear stresses prevent microbubbles from remaining attached to the target. therefore, peptides with higher eselectin affinity are needed to design probes capable of imaging these diseases. in this context, automated docking and molecular dynamics methodologies were combined and applied to different e-selectin binding peptides. these studies predicted the energetically more favorable binding mode as well as the key interactions between the peptide ligands and the e-selectin receptor. some of these peptides were prepared by solid-phase peptide synthesis and their interactions with e-selectin analyzed by surface plasmon resonance technique. the results showed that these peptides have different affinities for e-selectin. these data were correlated with the computational studies and evaluated to obtain crucial information of the key recognition elements needed for higher e-selectin affinity. these recent results will be presented. burkholderia pseudomallei is the causative agent of melioidosis, a serious invasive disease of animals and humans in tropical and subtropical areas. sedoheptulose- -phosphate isomerase from b. pseudomallei (bpgmha) is the antibiotics adjuvant target for melioidosis. in general, bpgmha converts dsedoheptulose- -phosphate to d-glycero-a-d-manno-heptopyranose- -phosphate (m p). this is the first step of the biosynthesis pathway of ndp-heptose responsible for a pleiotropic phenotype. therefore, this biosynthesis pathway is the target for searching novel antibiotics increasing the membrane permeability of gram-negative pathogens or adjuvants synergistically working with known antibiotics. the crystal of this enzyme has been solved at . Å resolution. there is an active site pocket where a putative metal binding site is located. to find out inhibitors of bpgmha, in-silico virtual screening with zinc, a free database of commercially-available compounds, has been performed. tens of thousands of chemical compounds were docked into the active site of bpgmha. a number of putative bpgmha binding compounds better than m p were found using surflex-dock included in the sybyl software package. characteristics of these compounds were surveyed and classified to identify common binding properties with bpgmha. mapping the structure of laminin using cross-linking and mass spectrometry gad armony , toot moran , yishai levin , deborah fass weizmann institute of science, department of structural biology, weizmann institute of science, israel center for personalized medicine laminin, a kda heterotrimer, is a major element in the extracellular matrix (ecm). within the ecm, laminin contributes to the adhesion and migration of cells, both in health and disease. the laminin trimer was observed by rotary shadowing electron microscopy to be cross shaped: the three short arms of the cross are formed by the amino-terminal halves of the three subunits, whereas the long arm of the cross holds the three chains together in a long coiled coil. the narrow and flexible arms of the laminin cross complicate studying its structure to high resolution by crystallography or electron microscopy single particle reconstruction. to advance our understanding of this remarkable quaternary structural assembly, we have used cross-linking and mass spectrometry to analyze the organization of the laminin trimer. this technique was validated by known crystal structures of isolated laminin domains. in all cases the crystal structure distances agree with the cross-linker length. the identified cross-links were particularly helpful in assigning the register and the subunit order of the long coiled coil due to the high content of cross-linkable residues in this region. using known x-ray crystal structures, homology modeling, and distance restraints provided by two cross-linker chemistries, a clearer picture of the laminin quaternary structure is obtained. non-sequential protein structure alignment program mican and its applications shintaro minami , george chikenji , motonori ota dept. of info. sci., nagoya univ., dept. of comp. schi. & eng., nagoya univ. in some proteins, secondary structure elements are arranged spatially in the same manner, but they are connected in the alternative ways. analysis on such non-sequential structural similarity in proteins is important because it provides a deeper understanding of the structural geometry of protein. this can be also observed even in the homologous proteins, indicating the non-sequential structural similarity is significant in the protein evolution. however, the non-sequential structural similarity in proteins is less investigated. we developed a novel non-sequential structural alignment program mican, which can handle multiple chains, inverse direction of chains, c$lpha$models, alternative alignments, and non-sequential alignments. we performed comprehensive non-sequential structural comparison among homologous proteins in the same scop superfamily by using the mican program. based on the result, we found that approximately % of superfamilies include at least one protein pairs showing non-sequential structural similarity. % nonsequential structurally similar pairs are aligned in a simple way, e.g. circular permutation, $$strand flip/ swap, but % are complicated. interestingly, most of such complicated non-sequential similarities can be explicable by combination of - simple non-sequential relationships. this result indicates that accumulation of simple structural changes in the course of protein evolution produces completely different fold homologs. as early as , ritter surmised that the cell's molecules cooperate to form a "special apparatus and an organised laboratory". despite supporting evidence from srere, mcconkey and others, efforts to understand molecular organisation in vivo are still in their infancy. however, important aspects of the cell interior have already been revealed. for example, weak molecular interactions structure the cytoplasm into time-evolving, functional zones. weak interactions are difficult to capture and can preclude protein detection in cells by many biophysical techniques, including nmr spectroscopy. , we explored the effects of cell-like milieus on the cytochrome c (cyt c)-flavodoxin (fld) interaction. these oppositely charged proteins interact weakly with a number of cognate partners. neither cyt c nor fld is detectable by nmr in escherichia coli confirming their "sticky" nature ( figure a) . the cyt c-fld interaction was assessed in buffer, % polyacrylamide gels and in solutions containing g/l of macromolecular crowders ( figure b) . h, n hsqc nmr revealed that the interaction was transient in buffer, proceeding via the known binding site for both proteins. substantial line broadening was effected in crowded and confined solutions suggesting that the cyt c-fld complex is stabilised under native-like conditions. the stabilising effect of macromolecular crowders was also observed by native gel electrophoresis and crystallization. these findings coincide with spitzer and poolman's model for cytoplasmic structuring, emphasising the role of charge-charge interactions and crowding in the formation of macromolecular "clusters". the implications for cytoplasmic structuring will be discussed alongside related investigations of cationic protein interactions in e. coli extracts. , detergent:protein ratio. the transmembrane b-barrel of bama is folded in either micelles, bicelles or nanodiscs, however an n-terminally attached single potra domain is flexibly unfolded, due to the absence of stabilizing contacts with other protein domains. measurements of backbone dynamics show distinct time scales of dynamic behavior for bama b-barrel and parts of its extracellular loop l , revealing high local flexibility within the the lid loop. this work presents the first high-resolution d solution nmr spectra of the bama barrel and establishes improved biochemical preparation schemes, which will serve as a platform for structural and functional studies of bama and its role within the bam complex. protein arginine methylation is a widespread and important posttranslational modification in eukaryotic cells, shown to be involved in the activation or repression of transcription, modification of the splicing machinery, signaling, and dna repair. mammalian protein arginine methyltransferases include a family of nine sequence-related enzymes that transfer one or two methyl groups onto the terminal guanidino groups on arginine residues, producing monomethylarginine only (mma, type iii), symmetric dimethylarginine (sdma) and mma (type ii), or asymmetric dimethylarginine (adma) and mma (type i). while prmt , , , , , and have been characterized as type i enzymes, and prmt as a type ii enzyme, the role and activity types of the two final members of this family of enzymes, prmt and prmt , had been unclear due to conflicting results in the literature, and the substrates for these enzymes had been elusive. both prmt and prmt are distinct members of the family with two methyltransferase or methyltransferase-like domains and containing acidic residues in otherwise well-conserved substrate double e binding motif, features not seen in the other prmt enzymes. recent work in our laboratory confirmed prmt as the only type iii mma-forming enzyme in the group, with a unusual low temperature optimum for activity, and a heretofore not seen preference for a basic stretch of residues in an r-x-r sequence for methylation. mutations of the acidic residues in the substrate-binding motif results in a loss of the specific r-x-r activity and the appearance of a g-r-g specificity typical of many of the other prmts. the physiological substrate of prmt has yet to be confirmed, although histone h b is an effective in vitro substrate. prmt , on the other hand, had no reported activity, until immunoprecipitation from hela cells showed it pulled down two splicing factors, sf b and sf b , in a complex. amino acid analysis showed that prmt methylates sf b to produce both mma and sdma, thus making it the second type ii enzyme in mammals. prmt knockdown results in modulation of alternative splicing events. this enzyme appears to be relatively specific for the sf b protein; a peptide containing the methylatable arginine residue was not found to be a substrate, and typical substrates of other prmts are not recognized by prmt . we found that the position of the methylated arginine residue in sf b is important, and the acidic residues in the substrate-binding motif also play an important role in substrate recognition. thus, prmt and prmt represent unique members of the mammalian prmt family. hydrogen peroxide levels, endogenous hormones (cytokinins, salycilic acid, as well as jasmonic acid and its conjugates), polyphenolics and terpenoids in a model system of a. alba in vitro with inhibition of rootng and stimulation of callusogenesis by means of individual and combined cytokinin and cytokinin/ auxin treatments. results: it was established that inhibition of rooting and stimulation of callusogenesis caused by benzyl adenine (ba) or combinations of ba and indole- -butiric acid (iba) in vitro were related to elevation of sesquiterpenoids in the essential oils, as well as polyphenolics content, accompanied by a drop of stress hormones, bioactive cytokinins and preservation of oxidative stress and lipid peroxidation levels, as compared with non-treated control. individual treatments with either iba or ba, also increased the sesquiterpenoid content in the essential oil of the plant, in a concentration related manner, this effect being more profound after ba treatment. in addition, ba treated plants exhibited a drop of protein levels of the aerial samples, as well as profound differences of enzymatic activity in the callus tissues, as compared with callus of plants treated with different combinations of ba and iba. conclusion: the results of the present work indicate that alterations of endogenous phytohormonal levels, caused by exogenous plant growth regulators treatment, might be the mediator between primary and secondary metabolism by means of affecting protein levels and activity of key enzymes in vitro. three different additives (( . % (v/v); formic acid, acetic acid, ammonium format with formic acid) have been investigated in response to ion intensity of esi-ms for individual hnp - in saliva. kinetex v r column separation efficiency was evaluated using two different column dimensions ( x . mm and x mm.) and two different stationary phases (c and c ). kinetex v r column (homogenous porous shell) performance was also compared to new ultra ace v r (encapsulated bonded phase) column. sample optimisation revealed that the spe method removes interference from salivary glycoproteins and consequently yields larger peak area ( - %) for all hnps. hnps were extracted by spe with a recovery of - %. the meoh: h o: acetic acid ( . %) provided enhanced (p> . ) hnp - ion intensities. the kinetex v r c ( x . mm, . mm) column facilitated a better separation efficiency of the four hnps as compared to the ultra core super c ace v r ( x . mm, mm) column, the kinetex v r c ( x . mm, . mm) and the kinetex v r c ( x . mm, mm) column. the relative levels of the hnps were determined in healthy volunteers before and after a rigorous exercise regime: it is possible that prolonged strenuous exercise will affect oral innate immunity and therefore also the level of salivary defensins. hnp - are traditionally detected in an enzyme-linked immunosorbent assay (elisa) which does not discriminate between the different hnps due to their structural similarities. there has therefore been a need to develop a mass spectrometry method that will discriminate between the defensins. as part of the method validation, the hnp - level was determined by elisa and the data was compared with the lc-ms data. here we present this cross-validation; the data revealed no significance difference between the two methods (r . ) which confirms that the developed lc-ms method is and equal sensitive method for the detection of these potential antimicrobial markers. this method can easily be adopted for similar molecular weight of peptides as hnps and also for any other biological matrix. moonlighting proteins: relevance for biotechnology and biomedicine luis franco serrano , sergio hern andez , alejandra calvo , gabriela ferragut , isaac amela , juan cedano , enrique querol institut de biotecnologia i biomedicina. universitat aut onoma de barcelona, laboratorio de inmunolog ıa, universidad de la rep ublica regional norte-salto multitasking or moonlighting is the capability of some proteins to execute two or more biochemical functions. the identification of moonlighting proteins could be useful for researchers in the functional annotation of new genomes. moreover, the interpretation of knockout experiments, in which the result of a gene knocking does not produce the expected results, might be enhanced. the action of a drug can also be facilitated because it might have an off-target or side effect with somewhat hidden phenotypic traits. it would be helpful that bioinformatics could predict this multifunctionality. in the present work, we analyse and describe several approaches that use protein sequences, structures, interactomics and current bioinformatics algorithms and programs to try to overcome this problem. among these approaches there are: a) remote homology searches using psi-blast, b) detection of functional motifs and domains, c) analysis of data obtained of protein-protein interaction databases (ppis), d) matches of the sequence of the query protein to d databases (i.e., algorithms like pisite), e) mutation correlation analysis between amino acids using algorithms like mistic. remote homology searches using psi-blast combined with data obtained from interactomics databases (ppis) have the best performance. structural information and mutation correlation analysis can help us to map the functional sites. mutation correlation analysis can only be used in very specific situations because it requires the existence of a multialigned family of protein sequences, but it can suggest how the evolutionary process of second function acquisition took place. we have designed a database of moonlighting proteins, multitaskprotdb (http:// wallace.uab.es/multitask/). from this database we determine the frequencies of canonical and moonlighting coupled functions (being an enzyme and a transcription factor the highest), the percentage of moonlighting proteins involved in human diseases ( % of the human moonlighting proteins in the database) and the percentage of moonlighting proteins acting as a pathogen virulence factor ( % of the moonlighting proteins in the database). correlation between potential human neutrophil antimicrobial peptides (hnp - ) and stress hormones in human saliva nadia ashrafi , frank pullen , birthe nielse , cris lapthorn , fernando naclario university of greenwich (faculty of engineering and sciene), university of greenwich (centre of sports science and human performance) numerous studies have investigated the effect of exercise on mucosal immunity but the focus has mainly been on salivary immunoglobulins lysozymes and hormones (cortisol, testosterone). this is not surprising given that iga and igg are the predominant immunoglobulins in saliva and there is a relationship between mucosal immunity and upper respiratory illness. it is well known that physical and mental stress provoke the release of cortisol from hypothalamic pituitary adrenal axis, by which stress can modulate various immune responses. in general, cortisol and growth hormones helps to induce the activation of neutrophils. to date, this study represents the first study that investigated the correlation between human neutrophil alpha defensins family against cortisol (stress hormone) and testosterone (growth hormone) in human saliva before and after exercise or training. twelve resistance trained athletes volunteered to participate in the study. participants consumed supplements during exercise and the hnp - , cortisol and testosterone response was investigated pre, post and minutes of the workout. the correlation between salivary antimicrobial peptide (hnp - ) and stress hormone (cortisol and testosterone) has been investigated using elisa. cortisol showed no significant (p . ) difference for (pre to min post) between cho and pl (cho: . . ng/ml; pl . . ng/ml) conditions but a strong trend (p . ) was observed for (pre to min post) post (cho: . . ng/ml; pl . . ng/ml) condition. testosterone showed no significant (p . ; p . ) difference for (pre to min post) between cho and pl (cho: . . ng/ml; pl . . ng/ml) and for (pre to min post) post (cho: . . ng/ml; pl . . ng/ml) condition. hnp - showed no significant (p . ) difference for (pre to min post) between cho and pl (cho: . . ; pl . . ) conditions but significant difference (p . ) was observed for (pre to min post) between cho and pl (cho: . . ; pl . . ) condition. the present findings suggested that there is no correlation between salivary hnp - and cortisol for (pl: r . and cho: r . ); hnp - and testosterone (pl: r . and cho: r . ). a worth note from previous study which suggested that using murine skin model (an increase in endogenous glucorticoids (cortisol) by physiological stress reduced mrna levels of antimicrobial peptide (cathelicidin). it is not clear that the correlation between hormones and antimicrobial peptide has been affected by the time interval of the exercise. both cortisol and antimicrobial peptide demonstrated a transient increase after exercise but it is surprising that they are not correlate to each other. one of the hypothesis from the present finding could be cortisol responses slow and it will be interesting to do further research with longer interval. the second hypothesis demands a further investigation to determine the synergism between substances. school of biomolecular and biomedical science, conway institute, ucd., king saud university, sciences, biochemistry department. the crystal structure of a human glucose -phosphate dehydrogenase (g pd) shows that each subunit has two nadp sites; in addition to a catalytic site there is a "structural" site which is distant from the catalytic coenzyme site. mutations causing severe deficiency tend to cluster round and close to the dimer interface and the structural nadp , indicating that the integrity of these areas is important for enzyme stability and therefore for maintenance of activity. in order to understand the molecular basis of g pd deficiency, and to have a clearer indication about the role of some features of the threedimensional structure, a fuller study of the second, "structural" nadp binding site is needed. human g pd controls the first committed step in the pentose phosphate pathway. it catalyses the oxidation of glucose -phosphate to gluconolactone -phosphate, generating nadph which is essential, amongst other things, for protection against oxidative stress. the human enzyme can be active in dimer or tetramer forms. human g pd of "structural" nadp per subunit of enzyme. this tightly-bound nadp can be reduced by g p, probably following migration to the catalytic site. the importance of nadp for stability is explained by the structural nadp site, which is not conserved in prokaryotes. after removing the tightly bound "structural" nadp the enzyme is still active but not stable. the effects of different nadp fragments on the stability of human recombinant g pd have been investigated. nadp is crucial for the long term stability of human g pd, and only one of nadp analogues which is adenosine diphosphate ribose - '-phosphate was able to slightly promote the stability of enzyme. . molecular characterization of specific positively selected sites in mammalian visual pigment evolution miguel a. fern andez-sampedro , eva ramon , brandon m. invergo , jaume bertranpetit , pere garriga grup de biotecnologia molecular i industrial., visual rhodopsin is a member of the g-protein coupled receptors superfamily. this membrane protein consists of a -cis-retinal cromophore bound to a seven transmembrane protein, opsin, by means of a protonated schiff base linkage. it has an important role as a dim light photoreceptor in the retina of the eye. by statistical models, where episodic selection in rhodopsin is tested on one branch of the phylogeny against a background of neutral or purifying selection on the rest of the tree, we have found some significant evidence of specific positively selected sites in early mammalian divergence. we have chosen the three amino acid sites identified with the highest posterior probability of having been targets of positive selection to perform experimental studies, i.e. (positively selected from m to f), (positively selected from r to q) and (positively selected from s to a). we have constructed, expressed, immunopurified and functionally characterized the proposed candidates, f m, q r and a s rhodopsin mutants located at the n-terminus, the transmembrane domain and the c-terminus region of the protein respectively. from the analysis of the molecular features of the f m mutant, we conclude that position is very important for protein folding and also for proper protein glycosylation, since we only could observe cromophore regeneration after its rescue in the double cysteine (n c/ d c) mutant background that stabilizes the n-terminal extracellular domain of the protein. our results also show that mutants q r and a s alter the g-protein activation rate, and hydroxylamine susceptibility in the dark-adapted state. in the case of q r, disrupting critical interactions with the neighbouring y of the conserved d/ery motif, critical in gt activation, could cause the lower gt activation ability. the mutant a s would create a potential additional phosphorylation site in the protein which could affect rhodopsin phosphorylation after photoactivation and, in turn, could affect the binding affinity of arrestin, a regulator of rhodopsin deactivation. this extra phosphorylation site could provide an evolutionary explanation for the enhanced response observed in the case of gt activation. in conclusion, these results highlight the importance of molecular investigations of positive selected sites in rhodopsin evolution and the relevance of structural and functional analysis of these sites in unravelling the molecular basis of visual pigment evolution. natural evolution sheds light on modern drug resistance in protein kinases marc hoemberger , christopher wilson , roman agafonov , dorothee kern the anti-cancer drug imatinib exhibits highly specific binding to the human kinase and oncogene abl with a three thousand fold weaker affinity for the structurally and functionally very similar kinase src. it has been shown recently that the major difference in binding of imatinib to abl and src stems from an induced fit after binding of the drug. to further understand the mechanism of imatinib binding to its target we used ancestral sequence reconstruction (asr) and resurrected enzymes along the node from the common ancestor of abl and src up to the extant kinases. we show that imatinib affinity is gained towards the evolution of extant abl while it is lost towards evolving src. the combination of asr and crystallographic data of the ancestors in addition to kinetics data allowed us to identify a subset of residues involved in imatinib specificity sufficient to switch from an intermediate binder to a tight binder. preliminary data shows that a network of hydrogen bonds and packing interactions stabilize the kinked p-loop conformation for tight binders thus allowing for more interactions between the kinase and the drug. strikingly, many of these residues were identified in human cancer patients as "hot spots" for the development of resistance mutations. further investigation into the identified subset of residues in combination with these commonly found imatinib resistance mutations will allow us to understand emerging drug resistances better. an evolutionary view of the cold adapted catalysis of enzymes vy nguyen , christopher wilson , dorothee kern the diversity in protein function that we see today arose as a result of life adapting to a cooling earth. how did enzymes, the catalysts of many crucial cellular processes, achieve this cold adaptation? this is a challenging question to answer because ancient sequences of proteins that existed billions of years ago are not available. to address this question we used ancestral sequence reconstruction to create adenylate kinase (adk) enzymes from the divergence of anaerobic and aerobic firmicutes towards modern day thermophilic, mesophilic and psychrophilic organisms. adk is a phosphotransferase that catalyzes the conversion of two adp molecules into atp and amp. we make the following observations. first, all ancestral enzymes are active with optimal catalytic rates linearly corresponding to the temperature of the environments where these proteins would have been found. most strikingly, the catalytic rate of our oldest adk ancestor exhibits a higher enthalpy of activation at low temperatures as compared to the modern thermophilic adk. this suggests a large enthalpic penalty had to be paid for reactions to occur at cold temperatures in an ancestor that existed in a hot environment. second, several high resolution crystal structures of extant proteins that we solved ( . Å - . Å), show that the oldest ancestors were more rigid than the modern adks due to an intricate salt-bridge network. this work, thus shows for the first time, the molecular and thermodynamic determinants of cold adaptation in an enzyme over a time period that spans billions of years. induced oxidative modification of plasma and cellular fibrin-stabilizing factor anna bychkova , tatiana danilova , alexander shchegolikhin , vera leonova , marina biryukova , elizaveta kostanova , alexey kononikhin , anna bugrova , evgeny nikolaev , mark rosenfeld n. m. emanuel institute of biochemical physics, russian academy of sciences, institute for energy problems of chemical physics, russian academy of sciences the main function of plasma fibrin-stabilizing factor pfxiii is to catalyze the formation of the intermolecular covalent cross-links between both gand afibrin polypeptide chains. the crosslinking crucially affects mechanical strength of fibrin and its resistance against fibrinolysis. the precise role of cellular fibrin-stabilizing factor cfxiii remains poorly understood. pfxiii is a heterotetramer (fxiii-a b ) consisting of two single-stranded catalytic a subunits (fxiii-a ), and two identical single-stranded inhibitory/ carrier b subunits (fxiii-b ). the subunits are held together by weak non-covalent bonds. contrary to plasma fxiii, cfxiii is a dimer (fxiii-a ) devoid of b subunits. as well as many other proteins circulating in the bloodstream, pfxiii is known to be a target for reactive oxygen species (ros) causing processes of protein oxidative modification. since the conversion of pfxiii to the active form of the enzyme (fxiiia) is a multistage process, ozone-induced oxidation of pfxiii has been investigated at different stages of its enzyme activation. the biochemical results point to an inhibition of enzymatic fxiiia activity depending largely on the stage of the pfxiii conversion into fxiiia at which oxidation was carried out. uv-, ftir-and raman spectroscopy demonstrated that chemical transformation of cyclic, nh, sh and s-s groups mainly determines the oxidation of amino acid residues of pfxiii polypeptide chains. conversion of pfxiii to fxiiia proved to increase protein susceptibility to oxidation in the order: pfxiii < pf-xiii activated by thrombin < pfxiii in the presence of calcium ions < fxiiia. with the aid of massspectrometry it has been demonstrated that oxidation leads to decreasing fxiii-a and fxiii-b coverage both in the forms of zymogen and in the presence of calcium ions. a group of amino acid residues involved in oxidation modification of pfxiii is identified in this study. the oxidation of either cfxiii or cfxiiia has revealed an almost complete loss of enzyme activity caused by dramatic changes in the primary and secondary structure of the proteins detected by the ftir data. taking into account these new findings, it seems reasonable to assume that the inhibitory/carrier fxiii-b subunits can serve as scavengers of ros. hypothetically, this mechanism could help to protect the key amino acid residues of the fxiii-a subunits responsible for the enzymatic function of fxiiia. the study was supported by rfbr, research project no. - - - a. mass spectrometry study was supported by the russian scientific foundation grant no. - - . performance and quality. making microcalorimetry simple with microcal peaq-itc natalia markova , ronan o'brien , mark arsenault microcal, malvern instruments ltd. dynamic interactions involving biomolecules drive and regulate all biological processes. studies of biomolecular interactions are fundamentally important in all areas of life sciences. data provided by isothermal titration calorimetry (itc) enables scientists in academia and industry to directly and quantitatively characterize these interactions in solution. microcal peaq-itc, the latest generation of microcal itc instrumentation, offers a whole range of solutions for addressing current bottlenecks associated with interaction analysis. among the most recognized challenges are the needs to adequately address a broad range of binding affinities and to reliably interpret binding data complicated by the presence of inactive protein fraction or inherent uncertainty in the concentration of a ligand. consistently high performance of microcal peaq-itc enables increased confidence and data resolution when measuring low heats at low or uncertain sample concentrations and complex binding modes. the new microcal peaq-itc analysis software allows for utomated data analysis, minimizing analysis time and user subjectivity in assessing data quality. data quality is determined and advanced fitting performed in a few seconds per experiment allowing for analysis of large data sets of or more experiments in a matter of seconds. glutamine-rich activation domain of transcription factor sp -biochemical activity and structure jun kuwahara , chisana uwatoko , emi hibino , katsumi matsuzaki , masaru hoshino faculty of pharmaceutical sciences, doshisha women's university, graduate school of pharmaceutical sciences, kyoto university transcription factor sp is ubiquitously expressed in a mammalian cell, activates reasonably large subset of mammalian genes, and is involved in the early development of an organism. the protein comprises two glutamine-rich (q-rich) regions (a and b domains) located in its n-terminal half, while three tandem repeats of c h zinc finger motif at its c-terminus binds directly to a gc-rich element (gc box) of dna. in general, q-rich domain is one of the typical motifs found in trans-activation domain of transcription factors together with acidic and proline-rich domains. transcriptional signal of sp are transmitted via interaction between q-rich domains of sp and different classes of nuclear proteins, such as tata-binding protein (tbp) associated factors (tafs) in components of basic transcription factor complexes (tfii). in addition, self-association of sp via q-rich domains is also important for its regulation of transcriptional activity. it has been considered that an sp ! molecule bound to a 'distal' gc-box synergistically interacts with another sp molecule at a 'proximal' binding site. although formation of multimers via q-rich domains seems functionally important for sp , little is known about relevance between biological activity and structural nature of q-rich domains. we analyzed nature of glutaminerich domains of sp by biochemical and physicochemical methods. we found that q-rich domains do not have clear secondary structure whereas they can indicate biochemical activity. detailed analysis of nmr spectra indicated interaction between the domains. the q-rich domains of sp might be one of the intrinsically disordered proteins (idp). chipping away at the yeast proteome: redesigning an e ubiquitin ligase for targeted protein degradation michael hinrichsen , lynne regan one of the central goals of synthetic biology is to exploit biological systems in order to produce compounds of therapeutic or industrial value . often, these efforts are complicated by the many natural biochemical pathways in cells that can compete for the same small molecule precursors. currently, the most common solution is to simply delete the genes coding for the competing enzymes . while such an approach has been successful, it is only applicable to nonessential genes and can produce unintended off-target effects such as decreased cell viability . an alternative strategy is to instead target proteins directly for degradation. using this strategy, scientists would first grow cultures of engineered cells to high densities under permissive conditions (i.e. targeted proteins are stably expressed). then, once sufficient cell density has been reached, enzymes of competing pathways would be rapidly degraded, resulting in the rapid production of high concentrations of the compound of interest. we propose to create such a tool by reengineering the c-terminus of hsp interacting protein (chip), an e ubiquitin ligase. chip recognizes substrate proteins through a short c-terminal peptide tag on target proteins . we have shown that fusing this tag to non-native substrates is sufficient for ubiquitination in vitro (data not published). cellular assays have also been performed in s. cerevisiae, a model organism commonly used in metabolic engineering applications . as a number of native yeast proteins possess c-termini similar to that of chip's native substrates (data not published), it was necessary to develop an orthogonal chip-peptide pair. this was achieved by replacing chip's natural tpr ligand-binding domain with a ligand-binding domain engineered previously in the regan lab . the altered chip construct has been shown to be active both in vitro and in vivo, and produces an altered growth phenotype when targeted against an enzyme involved in uracil biosynthesis. future work will focus on further kinetic characterization of the engineered enzyme, increasing its activity, and introducing the system into a proof of concept synthetic biology application. advances in modern sequencing techniques have resulted in an explosion of genomic data. correctly classifying this new wealth of information can be daunting not only because of the sheer volume of sequence data, but also because the propagation of erroneous and less-than-ideal names and functional characterizations in the current databases gets in the way of functional classification by mere sequence similarity. we are investigating the extent to which protein domain architecture can be utilized to define groups of proteins with similarities in molecular function, and whether we can derive corresponding functional "labels", starting with some of the most common domain architectures found in bacteria. to this end, we have developed an in-house procedure called sparcle ('specific architecture labeling engine') that lets us track and examine specific or sub-family domain architectures, resulting from annotating protein sequences with domain footprints provided by the conserved domain database (cdd), which includes hierarchical classifications for many common domain families. we will discuss how the proteins are grouped into specific architectures, our successes in assigning functional labels, and the major limitations we have encountered to date. while we will be able to assign functional labels to a large fraction of protein models derived from genome sequences, this effort has the added benefit of pointing out insufficient coverage and resolution of the current protein domain model collections that constitute cdd. we will also discuss alternative procedures that utilize pre-computed domain annotation for clustering protein sequences at a level that is well suited for functional labeling. we hope that this preliminary study will help to identify approaches that facilitate rapid and accurate annotation of genomes with a minimum of manual intervention. pegylated amyloid peptide nanocontainer delivery and release system self-assembly of telechelic peg end-capped with hydrophobic dipeptides collagen stimulating effect of peptide amphiphile c -kttks on human fibroblasts self-assembly of palmitoyl lipopeptides used in skin care products bioactive films produced from selfassembling peptide amphiphiles as versatile substrates for tuning cell adhesion and tissue architecture in serum-free conditions influence of elastase on alanine-rich peptide hydrogels interaction between a cationic surfactant-like peptide and lipid vesicles and its relationship to antimicrobial activity self-assembled arginine-coated peptide nanosheets in water toll-like receptor agonist lipopeptides self-assemble into distinct nanostructures approved drugs containing thiols as inhibitors of metallo-ß-lactamases: a strategy to combat multidrug-resistant bacteria references leukotriene a hydrolase -an envolving target. inflammatory diseases -immunopathology, clinical and pharmacological bases the bifunctional enzyme leukotriene-a, hydrolase is an arginine aminopeptidase of high efficiency and specificity lipoxygenase and leukotriene pathways: biochemistry, biology, and roles in disease a critical role for lta h in limiting chronic pulmonary this work was supported by the czech science foundation (project p / / ) and czech academy of sciences the tn antigen-structural simplicity and biological complexity bel b-trefoil: a novel lectin with antineoplastic properties in king bolete (boletus edulis) mushrooms acknowledgements: cynthia leyva-arg€ uelles is supported by a personal grant from conacyt, mexico. this work is supported by conacyt grant ' : and papiit grant in sequence information-based deciphering of biofunctionalities using ism-based techniques has fetched calculation of biological functionalities, designing of biomedical device called computer-aided drug resistance calculator, the understanding of the mechanism of hiv progression to aids [ ], and others. they have compared the efficacies of drugs and vaccines, which formed the basis for the innocentive award (id ) for assessing vaccine potency. conclusions: deciphering biological features without engaging reagents, equipments and animal tissues but biological data such as sequence information is one novel, feasible genotypic hiv-coreceptor tropism prediction with geno pheno [coreceptor]: differences depending on hiv- subtype a reliable phenotype predictor for human immunodeficiency virus type subtype c based on envelope v sequences available: http:// istree.bioprotection.org signal processing-based bioinformatics methods for characterization and identification of bio-functionalities of proteins an empirical framework for binary interactome mapping estimating the size of the human interactome coming to peace with protein complexes? th capri evaluation meeting pj- cabs-dock web server for protein-peptide docking with significant conformational changes and without prior knowledge of the binding site while other docking algorithms require pre-defined localization of the binding site, cabs-dock doesn't require such knowledge. given a protein receptor structure and a peptide sequence (and starting from random conformations and positions of the peptide), cabs-dock performs simulation search for the binding site allowing for full flexibility of the peptide and small fluctuations of the receptor backbone cabs-flex: server for fast simulation of protein structure fluctuations cabs-fold: server for the de novo and consensus-based prediction of protein structure cabs-dock web server for the flexible docking of peptides to proteins without prior knowledge of the binding site mechanism of folding and binding of an intrinsically disordered protein as revealed by ab initio simulations modeling of protein-peptide interactions using the cabs-dock web server for binding site search and flexible docking cabs-fold: server for the de novo and consensus-based prediction of protein structure cabs-flex: server for fast simulation of protein structure fluctuations aggrescan d (a d): server for prediction of aggregation properties of protein structures cabs-dock web server for the flexible docking of peptides to proteins without prior knowledge of the binding site staphylococcal pathogenicity island dna packaging system involving cos-site packaging and phage-encoded hnh endonucleases the etiology of asdis unknown, but it is believed that it involves genetic and environmental components. the purpose of this work is to assess the possible involvement of food contaminants, such as mycotoxins, in the etiology of asd. the hypothesis is that the mycotoxins ingested with the diet could bind to proteins and expose the entire organism,including cns, to the negative effects of xenobiotics, in genetically predisposed patients. in this study some possible protein targets for the mycotoxinswere identified to evaluate if the bond between any protein target and the mycotoxin in exam could play a role in asd. twelve mycotoxins were selected (ochratoxin a, gliotoxin, aflatoxin b , aflatoxin b , aflatoxin m , aflatoxin m , aflatoxicol, a-zearalanol, b-zeralanol, zearalenone, deoxynivalenol, patulin),which are contaminants of milk and cereals.for each of these molecules,possible protein targets were searched by a reverse docking approach using the idtargetserver[ ].from the results given by idtarget, human protein targets expressed in the brain or involved in brain diseaseswere selected. subsequently, a direct docking was made using auto-dock . [ ], in orderto verify the strength of the interaction between selected proteins and each mycotoxin, and to identify the mycotoxins' binding site on each of the selected protein. finally, the bond of some mycotoxins to selected protein targets has been experimentally tested. for each mycotoxin, idtarget returned thousands of possible protein targets,and only those with the best binding energy were selected and evaluated. among them, human protein targets that are expressed in the brain or that are involved in cerebral diseases,have been selected; moreover the protein targets that were not human but that idtargetselected for five or more mycotoxins, were replaced with their human counterparts. at the end of the procedure, nineteen protein targets have been identified for the following direct docking approach. from the docking results, eight proteins have been selected for experimental tests, having a predicted binding energy lower than kcal/mol. finally, the interactions between acetylcholinesterase (ache), b-secretase (bace ) and neuroligin- , x-linked (nlg x) with aflatoxin b , aflatoxin b , gliotoxin, ochratoxin a and deoxynivalenol, were evaluatedusing fluorescence spectroscopy and microscale thermophoresis. these experiments confirmed the presence of an interaction between bace and aflatoxin b idtarget: a web server for identifying protein targets of small chemical molecules with robust scoring functions and a divide-and-conquer docking approach the calculation of spatial structure and "assembling" of the whole protein from the obtained peptide structures were performed by using molecular dynamics of the protein in the fully hydrated -palmitoyl- -oleoyl-sn-glycero- -phosphatidylcholine (popc) [ ]. the obtained structural model may contribute to identification of ul . active sites and elucidation of its mode of action nmr structural studies of membrane proteins acknowledgments: polish national centre for research and development -grant number pl- functional and mechanistic studies of dysferlin, an essential protein in cell membrane repair references moreover the 'm' parameter, which represents the denaturant effect on the protein stability, is cal•mol- for bgla and cal•mol- for bglb albert einstein college of medicine protein structure modeling, protein-protein interaction computational modeling of ini /smarcb and novel insights into its interaction with hiv- integrase savita bhutoria epsteain bar virus, nuclear antigen) . ini /smarcb has no known structural homologues, and its amino-acid sequence yields little insight into its function. a detailed understanding of structure-function relationships is hampered by the lack of structural information for ini . computational methods that model protein/peptide structures with sufficient accuracy to facilitate functional studies have had notable successes. we carried out combination of sequence analysis ab initio structure modeling and dynamics studies of integrase binding domain of ini and found it to be similar to that of phospholipase a activating protein, plaa. structural similarity with this distant protein suggests divergent evolution of the two proteins. the modeled structure sheds light on various protein-protein interactions of ini . by integrating the experimental studies about the binding, we have shown through docking, how a fragment of ini binds to the hiv- in. molecular docking and experimental studies indicated that two proteins bind tightly through charged/polar residues surrounding a hydrophobic cleft. these studies provide first modeled structure of ini /smarcb or any component of the swi/snf complex, and provide structural basis for in-ini interactions. this molecular interpretation of the intermolecular interactions is expected to facilitate design of inhibitors as novel class of anti-hiv- therapeutic agents ): e . with their catalytic activity towards rna substrates, other biological properties have been reported and evolution studies suggest an ancestral host-defence function in vertebrates. indeed, genetic studies confirmed a rapid molecular evolution within the family, a distinctive trait for host defence proteins exposed to a changing pathogen environment. previous studies from our laboratory characterized the wide spectra antimicrobial activity of two highly cationic human rnases: the eosinophil rnase and the skin derived rnase ribonucleases and have antimicrobial function in the human and murine urinary tract structural determinants of the eosinophil cationic protein antimicrobial activity two human host defense ribonucleases against mycobacteria, the eosinophil cationic protein (rnase ) and rnase the regulatory mechanism that we are reporting will contribute to prevent both nad exhaustion and accumulation of the toxic bal. to the best of our knowledge, this is the first report of a novel reversible covalent modification of an aldh enzyme involving its own substrate anna lewandrowska , aldona jeli nska , agnieszka wi sniewska acknowledgement: this research was supported by the inactivation of the fxr gene reduces aqp expression and impairs urine concentrating ability, which leads to a polyuria or urine dilution phenotype. we have previously found that pon -/-mice exhibit a polyuria phenotype and produce twice as much -h urine as their wild type pon / littermates (borowczyk k et al. metabolism and neurotoxicity of homocysteine thiolactone in mice: evidence for a protective role of paraoxonase development and application of novel non-ewald methods for calculating electrostatic interactions in molecular simulations ikuo fukuda , narutoshi kamiya the most time-consuming part of molecular simulation is the calculation of long-range interactions of the particles. in particular, appropriate treatment of the electrostatic interaction is critical, since the simple truncation cannot be used due to the slow decay of the coulombic function. thus, it is highly demanded to calculate the electrostatic interactions with high accuracy and low computational cost. for this purpose we have developed the zero-multipole (zm) summation method [ ]. in this method the artificial periodic boundary conditions are not necessary and the fourier part evaluations are not needed, in contrast to the conventional ewald-based methods. instead, a pairwise function that is suitably redefined from the coulombic function is used with a cutoff scheme. the underling physical idea is simple: (a) in a biological system, a particle conformation for which the electrostatic interactions are well cancelled is more stable than other conformations [ ]; (b) since such well-cancelled conformations are essentially physical, we should clip a subset of such a conformation out of the conformation within an ad-hoc given cutoff sphere and calculate the interactions only from this subset. this idea is realized by a rigid mathematical consideration that leads to the deformation of the coulombic function. the efficiency of the zm method has been validated in applications to fundamental systems sema a) is a protein originally described as an axonal chemorepellent cue involved in many physiological processes ranging from embryonic development to bone homeostasis or immune responses sema a signal transduction requires the formation of a heteromeric complex with neuropilin- (nrp ) and plexina [ ]. in addition, sema a interaction with nrp is modulated by the furin protease cleavage at its c-terminal basic domain this c-terminal basic domain has also been suggested to mediate the binding to glycosaminoglycans (gags), an association that locates sema a to perineuronal nets and enhances its function in restricting neuronal plasticity and inhibiting axonal regeneration in the central nervous system two peptides corresponding to the highly positively charged regions on the domain were shown to bind to immobilized heparin by surface plasmon resonance (spr) and the affinity dramatically increased when the complete domain was assayed. the binding was confirmed by nuclear magnetic resonance (nmr) and circular dichroism (cd) the conserved cysteine within this motif, necessary for the dimerization of sema a [ ], is also critical for the helix formation. in addition, fluorescence spectroscopy studies showed that the n-terminal region also has a contribution in the binding to gags. we acknowledge the financial support from the european union seventh framework programme (fp / - ) under the project vision semaphorin a: a new player in bone remodeling neuropilins lock secreted semaphorins onto plexins in a ternary signaling complex furin processing of semaphorin f determines its anti-angiogenic activity by regulating direct binding and competition for neuropilin semaphorin a displays a punctate distribution on the surface of neuronal cells and interacts with proteoglycans in the extracellular matrix semaphorin a binds to the perineuronal nets via chondroitin sulfate type e motifs in rodent brains mechanistic basis for the potent anti-angiogenic activity of semaphorin f. biochemistry collapsin- covalently dimerizes, and dimerization is necessary for collapsing activity prior attempts to create functionally relevant groupings of proteins in the crotonase superfamily suggest that this superfamily is difficult to cluster functionally due in part to the functionally diverse nature of the protein superfamily. we have developed two novel procedures to combat this difficulty: tulip (two-level iterative clustering process), a process that utilizes structural information from active sites to cluster protein structures into hypothesized functional groupings, and misst (multi-level iterative sequence searching technique), a process that uses the protein groupings created in tulip as a starting point for iterative genbank searches and further clustering after each search. through these two methods, the total coverage of the crotonase superfamily has increased, and the generated groups contain proteins from subgroups and families that did not have a structural representative. novel hypothesized functional protein groupings have been created, most notably for a large number of proteins that lack annotation data at the subgroup or family level, and for proteins of the enoyl-coa hydratase family fernandes , teresa sorbo , ivan duka , lia christina appold , marianne ilbert , fabian kiessling cnrs, umr , ucibio-requimte, faculdade de ciências e tecnologia e-selectin is a cell-adhesion molecule induced on the surface of endothelial cells in response to cytokines. its upregulation has been reported in many disorders, including inflammatory and cardiovascular diseases, tumor angiogenesis and metastasis [ ]. this profile suggests e-selectin as a promising target to develop molecular imaging probes for the detection of these diseases cyt c with equivalent of fld in media mimicking the cytoplasm. these include % polyacrylamide gel, g/l bovine serum albumin or polyvinylpyrrolidone , and buffer alone for comparison. electrostatic surface representations of the proteins are shown with their in-cell spectrum pl- a search for anti-melioidosis drug candidates targeted to d-glycero-d-manno-heptose- , -bisphosphate phosphatase from burkholderia pseudomallei bpgmhb converts dglycero-d-manno-heptose- b, -bisphosphate to d-glycero-d-manno-heptose- b-phosphate. this is the third step of the biosynthesis pathway of ndp-heptose responsible for a pleiotropic phenotype. therefore, this biosynthesis pathway is the target for inhibitors increasing the membrane permeability of gram-negative pathogens or adjuvants synergistically working with known antibiotics. to find inhibitors of bpgmhb, we performed homology modeling of bpgmhb and in-silico virtual screening with zinc, a free database of commerciallyavailable compounds. tens of thousands of chemical compounds were docked into the active site of bpgmhb. a number of putative bpgmhb binding compounds better than d-glycero-d-manno-heptose- b, -bisphosphate were found using surflex-dock included in the sybyl software package crystal structure of dimeric d-glycero-d-manno-heptose- , -bisphosphate phosphatase from burkholderia thailandensis ewha womans university we have solved the crystal structures of d-glycero-d-manno-heptose- , -bisphosphate phosphatase from burkholderia thailandensis (btgmhb) catalyzing the removal of the phosphate at the position of d-glycero-d-manno-heptose- , -bisphosphate. it belongs to the haloacid dehalogenase (had) superfamily with an a/b rossman fold composed of six parallel b-strands sandwiched between two sets of three a-helices it reveals a conventional rossman-like a-b-a sandwich fold with a novel b-sheet topology. its c-terminus is longer than its closest relatives and forms an additional b-strand whereas the shorter c-terminus is random coils in the relatives. interestingly, its core structure is similar to that of enzyme iib(cellobiose) from e. coli (eciib(cel)) transferring a phosphate moiety. in the active site of the closest eceiib(fruc) homologues, a unique motif cxxgxaht comprising a p-loop like architecture including a histidine residue is found. the conserved cysteine on this loop may be thiolated to act as a nucleophile similar to that of eciib(cel). the conserved histidine residue is presumed to accommodate negatively charged phosphate during enzymatic catalysis leonor morgado , kornelius zeth , , , bj€ orn m. burmann , timm maier bama is a b-barrel membrane protein with five periplasmic n-terminal polypeptide transport associated (potra) domains. the bama structure has been determined recently by x-ray crystallography ( , ), however its functional mechanism is not well understood. this mechanism comprises the insertion of substrates from a dynamic, chaperone-bound state into the bacterial outer membrane, and nmr spectroscopy is thus a method of choice for its elucidation we demonstrated that knocked down autophagy by shrna (shatg , shbecn , and shatg ) and chloroquine (cq) could enhance high dose of uvb induced cell death in odc overexpressing hela and mcf- cells. here, we also observed that knocked down odc in odc overexpressing hela and mcf- cells inhibited autophagy and enhanced high dose of uvb radiation. because of atg can regulate cell apoptosis and utophagy. site directed mutagenesis was used to mutant the amino acid which can regulate cell apoptosis and autophagy on atg , respectively in these two odc overexpressing cells. according to the results fish ß-parvalbumin acquires allergenic properties by amyloid assembly using atlantic cod b-parvalbumin (rgad m ) displaying high ige crossreactivity, we have found that formation of amyloid fibers under simulated gastrointestinal conditions accounts for the resistance to acid and neutral proteases, for the presence of membrane active species at gastrointestinal relevant conditions and for the ige-recognition in allergic patient sera. incorporation of the anti-amyloid compound epigallocathequin gallate prevents rgad m fibrillation, facilitates its protease digestion and impairs its recognition by ige. conclusions: rgad m amyloid formation explains its degradation resistance, its facilitated passage across the intestinal epithelial barrier and the epitope architecture as allergen autophagy could degrade the citrullinated and unfolding protein. herein, padi could enhance autophagy in jurkat t cells and lead to a degradation of p and the accumulation of lc -ii. autophagy and apoptosis are two critical mechanisms which participate against cellular stress, cell activation, survival and homeostasis. pad -overexpressed jurkat t cells caused the activation of th cells to increase mrna expression of cytokines, such as il- , il- , il- and tnfa. cytokines provoked caspase expression and led to caspase-mediated cleavage of beclin- which was an important factor of apoptotic signaling. knockdown of bcen rescued cell survival due to the increase of bcl-xl and the decrease of caspase- . we suggested that padi participated in the activated t cell-induced autonomous death through triggering er stress pathway studies on secondary metabolites production and proteins and enzymes of in vitro cultivated artemisia alba turra and relations with some endogenous phytohormones yuliana raynova , krassimira idakieva , vaclav motyka , petre dobrev , yuliana markovska , milka todorova , antoaneta trendafilova , ljuba evstatieva switzerland aim: artemisia alba turra is an essential oil bearing shrub, characterized with great variability of the essential oil profile of wild grown plants, related to genetic, geographic and environmental factors. it was previously established that inhibition of rooting in vitro caused by cytokinin/auxin treatment affected the essential oil profile of the plant and these changes were also related to bioactive endogenous cytokinin levels in vitro ( , ) cytokinin and auxin effect on the terpenoid profile of the essential oil and morphological characteristics of shoot cultures of artemisia alba terpenoid profile of artemisia alba is related to endogenous cytokinins in vitro salivary hnp - are conventionally measured using an enzyme-linked immunosorbent assay (elisa) which does not discriminate between individual hnps due to their structural similarities. considering the biological importance of salivary human neutrophil a-defensin (hnps), there is therefore, a need to develop an analytical method that will discriminate between the defensins. an lc-ms method has been established for the separation and detection of hnp - . the method has been optimised, validated and applied to examine the relative level of hnp - in participants undertaking a circuit resistance training workout. to date, no studies have systematically investigated the effect of acute (min to hours) and chronic (days to weeks) change in salivary adefensins family before and after exercise by lc-esi-ms systems and models calorimetry showed no difference in dissociation constants at these ph values, while the binding stoichiometry is increased . fold. furthermore, the binding stoichiometry varied fold among the two alginates corresponding to their difference in average molecular weight and in addition fold higher binding affinity was found with the high as compared to the low molecular weight alginate. in conclusion, the binding stoichiometry of b-lactoglobulin with alginate increases by a factor that correlates to the average molecular weight of the alginate and also a much higher affinity was found for the high molecular weight alginate. acknowledgements: this work is supported by the danish council for the presence of mucins and other high molecular weight glycoproteins in saliva makes the direct analysis of defensins difficult. the lc-ms method was linear for concentrations of hnp- between . and ng/ml (r . ) with a lod of . ng/ml. inter and intra assay precision was . - %, respectively. saliva sample were clean up by solid phase extraction (spe) and without-solid phase extraction (wspe) . mm internal diameter column in relation to the method transfer . during lc-ms optimisation genome-wide docking database (gwidd) provides the most extensive data repository of structures and models of ppi on a genomic scale. currently, we are expanding the gwidd dataset to , ppi in , organisms, up from , ppi in organisms in the previous release. the ppi data were imported from intact and biogrid databases and were subjected to in-house modeling pipeline. gwidd current implementation contains , experimentally determined complexes, and , sequence homology and , structure homology models of complexes. the user-friendly interface offers flexible organism-specific search with advanced functions for a refined search for one or both proteins. the new gwidd version includes also a new interactive visualization screen that allows to view search results in different residue representations with the emphasis on the ppi interface refolding and activation of recombinant trypsin i from sardine fish (sardinops sagax caerulea) amyloid is detectable in human dental plaque and is produced by both clinical and laboratory strains of s. mutans, further supporting a functional role. s. mutans lacking p demonstrates residual amyloid forming properties, however, a mutant lacking sortase, the transpeptidase which covalently links p and several other proteins to the peptidoglycan cell wall, is defective in cell-associated amyloid-like properties. the objectives of this study were to identify additional amyloid forming proteins of s. mutans and to evaluate the effects of buffering conditions and ph on the ability of the identified proteins to form amyloids. a p -deficient mutant strain was grown to stationary-phase in defined minimal media, and secreted proteins from spent culture supernatants were fractionated by ion exchange chromatography. partially purified protein fractions were tested for binding of the amyloidophilic dyes congo red (cr) and thioflavin t (tht), and for characteristic birefringent properties following staining with cr and visualization under crossed polarizing filters. proteins from fractions that tested positive for amyloid-like material were separated by sds page, and identified by lc/ms. these included wapa, gbpa, gbpb, smu_ c and smu_ c. recombinant proteins were expressed in escherichia coli, and purified for confirmation and characterization of individual amyloidogenic properties in vitro. recombinant wapa and smu_ c displayed all the biophysical characteristics of amyloid, including visualization of fibrillar aggregates when viewed by transmission electron microscopy. in contrast, gbpa and smu_ c produced amorphous aggregates. wapa and smu_ c form amyloid at different ph, smu_ c under acidic conditions and wapa under neutral to basic conditions. this suggests that the prevailing environmental ph may represent different in vivo triggers for amyloid fibrillization of different s like other small gtpases, the activity of rheb is dictated by its guanine nucleotide binding states: it is active in its guanosine -triphosphate (gtp) bound form and inactive in the guanosine diphosphate (gdp)-bound form. rheb proteins play critical roles in regulating growth and cell cycle, and this effect is due to its role in regulating the insulin/tor/s k signaling pathway rheb interacts directly with fkbp and prevents its association with mtor in a gtp-dependent manner. moreover, fkbp bound to gtp-g-s, a nonhydrolyzable gtp analogon, has a much higher binding affinity for rheb than the gdp-bound form the second study contradicted both studies, since they could not detect any interaction between rheb and fkbp [ ]. to clarify whether there is an interaction and if it is nucleotide dependent, nmr monitored interaction studies were performed employing a c-terminal truncated construct of human rheb ( - rhebdct) that cannot be farnesylated and the biochemically defined binding region on fkpb (fkbp -like fkbp -bd). based on our data rhebdct -gdp does not significantly interact with fkbp -bp. n-fkbp -bd titrated , we observed a weak interaction between rhebdct bound to a gtpanalogon (gppnhp) and fkbp -bd. mapping of the observed spectral changes on the structure of rheb-gtp suggests that fkbp targets the switch region, loop - and the neighboring b-sheet region. we further analyzed the backbone dynamics of rhebdct -gdp and -gppnhp using n relaxtion data (t , t and heteronuclear noe) .based on these data the phosphorylation loop, the switch regions and the loop around residues - show increased backbone dynamics that modulated by the nucleotide binding international centre for genetic engineering and biotechnology tdp- is an rna processing protein that can form inclusions of debatable nature implicated in neurodegenerative diseases. within the putative aggregation domain, repeats of residues - can recruit endogenous tdp- into aggregates inside cells . recently, we showed that a coil to b-hairpin transition in a short peptide corresponding to tdp- residues - enables oligomerization . we have used a broad battery of biophysical experiments, including chromophore and antibody binding, electron microscopy (em), circular dichroism (cd), solution and solid-state nmr, and x-ray to shed light on the nature of these aggregates. based on these findings, structural models for tdp- ( - ) oligomers have been constructed, refined, verified, and analyzed using computational methods, ranging from docking and molecular dynamics simulations to semiempirical quantum mechanics calculations. interestingly, tdp- ( - ) b-hairpins assemble into a novel parallel b-turn configuration showing crossb spine, cooperative h-bonding and tight side chain packing cellular model of tar dna-binding protein (tdp- ) aggregation based on its c-terminal gln/asn-rich region structural characterization of the minimal segment of tdp- competent for aggregation structural evidence of amyloid fibril formation in the putative aggregation domain of tdp- kyungpook national university scolopendin , aglqfpvgrigrllrk, is a -mer peptide derived from the centipede scolopendra subspinipes mutilans. to investigate its property against fungal and bacterial pathogens, antimicrobial tests were performed. we observed that this peptide exhibited antimicrobial activity in a salt-dependent manner and showed no hemolysis. the circular dichroism (cd) analysis observed that a-helical structure properties. we determined the mechanism(s) of action using flow cytometry and investigated the release of potassium. the results showed that the microbial membrane in escherichia coli o and candida albicans was permeabilized with loss of potassium ions. additionally, the bis-( , -dibutylbarbituric acid) trimethine oxonol [dibac ( )] and , '-dipropylthiacarbocyanine iodide [disc ( )] assay showed membrane depolarization. using calcein-encapsulating giant unilamellar vesicles (guvs) and fitc-dextran containing large unilamellar vesicles (luvs), scolopendin disrupted the cell membrane and the damage size is between . to . nm against composition of microbial plasma membrane of e. coli and c. albicans. thus, we demonstrated that a cationic antimicrobial peptide, scolopendin , possesses broad-spectrum antimicrobial effects that formed pore on the cell membrane. structural and functional investigation of the far c-terminal domain (ctd) of the bifunctional enzyme trai using nmr spectroscopy protein structural biology structural and functional investigation of the far c-terminal domain (ctd) of the bifunctional enzyme trai using nmr spectroscopy b.krishna chaitanya, evelyne schrank and klaus zangger institute of chemistry/organic and bioorganic chemistry university of graz, austria corresponding author email id: krishna.bhattiprolu@uni-graz.at bacterial conjugation is a complex process for the horizontal transfer of single stranded dna from one cell to another. this mechanism also leads, for example, to the spread of antibiotic resistance genes and virulence factors among bacterial species. multi-protein complexes formed at the origin of transfer (orit) region of dna and at the cytoplasmic membrane of the bacterial cell, initiate this process. inside the membrane, the relaxosome identifies the single strand for transfer in a plasmid dna, relaxes and unwinds it, whereas the transferosome is involved in pilus formation (type iv secretion system) and transferring the gene through the cytoplasmic membrane. these events take place in the donor bacterial cell along with several other auxiliary proteins [ ] the bifunctional enzyme trai of plasmid r plays a crucial role in the relaxosome activity, as it contains both a relaxase and helicase domain. to exert its functions on dna, trai works in close co-ordination with other relaxosome proteins like tray, tram and the integration host factor. trai is a residual protein and contains major domains: n-terminal relaxase domain, a central helicase domain and a c terminal domain (ctd). the structure of the c-terminal domain until residue has been solved by crystallography, while the structure and function of the remaining residues remained undetermined [ ]. there are saxs models and crystallographic structures for different parts of trai and also for the full length protein. prediction of cleavage specificity in hcv ns / a serine protease and adv cysteine protease systems by biased sequence search threading gonca ozdemir isik , a.nevra ozer department of bioengineering,faculty of engineering,marmara university proteases are enzymes which recognize specific substrate sequences and catalyze the hydrolysis of designated peptide bonds to activate or degrade them. due to the biological importance of proteases, it is particularly important to identify the recognition and binding mechanisms of protease-substrate complex structures in drug development studies. the assessment of substrate specificity in protease systems is crucial, where interpreting the adaptability of substrate residue positions can be useful in understanding how inhibitors might best fit within the substrate binding sites and aid in the design of potent selective inhibitors. substrate specificity is generally determined by the amino acid profile, structural features and distinct molecular interactions. besides experimental methods, computational tools for prediction of natural substrate cleavage sites, such as threading, have emerged as useful alternative approaches to provide valuable insights into complex enzyme-substrate interactions. in this work, the substrate variability and substrate specificity of the hepatitis c virus (hcv) ns / a serine protease and the adenovirus (adv ) cysteine protease was investigated by the biased sequence search threading (bsst) methodology. using available crystal structures of the proteases, the template structures for the substrate-bound proteases were created in silico by performing various peptide building and docking procedures followed by energy minimization and molecular dynamics (md) simulations. bsst was performed starting with known binding, nonbinding and some random peptide sequences that were threaded onto the template complex structures, and low energy sequences were searched using lowresolution knowledge-based potentials. then, target sequences of yet unidentified potential substrates were predicted by statistical probability approaches applied on the low energy sequences generated. the results show that the majority of the predicted substrate positions correspond to the natural substrate sequences with conserved amino acid preferences, while some positions exhibit variability. for ns / a serine protease cleavage, the significant selection for pro at p and cys at p positions is zearalenone is a mycotoxin produced by fusarium graminearum and related fusarium species. f. graminearum is a powerful plant pathogen and infects major crop plants around the world. acute toxicity of zearalenone is low, but due to its structural similarity to b-estradiol it has binding affinity to the estrogen receptor, which results in interference with hormonal balance. typical effects seen in animals include symptoms like hyperestrogenism and reproductive disorders (reduced fertility, reduced litter size or swelling of uterus and vulva). to reduce the risk for human and animal health posed by the ingestion of contaminated food or feed different decontamination strategies have been studied, including biotransformation. today many microorganisms are known to degrade zearalenone, but for most of them the degradation pathway and formed metabolites remained unknown, hence it is unknown if this degradation also means detoxification. only for the fungal strains trichosporon mycotoxinivorans and gliocladium roseum zen degradation has been studied in detail and loss of estrogenicity of reaction products has been confirmed. we screened for, and isolated zearalenone degrading bacteria from soil samples. the most promising new bacterial isolate was taxonomically assigned to the species rhodococcus erythropolis and designated pfa d - . the zearalenone catabolism pathway of pfa d - was found to be identical as known from g. roseum. the primary reaction product, hydrolysed zearalenone, has so far only been postulated in g. roseum. we prepared hydrolysed zearalenone by preparative hplc and showed loss of estrogenicity in assays with the breast cancer cell line mcf and the estrogen reporter yeast strain yzhb . a genomic library was prepared and screened in zearalenone degradation deficient r. erythropolis pr . the gene encoding zearalenone hydrolase was found and named zena. the hydrolase was identified as member of the a/b-hydrolase family and named zena. it was cloned, recombinantly expressed in e. coli and purified by x his-tag mediated immobilised metal affinity chromatography. activity of his-tagged and untagged enzyme zena was compared in cleared lysate and zena was purified for enzyme characterisation. the influence of ph and temperature on enzyme activity and stability was evaluated and kinetic parameters were determined. a new biding site for snake venom c-type lectins?maria cristina nonato costa , ricardo augusto pereira de p adua , marco aurelio sartim , suely vilela sampaio university of são paulo, fcfrp c-type lectins are proteins that bind different glycan molecules by interactions with a calcium atom present in a carbohydrate recognition domain (crd). many organisms (plants, bacteria, virus and animals) use these proteins in various biological events like lymphocyte adhesion, erythrocyte agglutination and extracellular matrix organization. the c-type lectin fold is plastic and possible for about different sequences, what promoted its adaptation to diverse functions, similarly to the observed for the immunoglobulin fold ( - sequences). it is comprised of about - amino acid residues that folds in two fourstranded b sheets sandwiched by two alpha helices. interestingly, c-type lectins present in snake venoms are possible anti-cancer agents since they are toxic to cancer cells and inhibit the adhesion and proliferation of various cancer cell lines. therefore, we have purified a lactose binding c-type lectin from the venom of bothrops jararacussu (bjcul) to study its structure and binding properties to different sugars. bjcul crystals were obtained by vapor diffusion and the structure solved by x-ray crystallography to . Å resolution. bjcul structure is a decamer formed by a pseudo fivefold axis rotation of a dimer hold by a disulfide bond. each monomer binds a calcium atom and possibly another metal at a second and opposed binding site. key: cord- -d gcfepx authors: fan, samuel w; george, richard a; haworth, naomi l; feng, lina l; liu, jason y; wouters, merridee a title: conformational changes in redox pairs of protein structures date: - - journal: protein science doi: . /pro. sha: doc_id: cord_uid: d gcfepx disulfides are conventionally viewed as structurally stabilizing elements in proteins but emerging evidence suggests two disulfide subproteomes exist. one group mediates the well known role of structural stabilization. a second redox-active group are best known for their catalytic functions but are increasingly being recognized for their roles in regulation of protein function. redox-active disulfides are, by their very nature, more susceptible to reduction than structural disulfides; and conversely, the cys pairs that form them are more susceptible to oxidation. in this study, we searched for potentially redox-active cys pairs by scanning the protein data bank for structures of proteins in alternate redox states. the pdb contains over unique redox pairs of proteins, many of which exhibit conformational differences between alternate redox states. several classes of structural changes were observed, proteins that exhibit: disulfide oxidation following expulsion of metals such as zinc; major reorganisation of the polypeptide backbone in association with disulfide redox-activity; order/disorder transitions; and changes in quaternary structure. based on evidence gathered supporting disulfide redox activity, we propose disulfides present in alternate redox states are likely to have physiologically relevant redox activity. emerging evidence supports the concept of two distinct types of disulfides in protein structures which have different functional roles. the best known group act as structural stabilizers in proteins, significantly lowering the gibb's free energy of the protein chain by crosslinking it, resulting in proteins that are more resistant to denaturation. the second group are redoxactive and best known for their catalytic role in proteins such as thiol-disulfide oxidoreductases. an increasing role for the redox-active group in protein redox regulation is emerging. , these disulfides act as redox-sensitive switches and are best viewed as posttranslational modifications akin to phosphorylation. it has now been established that oxidative regulation: important in processes such as the cell cycle, apoptosis, and response to oxidative stress; is an important control mechanism in the cytosol. transient oxidation mediates control of enzymes, signaling pathways, as well as transcriptional and translational control. however, these characterized cytosolic proteins likely only represent the tip of the iceberg. increasingly, reduction of disulfides is also emerging as a key regulator of protein function in oxidizing environments. [ ] [ ] [ ] importantly, ageing and several major diseases including neurodegenerative diseases such as alzheimer's disease, type ii diabetes, cancer, and cardiovascular disease have been associated with abnormal redox conditions. structural and redox-active disulfides can be distinguished by their redox potentials. disulfide redox potentials measured in thiol-disulfide oxidoreductases range from À to À mv. [ ] [ ] [ ] [ ] for disulfides serving structural purposes, the redox potential can be as low as À mv. computational methods for distinguishing the two groups would be a boon. reactivity of a redox-active cys is modulated by its immediate protein environment. in addition to the effects of the electrostatic environment, physical stress applied to a disulfide bond both through stretching and twisting enhances its reactivity. these forces can be applied to a protein dynamically or may be captured during the protein folding process, rendering the disulfide poised to act upon exposure to variations in redox conditions. because of their enhanced reactivity, redoxactive disulfides are also more susceptible to cleavage/ oxidation under nonphysiologic conditions. for instance, redox-active disulfides are prone to cleavage by synchrotron radiation during the process of x-ray structure determination. [ ] [ ] [ ] other factors such as the ph of crystallization or the presence of mutations that change the electrostatic environment near a disulfide may also influence reactivity. here, we mined the protein data bank (pdb) for highly similar proteins that have been solved in multiple redox states, that is, disulfide-bonded in one structure and reduced in another. we present evidence that most of the disulfides present in our redox pairs dataset are likely to be physiologically redox-active. many of the proteins have known thiol-based redox activity, while others have a high likelihood of thiol-based redox activity based on their involvement with redox processes. we also observed and classified types of conformational changes which occur during redox transitions. the redox pairs dataset contains , pairs of protein structures consisting of protein chains, of which ( . %) are high-resolution structures (< . Å). many structures are of the same protein solved in different redox environments. a filtered dataset consists of unique protein groups clustered at % sequence identity. further evidence of disulfide redox activity was sought based on experimental data in the literature, association of protein function with known redox activity, and implication of the disulfide in redox activity by independent computational prediction methods. for proteins, direct experimental evidence of redox activity for the particular disulfide was available (des-ignated by ''k'' in table s , supporting information). the proteins belong to several major groups known to have redox activity. unique redox pairs are associated with aerobic metabolism in eukaryotes or carbon fixation in plants (in groups d and e of table s ); a further five are associated with maturation of proteins of these pathways (in groups e and g). four redox pairs are associated with ion channel activity (group a): two of these, the chloride channel clic and the detoxification enzyme arsenate reductase, have known redox activity , ; while a third, aquaporin, was recently implicated as a pore for the reactive oxygen species hydrogen peroxide, as part of the redox signaling process. seven redox pairs are associated with the cell cycle, a process known to be redox regulated (group b). formation of disulfides involving the catalytic cys in the phosphatases pr- and cdc b regulate their resident protein's function. , redox pairs (group d) are involved in transcriptional control, replication, and dna synthesis including the oxidative stress response transcription factor oxyr and ribonucleotide reductases from several species in multiple redox states. redox pairs generated or destroyed reactive oxygen species (groups f and y). the latter comprised three different peroxiredoxins as well as superoxide dismutases from several species. four different amine oxidases generate peroxide while inducible nitric oxide synthase generates no. other large groups consist of proteins devoted to cell entry figure . to further assess the likelihood that the presence of proteins in the pdb with disulfides in both redox states is an indicator of physiological disulfide redox activity, we looked for gene ontology (go) terms overrepresented in the redox pairs dataset compared to the entire pdb. go is a dynamic controlled vocabulary of over , terms used to describe molecular function, process and location of action of a protein in a generic cell. the analysis revealed many go terms consistent with redox function, including cell redox homeostasis and multiple pathways of cysteine biosynthesis (supporting information table ). interestingly, several of the enriched go terms are linked to proteins mediating cell entry, including bacterial toxins and viral entry proteins. reduction of disulfides, often accompanied by irreversible conformational changes, have previously been implicated as a general mechanism in cell entry proteins of diverse types. experimental evidence directly supporting such a general process exists for hiv gp , newcastle disease virus, sars coronavirus and chlamydia. [ ] [ ] [ ] [ ] thus the go terms support redox roles for proteins in the redox pair dataset. disulfides were tested computationally for likely redox activity by screening the dataset for oxidized structures where the disulfide had particular properties previously associated with redox activity. flagged disulfides were those (a) with high torsional energies; (b) found in known redox-active sequence motifs; or (c) which disobeyed known rules of protein stereochemistry. studies of disulfide-bonds in model proteins have shown that those bonds with high torsional energies are more easily cleaved than disulfide-bonds with lower stored energy. [ ] [ ] [ ] [ ] disulfides in proteins have torsional energies > kj mol À : a con-servative threshold associated with redox activity. these are designated by ''h'' in supporting information table . redox-pair disulfides were also found in sequence motifs previously shown to be associated with redox activity. proteins, designated by ''m'' in table s , contain known redox-active motifs such as the cxxc motif: a redox-active disulfide motif commonly found in the thioredoxin fold, but which has also arisen convergently in other folds. a further proteins, designated by ''f'' in table s , contain disulfides in protein structures that disobey known rules of protein stereochemistry. , these disulfides, which table . key to groups: a, ion channel, ion pump modifier; b, protein folding, isomerization; c, cell cycle; d, calvin cycle; e, photosynthesis, respiration, transport, maturation of proteins involved in these processes; f, destruction of oxygen radicals; g, redox metal homeostasis; h, amino acid metabolism; i, sulfur metabolism; j, nucleotide metabolism/homeostasis; k, isoprenoid biosynthesis; l, rna-interacting; m, proteases; n, dna repair; o, actin-related; p, carrier proteins; q, other metabolism; r, nadp þ -dependent oxidoreductase; s, other oxidoreductase; t, signaling; u, development; v, cell growth pathways; w, enzyme inhibitors; x, redox homeostasis; y, ros generating; z, apoptosis; a, protein fate; b, cell entry; c, cell adhesion; d, dna-interacting proteins; e, translation; f, immunity; g, carbohydrate metabolism; h, nad þ -dependent oxidoreductases; i, ca handling; j, methyltransferase; k, other. fan et al. we refer to as ''forbidden'' disulfides, conform to canonical structural motifs. deformation of the resident structure enhances disulfide reactivity by inducing stress in the protein chain. , forbidden disulfides are proposed to have a functional role through redox activity. in summary, a total of proteins in the redox pair dataset contain disulfides predicted to be redox-active by a second independent bioinformatic analysis method. nonetheless, it is possible that some proteins with alternate cys-pair redox states are non-native, that is, not encountered under normal, or abnormal, physiological conditions. we reasoned that proteins demonstrated selective reduction of disulfides were more likely to represent physiological states. accordingly, for proteins with more than one disulfide, the dataset was partitioned into pairs where all the disulfides were reduced and those where some disulfides were selectively reduced while others remained intact, to further test the likelihood of involvement in physiological redox processes. the enriched go terms for both the partially and fully reduced sets contained terms previously associated with thiol-based redox regulation indicating both the fully and partially reduced sets have likely redox activity (supporting information table ). having established the redox-pair dataset is enriched in proteins involved in redox-regulated processes, we studied conformational changes between redox pairs of proteins. for many proteins in the data set there were negligible conformational differences between the sg atoms of each cys residue of the reduced and oxidized states (see fig. ). for this group, other factors may be required for, or preclude, conformational changes in these proteins. crystal packing forces, for example, may prevent a subsequent conformational change after cleavage of the disulfide by synchrotron radiation. for another group, the small distance changes of - Å observed are most likely due to rotation of one or both cys sidechains toward each other to form the sas bond. some of the proteins exhibiting these small conformational changes are oxidoreductases with catalytic thiols such as thioredoxin, thioredoxin reductase, and protein disulfide isomerase. for proteins which exhibit major conformational changes, four classes were identified based on the nature of the change. these groups were: proteins that oxidize disulfides following expulsion of metals such as zn; proteins that exhibited major reorganization or ''morphing'' of portions of the polypeptide backbone in association with disulfide redox-activity; proteins that exhibited order/disorder transitions; and proteins that exhibited changes in quaternary structure. redox regulation of proteins that form disulfides following expulsion of zn is an emerging area of signaling. for example, reversible redox regulation of the zn-binding site has recently been proposed for small tim proteins of the mitochondrial intermembrane space (ims). small tim proteins have an essential role in the transport of proteins into the ims. these proteins can exist in both a reduced zn-bound state figure . change in disulfide separation in protein redox pairs. excluding interchain disulfides, around % of redox structure pairs have a change of over Å in the sulfur atom separation between the oxidized and reduced structures. maximal intrachain differences of up to . Å were apparent in -deoxy-d-xylulose- -phosphate reductoisomerase (dxp reductoisomerase) and the rna sulfuration enzyme trmu. and an oxidized disulfide-bridged state. both states likely represent physiological forms of the proteins which are adopted in response to changing redox conditions in the ims. in addition, expelled zn acts as a second messenger to effect further cellular responses. proteins that form disulfides following expulsion of metals such as zn were identified by searching for ligation of metals by cys in reduced structures. a total of redox pairs in proteins were found (table i , z in s ). proteins identified include transcriptional regulators such as smad and anti-trap; transcriptional silencers such as sirt ; and the enzymes of sulfur metabolism, bhmt, mete, and meth. also notable were a number of rna-interacting proteins including amino-acid-trna synthetases, rna/dna polymerases, ribonucleotide reductase and the ribosome. in addition to zn, a few other bound metal atoms were represented including fe, ni, cu. there were several examples of inorganic iron-sulfur (fe-s) clusters associated with redox-active disulfides including: xanthine dehydrogenase, a major source of ros in the failing heart; and components of respiratory complexes (table i) . other proteins containing fe-s clusters contained other redox-active disulfides that were not associated directly with the fe-s cluster. all proteins containing fe-s clusters (designated with ''c'' for cofactor) are listed in table i regardless of whether the fe-s cluster is directly associated with a labile dithiol pair or not. crystallographers typically differentiate two types of zn sites: catalytic zn sites, which in their simplest, mononuclear form have three amino-acid sidechains and one water molecule as ligands; and structural zn sites, which have four amino acid ligands. from a redox point of view, a second variable -lability, is important. some structural zn sites expel zn upon variation of physiological redox conditions, whereas others bind zn tightly, and would not be expected to release zn under physiological conditions. we refer to this first redox-regulated group as ''labile'' zn sites; and the second, purely structural group, as ''inert.' ' we wished to determine whether zn sites associated with redox pair disulfides detected in the study were labile or inert. the number of zn ligands and their identity are key indicators of zn site lability. for catalytic mononuclear zn sites, three amino acids, which are often his, ligate the zn. in binuclear zn sites there are usually six ligands, and the carboxylated residues asp and glu predominate over his. , cys is more commonly found as a ligand in structural zn sites which generally have four ligands. on consideration of ligand type alone, s sites -those with four cys ligands, are the most labile type of structural zn site, and are almost as reactive as a free thiol. most of the structural zn sites identified in this study were of the s type (table i) , suggesting they are labile. four catalytic zn sites were also found in the dataset. these catalytic zn sites are highly unusual because of their utilization of cys as a ligand instead of the more usual his, glu, or asp. two of the catalytic sites are within bhmt and meth: enzymes of sulfur metabolism, which is redox-regulated in plants. selection of sulfur ligands for zn in these enzymes may confer an additional level of redox control on sulfur metabolism. as an additional control, we checked whether metal atoms were expelled from oxidized structures. a metal atom is not present in over a third of the oxidized structures. for another structure, aspartate transcarbamylase ( tug), the metal site was only partially occupied in the oxidized chain. for the remaining structures where the metal atom is retained in the oxidized structure, the shortened sg-sg distance may be caused by a heterogeneous mix of oxidized and reduced, metal-bound structures in the ensemble. we reasoned that if the metal was expelled from some members of the ensemble, or partially expelled (still in the site but held, for example, by only two of four potential ligating cys), then the b-factor should be higher than for chains where no shortened sg-sg distances are observed. although b-factors cannot be compared between different structures, for some of the redox pairs, different chains of the same structure in different disulfide redox states could be compared. our analysis confirmed that the b-factor of the metal was higher in the oxidized chains for % of structures (see fig. ). a similar result was obtained for proteins with fe-s clusters. there were no instances of expulsion of the fe-s cluster from the oxidized structure but oxidation of fe-ligating cys residues to form disulfides was generally associated with a higher b-factor of the relevant fe atom than fe atoms where the ligating cys remained reduced. in contrast, in reduced structures all fe atoms of the fe-s cluster had similar b-factors. further inspection of proteins with likely redoxactive zn sites reveals that two forbidden disulfide motifs, the jump strand disulfide (jsd) and a type of b-diagonal disulfide (bdd), are repetitively associated with redox-active zn-binding sites in the dataset. oxidized structures that contain a bdd include isoleucyl trna synthetase ( ile - , - ), the apoptosis-related proteins birc and survivin ( xb , - ; f h, - ), aspartate transcarbamylase ( r b, - ) and the dna repair enzyme recr ( vdd, - ). oxidized structures that contain a jsd include the amino-acyl trna synthetases for leu and ile ( obh, - ; jzs, - ), the enzymes of sulfur metabolism meth and bhmt ( q m, - ; lt , - ) and gtp hydrolase gtphi ( gtp, . a large group of proteins in the dataset were observed to undergo plastic deformations involving large scale rearrangements of the polypeptide backbone. we refer to these conformational changes as morphing transitions. these transitions, which typically involve unraveling portions of secondary structure, are illustrated in figure and in animations in the supporting information. the well known redox-sensitive protein oxyr belongs to this group. in oxyr, a protein of residues, significant refolding of the c-terminus occurs involving residues - . in the reduced structure, residues - form a short helix flanked by regions of coil. upon oxidation, residues - adopt a different fold with residues - and - forming two parallel b-strands joined by a loop with a disulfide-linkage between cys and cys . in the reduced oxyr structure, the two cys that form the disulfide are separated by almost Å. cys in oxyr is known to undergo alternate modifications such as glutathionylation. because of the spectacular nature of the transition between the reduced and oxidized structures, and the existence of an alternate mode of redox regulation, the physiological relevance of the disulfide-linked structure has been called into question. to determine whether the oxidized structure of oxyr is indeed an oddity, or if other proteins exhibit similar conformational changes, we systematically searched for morphing proteins in the dataset by scanning for protein structure pairs that exhibited large differences of the backbone torsional angles over at least seven contiguous residues. changes in pseudo-dihedral angles calculated using four consecutive ca atoms exceeding a summed threshold of degrees over seven consecutive residues were used to detect changes in secondary structure. twenty-two parents were found in the fully reduced set and four in the partially reduced set (designated m in supporting information table ). proteins showing the largest conformational changes are in table ii . these proteins include the cl À channel clic : a protein that forms ion channels by inserting itself into membranes in response to oxidation; the detoxification enzyme arsenate reductase, which undergoes multiple conformational changes associated with a disulfide relay as part there is partial oxidation of the metal site, the b-factor of the metal should be higher because it has fewer ligands and hence more conformational freedom. this is true for most structures (bars above the axis). standard errors are depicted where they could be calculated. key to structures: clostridium acetobutylicum thymidine kinase - xx ; e. coli aspartate transcarbamylase - r b, - tug; thermotoga maritima sir - h ; homo sapiens sir - j f; equus caballus alcohol dehydrogenase - axg; bacillus subtilis tryptophan rna-binding attenuator protein-inhibitory protein (antitrap) - bx ; severe acute respiratory syndrome coronavirus nonstructural protein - g t, - ga ; homo sapiens estrogen receptor - hcq; deinococcus radiodurans recombinational repair protein - vdd; norovirus c-like protease - wqs; moorella thermoacetica bifunctional carbon monoxide dehydrogenase/acetyl-coa synthase , and cyclic phosphodiesterase, a trna-splicing enzyme where the disulfide modulates access to the active site. like oxyr, several of these proteins exhibit large changes in ca separations of the two cys between alternate redox states (table ii) . thus large physical separation of the reduced cys pair seems to be a common feature of many of these proteins and does not preclude disulfide formation. another recognizable group of conformational changes involves order/disorder transitions. in these transitions, alternate redox states of the protein correlated with differing amounts of disorder in the protein structure, as evidenced by missing electron density. a total of redox pair structures in parents exhibited order/disorder transitions correlated with the redox state of the disulfide (table iii, fig. ). proteins were classified into four groups based on the location of disulfide-forming cys residues with respect to the disordered region in the protein chain. proteins in group a had a single cys residue in a disordered region, with the other cys residing on a more stable region of structure. in group b, both cys residues reside on a single disordered region. in group c, both residues lie on disordered regions which are not contiguous in the sequence. in group d, disulfide reduc-tion is associated with multiple regions of disorder in the reduced protein chain. disorder-to-order transitions have previously been observed upon binding of ligands. acquisition of order upon binding of ligands concomitant with disulfide formation was apparent for the group b oxidoreductase gdhb, where the disulfide straddled part of the pqq binding site, and the group a rna sulfuration enzyme ectrmu, where the disulfide straddles the trna-binding site. however, the reverse was true for the thermotoga maritima trna-processing enzyme, psi s, where significant disorder of the protein chain concomitant with disulfide reduction occurred upon binding of the trna substrate fragment. the introduction of disorder in this case may facilitate further co-operative binding of the rna and protein after the initial docking step. analyses of the composition of disordered sequences have detected amino acid biases from global frequencies. in particular, aromatic amino acids and cys are depleted in disordered sequences, whereas lys is enriched. , the disordered sequences in the redox pair dataset are also depleted in aromatics, in agreement with more general studies of disordered sequences, but are not depleted in cys, or enriched in lys; and thus do not conform entirely to previously detected sequence patterns. redox pair disordered sequences are instead enriched in oxygen-bearing sidechains such as asp, glu, ser and thr. some changes in secondary structure are also apparent during the order/disorder transitions. in the oxidized state, both cys residues generally reside on coil in their proteins, but for a small number of pairs the oxidized form has a secondary structure that ''melts'' upon reduction of the disulfide. examples include vitamin d-binding protein , and the e. coli oxidoreductase ghdb, , where helices are lost upon reduction; and the bacteriophage cell entry protein prd where a small b-sheet disappears upon reduction. the amount of disorder in the chains is also of interest. for most reduced structures, the disorder introduced upon reduction of the disulfide affects less than % of the polypeptide chain. however, three of the four group d proteins exhibited large regions of disorder in excess of % of the involved protein chain. interestingly, all were associated with a second more-ordered monomer that was oxidized. for example, the thermotoga maritima trna-processing enzyme, psi s undergoes a spectacular loss of secondary structure in one monomer upon binding of rna. it is possible that the fully reduced state of these protein dimers is uncrystallisable and that only partially and fully oxidized oligomers are sufficiently ordered to enable structure solution. we also investigated whether formation of interchain disulfide bonds altered the quaternary state of proteins. different redox states of the interchain disulfides are associated with two general types of quaternary interaction: those where the number of oligomers differs between the reduced and oxidized state; and those where the number of subunits involved in the oligomer stays constant, but the oligomerization interface changes. some redox pairs exhibit a mixture of the two types of quaternary change. for example, disulfide-bonded dimers of the macrophage sialoadhesin in the more ordered oxidized structure: a disulfide is formed between cys and cys which straddles a key phosphorylation site at thr . formation of the disulfide is proposed to inhibit kinase activity by recruiting the cognate phosphatase. the t-loop was recently identified as a target for selective cancer drugs. b: a larger order/disorder transition associated with expulsion of zn from the oxidized structure of leucyl-trna synthetase. , in the more ordered reduced structure, several new secondary structures condense enabling rigid co-ordination of the zn. zn is represented by red spheres in the reduced structure. oxidized structures are shown on the left, reduced structures on the right. fan et al. form an interface distinct from that found in trimers formed upon binding of sialosides, and may negatively regulate ligand binding. twenty-nine redox pair protein clusters with intermolecular disulfide bonds exhibit changes in quaternary structure upon oxidation/reduction. redox regulation of the quaternary state has previously been associated with cooperative behavior in proteins. for example, the hoxb protein binds dna in vitro when either oxidized or reduced but formation of an intramolecular disulfide between hoxb monomers is necessary for co-operative binding. other well-known examples are oxyr and p . several other proteins in the dataset exhibit co-operative behavior. cytoglobin is a member of the vertebrate haemoglobin family residing in the nucleus and cytosol that is upregulated figure . quaternary changes between different redox states. a: changes in the number of subunits in the multimer between different redox states of chloroplast gapdh. the a subunit of spinach chloroplast gapdh in a reduced, monomeric form (pdb hki) is shown on the right and the oxidized homotetramer in complex with nad þ (pdb nbo) is shown on the left. in higher plants, photosynthetic gapdh exists in vivo mainly as heteromeric isoforms, a b and a b being the most abundant, interconvertible conformations. the b subunit is homologous to the a subunit but has an additional nonhomologous cterminal extension. stable homotetramers of a subunits (a -gapdh) are found at low levels in chloroplast preparations and may be the only form of photosynthetic gapdh present in unicellular green algae and cyanobacteria. calvin cycle enzymes are known, as a group, to be redox regulated. the activity of the ab isoform of chloroplast gapdh is redox regulated by formation of a disulfide between c-terminal thiols in the nonhomologous part of the b chain in light, and its reduction in the dark. the interchain disulfide between a subunits may be important for redox control of the a isoform, which is known to form complexes with the small chloroplast protein cp and phosphoribulosekinase in the dark. the disulfide-forming cys ( nbo numbering) is not present in the b subunit of chloroplast gapdh which is transcribed from a different gene, or in cytosolic gapdhs. b: changes in the oligomeric interface in cytoglobin, a member of the vertebrate haemoglobin family residing in the nucleus and cytosol that is upregulated under conditions of hypoxia and certain types of stress. intermolecular disulfide bonds in cytoglobin modulate the quaternary structure and may regulate the ligand-binding properties. both the reduced and oxidized states are dimers. under conditions of hypoxia and certain types of stress (see fig. ). it has recently been implicated as a tumor suppressor. intermolecular disulfide bonds in cyto-globin modulate the quaternary structure and may regulate the ligand-binding properties (table iv) . the homologous lamphrey haemoglobin is in an equilibrium italicized prefixes are species abbreviations. protein names in roman font. ao, aminooxidase; bcp, bacterial comigratory protein peroxiredoxin; bmp , bone morphogenetic protein ; dna pol iii k, dna polymerase iii k; ef-ts, elongation factor ts; gapdh, glyceraldehyde- -phosphate dehydrogenase; gef, guanine nucleotide exchange factor; gga , golgi-localized c adaptin; grp , guanine nucleotide exchange factor and integrin binding protein homolog grp ; grx, glutathione reductase; pcbp , poly(c)-binding protein- ; phsam domain, polyhomeotic-proximal chromatin protein sterile alpha motif domain; pnmt, phenylethanolamine n-methyltransferase; rubiscol, ribulose- , -bisphosphate carboxylase/oxygenase large subunit; sam-s-adenosyl-l-methionine; vegf, vascular endothelial growth factor; ae, aeropyrum pernix; af, archaeoglobus fulgidus; bpv, bovine papilloma virus; cr, crithidia fasciculata, dm, drosophila melangaster; fmdv, foot and mouth disease virus; hpv, human papilloma virus; hs, homo sapiens; me, methanobacterium thermoautotrophicum; mm, mus musculus; mp, mycoplasma pneumoniae; nt, nicotiana tabacum; ps, pisum sativum (pea); rn, rattus norvegus; sp, spinacia oleracea; td, themiste dyscritum; tt, thermus thermophilus. a nd cys in different monomer. state between a low o affinity oligomer and a high-affinity monomer which contributes to its co-operativity. there was also a small group of transmembrane receptors which form covalent dimers in a neck region close to the membrane. these include the low-density oxidized ldl receptor, and the asialoglycoprotein receptor. covalent dimer formation may promote further quaternary changes such as higher oligomer formation in response to bound ligands as has been recently demonstrated for the metabotropic glutamate receptors. relevance to disease importantly, ageing and several major diseases including neurodegenerative diseases such as alzheimer's disease, type ii diabetes, cancer and cardiovascular disease have been associated with abnormal redox conditions. inadvertent triggering of redox-active disulfide switches is a likely contributor to these phenotypes. this view is supported by the association of several redox pairs in the dataset with disease. sod (also known as cuzn-sod) is an enzyme mutated in familial lateral sclerosis (fals), an age-dependent degenerative disorder of motor neurons in the spinal cord, brain stem and brain. sod is largely localized to the cytosol but is also found in the intermembrane space of mitochondria. sod is activated by formation of the disulfide and insertion of cu by the copper carrier ccs. both of these proteins are represented in the redox pair dataset (tables i and s ). the conserved disulfide in sod is essential for activity and unusual because it remains oxidized in the cytosol. although the disulfide is intrachain, it is thought to play a critical role in stabilizing the sod homodimer. formation of the disulfide is part of the sod maturation process which is regulated by physiological oxidative stress. the three reduced proteins in the dataset are three distinct mutants associated with familial lateral sclerosis (fals): g r, h r, and i t. the exclusive association of the reduced proteins with the disease mutants suggests redox imbalance of this disulfide is implicated in the disease. this hypothesis is supported by the finding that fals mutants have the common property of being more susceptible to disulfide reduction than wild-type protein. toxicity associated with disulfide-linked multimerization of sod mutants has recently been implicated in the disease mechanism. several types of conformational change were observed in the redox pair dataset suggesting there may be common structural modes of redox regulation. proteins exhibited disulfide oxidation following expulsion of metals such as zn, morphing, order/disorder transitions, and changes in quaternary structure. in an individual protein more than one mode may be employed. for example in leu-trna synthetase, part of the protein chain becomes disordered upon zn expulsion but this is not the case for all proteins with labile zn. redox regulation of proteins that form disulfides following expulsion of zn is an emerging area of signaling. well known and characterized examples including hsp which is activated by zn expulsion as part of the oxidative stress response ; and the small tim proteins which are kept in a transport-competent state in the cytosol by conjugation with zn. upon delivery to mitochondria, zn is expelled from small tim proteins which are activated by disulfide formation. zn is not expelled from all protein zn-binding sites with equal facility, and is probably not expelled from some sites at all under physiologic conditions. for structural zn sites an analogous situation may exist to the bipartite distribution of redox-active and structural disulfides in proteins, with labile zn sites being redox-active and inert zn sites corresponding to the purely structural group. key to zn expulsion under oxidizing conditions is the presence of oxidation-prone cys as a zn ligand. sites with more cys are likely to be more facile. based on the number and nature of ligands, the zn sites detected in the redox pair dataset likely belong to the redox-active group. the repeated association of forbidden disulfides of the bdd type with labile zn binding sites is particularly interesting because of the recent characterization of the oxidative process that leads to activation of the bacterial chaperone hsp . the presence of the zn center keeps hsp monomeric and functionally inactive. upon exposure to elevated temperatures, the cterminal domain swings away from the protein's center of mass, exposing the zn centre. the n-terminal thiols in the zn site, cys and cys , are preferentially oxidatively modified. the remaining two cys, and , form a disulfide of the bdd type, releasing zn and unfolding the c-terminal domain. unfolding the c-terminus removes a steric barrier to dimerization, activating the protein's chaperone function. the function of disulfide formation between cys and in the zn binding site has not been fully delineated. potential benefits are protection from other oxidation modifications and restriction of the conformational freedom of the protein chain allowing easier refolding upon return of the protein's milieu to normal conditions. however, these benefits apply to any disulfide and the repeated appearance of the bdd in labile zn sites of proteins of the redox pair dataset suggests a more specific function, possibly recognition by a foldase or metallochaperone. we hypothesize the forbidden disulfide would become stressed upon condensation of the b-sheet in its vicinity, possibly allowing easier re-insertion of zn. without any detailed experiments as a guide, the significance of the other repeated forbidden disulfide motif, the jsd, is not clear, but the same arguments relating to its forbidden disulfide nature would apply. the jsd-containing proteins meth and bhmt are known to be reversibly inactivated by disulfide formation. in vitro, bhmt can be reactivated by dialysis against dtt and zn, but the specific method of reactivation in vivo has not been characterized. for most structures exhibiting order/disorder transitions in the dataset, the reduced structure is associated with more disorder than the oxidized structure. energetically, formation of a disulfide bond between cys residues would have a favorable entropic contribution to the stability of the chain. a notable exception is leu-trna synthetase where the more ordered reduced structure binds zn (fig. (b) ). this may be generally true for order/disorder transitions associated with labile zn sites: the zn-bound state is entropically favored over a single disulfide because the polypeptide chain is constrained at three or four points instead of two. a subset of redox pair proteins exhibiting order/ disorder transitions correlated with disulfide redox status contain regions of disorder in excess of % of the protein chain. all the proteins in this group exist as dimers with the other monomer being more ordered. the disordered monomers in these class d structures may correspond to the molten globule state. for these proteins, the molten globule state adopted may be physiologically relevant to their function. molten globules are collapsed forms of the protein chain which have some native-like secondary structure but a dynamic tertiary structure as seen by far and near circular dichroism spectroscopy, respectively. the radius of gyration of proteins in the molten globule state is $ % larger than the native state but precise tertiary structures at atomic resolution have not yet been obtained for commonly studied molten globule proteins such as lactoglobulin a. however, one of the proteins in group d: platelet factor , which like hepatocyte growth factor, binds heparin and regulates angiogenesis, has been shown to adopt the molten globule state upon reduction of its disulfides. it seems likely proteins in group d adopt a molten globule state in vivo but can also adopt more ordered states upon binding of appropriate ligands. several of the proteins bind large polymers such as rna and heparin and the order/disorder transitions may mediate co-operative behavior between monomers. general studies of disordered sequences suggest there may be several different types of disordered sequence, three of which have been previously detected using an automatic classifier. the oxygen-rich sequences of the disordered regions of proteins of the redox pair dataset seem to represent a novel type of disordered sequence not previously recognized. oxygen-rich sequences may confer bistable switch-like properties on the sequence: an oxygen-rich environment stabilizes the disulfide in the oxidized state by increasing its pka ; while the subset of negatively charged asp and glu residues in oxygen-rich sequences may contribute to disorder in the reduced state via likecharge repulsion. disordered regions are known to undergo post-translational modifications including acetylation, glycosylation, methylation, and phosphorylation which may regulate the order/disorder transition. redox-activity of resident cys residues should be added to this list of post-translational modifications. perhaps the most astonishing conformational changes associated with disulfide reduction are the morphing transitions. the current paradigm, due to anfinsen, is that the primary sequence of a protein encodes a unique three-dimensional structure. various discoveries that have challenged this notion include the spectacular ph-induced conformational change in influenza a haemagglutinin (ha), molecular chaperones, and the previously discussed disordered sequences. all of these discoveries have been difficult to reconcile with the concept that protein sequences adopt a unique minimally frustrated fold. ha is believed to be kinetically trapped in a higher energy fold by an n-terminal domain which is subsequently cleaved, allowing it to cascade into a minimally frustrated fold. the conformations of disordered sequences appear to be principally governed by entropic factors: weak sequence preferences for a variety of conformational substates or modes appear to be co-selected by ligands. morphing structures appear to be a new type of challenge to the concept of a unique minimally frustrated fold. for morphing sequences, the structure appears to be dependent on the environment. unlike influenza a ha, where kinetic trapping during the folding process clearly plays a part in the structure adopting two different folds, for morphing sequences the two folds are energetic minima for the sequence in a particular environment. as such structural changes are reversible and dependent on environment. we were previously aware of two instances where subdomain morphing of proteins has been associated with reversible disulfide reduction: a redox-controlled structural reorganization of the ion channel clic proposed to regulate its insertion into membranes, and sequential oxidation of the transcription factor oxyr in response to oxidative stress which modulates its quaternary structure and dna-binding properties. here we showed that morphing is a common type of major structural change associated with disulfide reduction. the most spectacular morphing transition is that of clic , where an entire subdomain of the protein rearranges in response to different redox conditions. several redox pairs were found exhibiting large morphing conformational transitions of similar magnitude to oxyr, supporting the notion that these types of transitions may be reasonably common. large separations of the cys residues in the reduced state are a typical feature of these proteins, and do not preclude disulfide linkages forming. possibly attack by a helper molecule, such as glutathione, may be required as an intermediate in disulfide formation to bridge the intervening distance. large physical separation of partner cys residues may represent an example of negative morphing transitions are not limited to changes in disulfide redox status. other morphing transitions not associated with disulfide redox state have been noted in mad and lymphotactin. [ ] [ ] [ ] ph and redox are probably only two of multiple environmental changes associated with structural morphing. the relative importance of these and other environmental changes in protein morphing remain to be determined. morphing transitions may also have some relevance to evolutionary bridging states. in , based on structures of arc repressor, sauer proposed the existence of evolutionary bridging states. a substructure in wild-type arc repressor adopts a b-strand conformation which forms a two-stranded b-sheet with the adjacent monomer. in the n l mutant of arc repressor, the substructure was shown to fluctuate between the native state and a helical state on a millisecond time scale. a double mutant known as ''switch arc'' (n l, l n) was shown to be stabilized in the alternate helical conformation. sauer proposed the fluctuating n l mutant represented an evolutionary bridge between two stable low energy folds. proteins that can adopt two different folds from the same sequence could evolve two different functions upon gene duplication and mutation hence acting as an evolutionary bridge between two different folds. it is clear morphing proteins could also act as bridge states: deselection of one state over the other by point mutation may lead to loss of the ability to morph in the daughters resulting in proteins with different but related folds. although we have concentrated solely on disulfide reduction in this study, several anecdotal examples of disulfide isomerization should be mentioned in the context of the aforementioned discussion. a second example of an evolutionary bridging state was proposed based on a study of cnidarian collagen. the nterminal domain of minicollagen has % sequence identity with the c-terminal domain, but the structures show a different disulfide bonding pattern and fold. at this stage it is not clear if the two forms are interconvertible or represent true one-state endpoints evolved from an evolutionary bridging state. a different example where major structural organization is regulated by disulfide isomerization is the activation of p lysozyme. this conformational change is irreversible and associated with release of a hydrophobic transmembrane sequence by membrane depolarization, suggesting the latent state may be a kinetically trapped state rather than a true morphing protein. our study mined pairs of protein structures which contain disulfides in both reduced and oxidized states and represents the first systematic compilation of such a dataset. quaternary changes, morphing and expulsion of transition metals, particularly zn, have previously been associated with redox-related changes in proteins and here we showed these conformational changes are more common than anecdotal examples suggest. to our knowledge order/disorder transitions have not previously been linked with redox changes but also seem to be a common type of conformational change. like morphing transitions, changing redox conditions appear to be only one of many mechanisms regulating order/disorder transitions. the oxygen-rich sequences of the disordered regions of proteins of the redox pair dataset seem to be a novel type of disordered sequence not previously recognized. finally, two forbidden disulfide motifs are associated with redoxregulated zn binding here for the first time. the physiological significance of redox-controlled structural changes is only beginning to be appreciated. previously, all oxidative changes were thought to be irreversible and damaging. it is now known that redox signaling pathways can modulate homeostatic and adaptive responses. however, continued stimulation of adaptive responses may lead to maladaptive responses. the redox pair dataset is a valuable resource for the investigation of physiological and pathological processes associated with disulfide redox activity. the pdb was mined for pairs of structures with cys in different redox states. first, all disulfide-containing structures were identified by querying their dssp records, as ssbond records in the pdb header were found to be unreliable. for each disulfide the atomic residue positions were mapped to the sequence position. the connectivity of the disulfides in the sequence was then used to assign a unique identifier, d mn score [eq. ( )], to each protein. the d mn score allows direct comparison of the disulfide connectivity between structures as structures with the same disulfide bonding will have the same d mn score. where i and j are the sequence positions of disulfidebonded cys. all protein sequences in the pdb were clustered at % sequence identity using blastclust. the % threshold was chose to capture the same protein with some natural and engineered mutations allowed. some highly conserved orthologs from closely related species were also captured in the same cluster, for example, the nf-jb orthologs in human and mouse. proteins within each group were compared to detect subgroups with different d mn scores-indicating alternative disulfide connectivity. for fully oxidized proteins, a set of ''pseudo-reduced'' d mn s were calculated by iteratively ignoring one or more disulfides. the set of pseudoreduced d mn s for the oxidized protein were matched against d mn s for the reduced proteins in the dataset to select redox pairs. as an additional control, proteins were split into two groups based on whether some or all disulfide bonds in the protein were reduced. proteins in which cys residues were engineered into proteins were filtered from the list. analysis of gene ontology term enrichment go terms that are over and under-represented in the oxidized proteins in the redox-pairs dataset compared to the pdb as a whole were identified using fisher's exact test, in the same way as applied in tools that analyze gene-expression data. go annotations for the entire pdb were taken from the goa resource, downloaded from the ebi. to ensure a fair comparison, the go annotations of a nonredundant set of the oxidized structures ( % sequence identity) were compared to go annotations of a nonredundant pdb data set ( % sequence identity). to improve overall annotation coverage, a representative pdb structure is assigned all the go terms belonging to the pdbs within its cluster group. because go is a hierarchical data structure, go terms are set to a single level in the hierarchy to enable a fair comparison. here we used go level three. at this level there is a compromise between information quality and the number of annotations available. where a go term is below this level we move up the hierarchy until we reach the third level go term(s). go terms above level three cannot be used. a p-value was calculated representing the probability that the observed number of each go term (n) could have resulted from randomly selecting this go term in the pdb as a whole (n). fisher's exact test was used in order to approximate this p-value. go terms that are most specific to the redox pairs set will have p-values near zero, and those that are under-represented will be near one. protein conformational changes were examined using a variety of complementary approaches. we used profit (www.bioinf.org.uk/software/profit) to search for large conformational changes such as hinge movements. profit superimposes two structures using ca atoms and was used to calculate rmsd and distances between residues. large movements were indicated by the change in distance between the sulfur groups of the cys residues in a disulfide. because rmsd is averaged over the entire molecule, large changes confined to a subdomain of protein may not be obvious using rmsd. large conformational changes between structure-pairs corresponding to rearrangements of the polypeptide backbone were also identified using an implementation of a method by flocco and mowbray. for pairs of structures, the method compares the change in a pseudo-torsional angles defined by four consecutive ca-atoms. conformational changes were identified by running a smoothing window (five residues long) over the torsional-difference plot. stretches of contiguous residues with seven or more pseudo-dihedral angles that exhibited individual movements in excess of were initially filtered from the dataset. these conformational changes were further classified by comparing dssp annotations, solvent accessibility and secondary structure, between the protein pairs. proteins with conformational changes where the summed changes in the pseudo-torsional angles exceeded a threshold of degrees with accompanying secondary structure changes were classified as morphing. the presence or absence of multiple chains in the pdb structure could cause errors when calculating changes in solvent accessibility; therefore, dssp was run on individual chains only. all solvent accessibility measures were normalized using values derived by chothia. each amino acid was assigned to one of two states; core or exposed. core residues are those with a solvent accessibility of less than % and exposed residues are those with a solvent accessibility equal to % or more. secondary structures were assigned to one of three states: helix, strand and coil. regulation of the protein disulfide proteome by mitochondria in mammalian cells redox regulation: a broadening horizon disulfide isomerization switches tissue factor from coagulation to cell signaling disulfide exchange in domain of cd is required for entry of hiv-i redox regulation facilitates optimal peptide selection by mhc class i during antigen processing a single dipeptide sequence modulates the redox properties of a whole enzyme family substitution of the conserved tryptophan- in escherichia coli thioredoxin by site-directed mutagenesis and structure-function analysis urea dependence of thiol disulfide equilibria in thioredoxin-confirmation of the linkage relationship and a sensitive assay for structure redox properties of protein disulfide isomerase (dsba) from escherichia coli molecular and cellular aspects of thiol disulfide exchange force-dependent chemical kinetics of disulfide bond reduction observed with single-molecule techniques forbidden'' disulfides: their role as redox switches tryparedoxins from crithidia fasciculata and trypanosoma brucei -photoreduction of the redox disulfide using synchrotron radiation and evidence for a conformational switch implicated in function oxidized and synchrotron cleaved structures of the disulfide redox center in the n-terminal domain of salmonella typhimurium ahpf specific chemical and structural damage to proteins produced by synchrotron radiation the protein data bank all intermediates of the arsenate reductase mechanism, including an intramolecular dynamic disulfide cascade the intracellular chloride ion channel protein clic undergoes a redox-controlled structural transition membrane transport of hydrogen peroxide a combined in vitro/bioinformatic investigation of redox regulatory mechanisms governing cell cycle progression structure and biochemical properties of prl- , a phosphatase implicated in cell growth, differentiation, and tumor invasion structural mechanism of oxidative regulation of the phosphatase cdc b via an intramolecular disulfide bond structural basis of the redox switch in the oxyr transcription factor a model for the role of multiple cysteine residues involved in ribonucleotide reduction-amazing and still confusing substrate specificity and redox potential of ahpc, a bacterial peroxiredoxin structure of superoxide reductase bound to ferrocyanide and active site expansion upon x-ray-induced photo-reduction cross-strand disulphides in cell entry proteins: poised to act the gene ontology annotation (goa) database: sharing knowledge in uniprot with gene ontology chlamydia attachment to mammalian cells requires protein disulfide isomerase thiol/disulfide exchange is required for membrane fusion directed by the newcastle disease virus fusion protein inhibitors of protein-disulfide isomerase prevent cleavage of disulfide bonds in receptor-bound glycoprotein and prevent hiv- entry protein-disulfide isomerase-mediated reduction of two disulfide bonds of hiv envelope glycoprotein occurs post-cxcr binding and is required for fusion the crystallographically determined structures of atypical strained disulfides engineered into subtilisin a new force-field for molecular mechanical simulation of nucleic-acids and proteins in vivo formation and stability of engineered disulfide bonds in subtilisin harnessing disulfide bonds using protein engineering the anatomy and taxonomy of protein structure disulfide bridges in globular proteins snapshots of trna sulphuration via an adenylated intermediate zinc coordination environments in proteins as redox sensors and signal transducers a disulfide relay system in the intermembrane space of mitochondria that mediates protein import betaine-homocysteine methyltransferase: zinc in a distorted barrel identification of plant glutaredoxin targets conformational changes in protein redox pairs protein thiol modifications visualized in vivo zinc plays a key role in human and bacterial gtp cyclohydrolase i nuclear and mitochondrial compartmentation of oxidative stress and redox signaling crystal structure of the atx metallochaperone protein at . Å resolution engineering, characterization and phage display of hepatitis c virus ns protease and ns a cofactor peptide as a single-chain protein mutational and structural analysis of cobalt-containing nitrile hydratase on substrate and metal binding crystal structures of zinc-free and -bound heme domain of human inducible nitric-oxide synthase: implications for dimer stability and comparison with endothelial nitric-oxide synthase reversible interconversion of two forms of a valyl-trna synthetase-containing protein complex thioredoxin-linked processes in cyanobacteria are as numerous as in chloroplasts, but targets are different mutational analysis of the redox-sensitive transcriptional regulator oxyr: regions important for oxidation and transcriptional activation chloroplast transcription at different light intensities. glutathione-mediated phosphorylation of the major rna polymerase involved in redoxregulated organellar gene expression mutational analysis to define an activating region on the redox-sensitive transcriptional regulator oxyr zinc coordination, function, and structure of zinc enzymes and other proteins changes in zinc ligation promote remodeling of the active site in the zinc hydrolase superfamily reactivity of zinc finger cores: analysis of protein packing and electrostatic screening crystal structures of the semireduced and inhibitor-bound forms of cyclic nucleotide phosphodiesterase from arabidopsis thaliana oxyr: a molecular code for redox-related signaling evaluating the stability of disulfide bridges in proteins: a torsional potential energy surface for diethyl disulfide redox signalling in the chloroplast: structure of oxidized pea fructose- , -bisphosphate phosphatase identification of the cysteine residues involved in redox modification of plant plastidic glucose- -phosphate dehydrogenase trpc channel activation by extracellular thioredoxin structural basis of redox-coupled protein substrate selection by the cytochrome c biosynthesis protein resa discovery of lactoquinomycin and related pyranonaphthoquinones as potent and allosteric inhibitors of akt/pkb: mechanistic involvement of akt catalytic activation loop cysteines crystal structure of an inactive akt kinase domain biological functions of the disulfides in bovine pancreatic deoxyribonuclease multiplexed proteomic analysis of oxidation and concentrations of cerebrospinal fluid proteins in alzheimer disease the redox regulation of pi -kinasedependent signaling structural and mechanistic basis of pre-and posttransfer editing by leucyl-trna synthetase the crystal structure of leucyl-trna synthetase complexed with trnaleu in the post-transfer-editing conformation intrinsic disorder and protein function structure and mechanism of soluble quinoprotein glucose dehydrogenase conformational change of pseudouridine synthase upon its association with rna substrate identifying disordered regions in fan et al. protein science proteins from amino acid sequence sequence complexity of disordered protein crystal structure of the complex between actin and human vitamin d-binding protein at . Å resolution a structural basis for the unique binding features of the human vitamin dbinding protein the . a crystal structure of the apo form of the soluble quinoprotein glucose dehydrogenase from acinetobacter calcoaceticus reveals a novel internal conserved sequence repeat the receptor binding protein p of prd , a virus targeting antibiotic-resistant bacteria, has a novel fold suggesting multiple functions crystallographic and in silico analysis of the sialosidebinding characteristics of the siglec sialoadhesin cooperative dna binding of the human hoxb (hox- . ) protein is under redox regulation in vitro formation of disulfide bond in p correlates with inhibition of dna binding and tetramerization role of light in the regulation of chloroplast enzymes molecular mechanism of thioredoxin regulation in photosynthetic a b -glyceraldehyde- -phosphate dehydrogenase redox regulation of chloroplast enzymes in galdieria sulphuraria in view of eukaryotic evolution cytoglobin, the newest member of the globin family, functions as a tumor suppressor gene structural basis of human cytoglobin for ligand binding oxidative stress causes rapid membrane translocation and in vivo degradation of ribulose- , -bisphosphate carboxylase/oxygenase crystallization and structural analysis of gadph from spinacia oleracea in a new form the intermolecular disulfide bridge of human glial cell line-derived neurotrophic factor: its selective reduction and biological activity of the modified protein a closed conformation for the polk catalytic cycle crystal structure of the apoptosis-inducing human granzyme a dimer crystal structure of the ef-tu.ef-ts complex from thermus thermophilus structures of the extracellular regions of the group ii/ iii metabotropic glutamate receptors mutant sod instability: implications for toxicity in amyotrophic lateral sclerosis disulfide cross-linked protein represents a significant fraction of als-associated cu, zn-superoxide dismutase aggregates in spinal cords of model mice the redox-switch domain of hsp functions as dual stress sensor liver betainehomocysteine s-methyltransferase activity undergoes a redox switch at the active site zinc human betaine-homocysteine methyltransferase is a zinc metalloenzyme molten globule monomer to condensed dimer: role of disulfide bonds in platelet factor- folding and subunit association flavors of protein disorder determination of the pk a of the four zn þ -coordinating residues of the distal finger motif of the hiv- nucleocapsid protein: consequences on the binding of zn þ structure of influenza hemagglutinin at the ph of membrane-fusion interconversion between two unrelated protein folds in the lymphotactin native state the mad conformational dimer: structure and implications for the spindle assembly checkpoint metamorphic proteins protein structure: evolutionary bridges to new folds the two cysteine-rich head domains of minicollagen from hydra nematocysts differ in their cystine framework and overall fold despite an identical cysteine sequence pattern disulfide isomerization after membrane release of its sar domain activates p lysozyme dictionary of protein secondary structure-pattern-recognition of hydrogenbonded and geometrical features a novel database of disulfide patterns and its application to the discovery of distantly related homologs basic local alignment search tool ontological analysis of gene expression data: current tools, limitations, and open problems supervised neural networks for clustering conditions in dna array data after reducing noise by clustering gene expression profiles ca-based torsion angles: a simple tool to analyze protein conformational changes scop-a structural classification of proteins database for the investigation of sequences and structures the authors thank dr. kieran scott for interesting discussions. disulfide torsional energy calculations were performed at the nci national facility. key: cord- -ak pq authors: nan title: th european congress of intensive care medicine athens - greece, october – , abstracts date: journal: intensive care med doi: . /bf sha: doc_id: cord_uid: ak pq nan objectives: evaluate the levels of tnf, il- and pai-i in different moments of the ards and the possible relationships among them. methods: septic patients with ards were studied. also significant differences for: tnf, pai-i and il- in septic patients and both evaluations of ards with control gropup; pai- between septics and nd evaluation in ards, and between the ist and nd evaluation in ards; il- between septics and both evaluations in ards; and il-~ in both evaluations in ards patients in relation to mortality. conclusions: i) elevations of tnf, pai-i and il- , with clinical signs, are suggestive of infection; ) the persistent and progressive elevation of pai-i with any clinical criteria may suggest evolution to ards; ) due to its own kynetics, il- takes part later in the acute phase, its levels being related to the magnitude of the injury in the tissues. objectives: the influence of long-term volume therapy with different solutions on plasma levels of circulating adhesion molecules was studied. methods: according to a randomized sequence, patients with sepsis secondary to major surgery exclusively received either hydroxyethylstarch solution ( % hes, mean molecular weight (mw) , daltons, degree of substitution (ds) . ) or human albumin % (ha) for volume therapy for days. plasma levels of circulating (soluble) adhesion molecules (endothelial leukocyte adhesion melecule- [selam -i] , intercellular adhesion molecule- [sicam -i] , vascular cell adhesion molecule- [svcam -i] , and p-selectin ) were serially measured on the day of admission to the intensive care unit (='baseline ' value) and during the next days. results: selam-i, sicam-i, and svcam-i plasma levels were markedly higher than normal at baseline in both groups. in the hes-patients, selam-j decreased to normal range, whereas it further increased in the ha-group (from • to • during the study period, sicam-i and svcam-i plasma levels remained unchanged in the hes-patients, but further increased in the ha-group (from • to , • sgmp- increased significatly only in the ha-group ( • to • only pao /fio was significantly correlated to plasma levels of adhesion molecules. conclusions: sepsis is associated with markedly elevated plasma levels of adhesion molecules indicating endothelial activation or damage. by long-term volume therapy with hes, these levels remained unchanged or even decreased, whereas volume therapy with human albumin did not have any beneficial effects on soluble adhesion. central venous catheters are frequently used in the care of the critically ill patient. the incidence of catheter related sepsis varies in the literature. we investigated the occurrence of contamination and sepsis compared to results of the epic study as part of quality assesment in our intensive care unit. from january until august all removed central venous catheters were examined for microbiological culture. the patients who showed signs of sepsis were also registered. the results of the contaminated catheters and septic patients were compared with results from the epic study. during the month period , patients were hospitalized on our intensive care unit. central venous catheters were examined for microbiological culture. specimens appeared to be possitive ( %). patients showed clinical signs of sepsis. the incidence of sepsis due to contaminated central venous catheters was / ( %). the incidence of sepsis due to the presence of all central venous lines was / ( %). the microorganisms responsible for the sepsis syndrom were : stapylococcus aureus (n= ), escherichia colt (n= ), others (n= ). in the epic study the percentage for sepsis on the icu was . % for the netherlands and . % for europe. despite a high number of positive culture from removed intravascular lines, a low percentage of sepsis was seen compared to results of the epic study. we recommend routine bacteriological culture of all removed central venous lines and recommend to look at colonization and sepsis due to intravascular lines as a measure of quality control in the intensive care unit. objectives: prognostic assessment of simplified acute physiology score (saps) in granulocytopenie patients with septic shock (ss). methods: the medical records of admissions to an intensive care unit (icu) of granuloeytopenic patients with ss are reviewed. fiftytwo patients had haematological malignancies. seven patients had aplastie anaemia. patients were categorised as survivors (discharged from icl and non-survivors (died in the icu). saps index was calculated for patients daily during their stay in icu. all patients were severe granulocytopenic (total white cell count less than , ] ] ). results: five patients ( , %) were discharged from icu. fifty-four patients died in icu. non-survivors had saps on admission higher than survivors ( . + . and . + . , respectively, p< , , mann-whitney u test). no patient with a saps greater than survived. mortality among the patients with saps from to was , %o. the evolution of ss was rapid. the mean stay in icu among non-survivors was only hours. an analysis of the saps index on admission of non-survivors showed an inverse correlation with the duration of their stay in icu (r=- , , p= . ). all survivors recovered from granulocytopenia. they had normal white cell counts at the time of discharge from icu. there was inverse correlation in survivors between saps and white cell counts, when these parameters were evaluated daily. however, the saps index alone cannot be considered to be on individual predictor factor of mortality. patients who had failure of the malignancy to respond to chemotherapy and who had persistent granuloeytopenia died in icu despite saps index on admission and recovery from ss. conclusion: saps index greater than , failure of the malignancy to respond to chemotherapy and persistent leueopenia all point to a poor outcome of granulocytopenie patients with ss. introduction: antipyretics sometimes are used for fever control in febrile neutropenic patients with hematological malignancies(hm). we observed a dramatic fall of blood pressure(bp) and development of septic shock(ss) in some of the patients who received antipyretics. aim: to clarify can antipyretics provoke ss in neutropenic patients with infection. methods: retrospective review of medicat records of neutropenic(wbc < , / )patients with hm, admitted to the intensive care unit for ss, was performed. there was selected group of patients receiving antipyretics shortly before a fall of bp. results: there was a definite causal relationship between receiving antipyretics and fall of bp in from patients. all patients had fever due to infection and had normal level of bp before receiving antipyretics. hypotension developed within minutes up to , hours after administration of antipyretics. three patients received , g of metamisol and one , g ofparacetamol per os. in all cases we observed dramatic diaphoresis and the temperature fall to subnormal level ( . + . ~ accompanied'by hypotension. but in - hours the fever was coming back without blood pressure elevation. the fluid replacement was controlled by central venous or wedge pressures. there were required + ml colloid and cristalloid solutions for volume loading. in spite of fluid administration the hypotension persisted and all patients required inotropic therapy. only one patient survived and is alive now. conclusion: it seems to us that our data offer to state that antipyretics administration can initiate ss in febrile neutropeuic patients with infection. objectives: to assess the agreement between cardiac output (co) measured by odm t and by other methods used in icu patients. methods: we prospectively studied adu t patients requiring hemodynamic monitoring with a pulmonary artery catheter. an esophageal doppler monitor provided measurements of co (odm), stroke volume and flow time (ft) used as an indirect evaluation of patient's volume status. patient hemodynamic status was evaluated by a modified fast response pulmonary artery catheter (baxter health care corporation, santa ana, ca), allowing co measurements by thermodilution "d) and an evaluation of right ventricular ejection fraction and end diastolic volume (rvef and rv-edv). in the last six patients co was measured by transthoracic echocardiography (echo) and oxygen consumption was measured by a deltatrack ii metabolic monitor (datex) allowing co calculation according to the fick formula (fick). the agreement between methods measuring co and their reproducibility, were evaluated by bland and altman analysis. results: agreement between co measurements is expressed as bias (d) and % limits of agreement (l of a = d_+ sd . td-fick - . - . to . fick-echo . - . to . there was no correlation between ft and rv-edv. conclusions: although co measurements by odmil had the best reproducibility, the limits of agreement between the four methods tested were unacceptable for clinical purposes. further investigation is required in order to improve the accuracy of co measurement in the icu. phd, a. paltzev, v.bajbikov, b.dobryakov d.sc., a.ostanin phd, o.leplifia phd, h.chernykh phd munieip. hosp. n l, n ; inst. of clin. immunol., novosibirsk, russia objectivies: efficiency of native cytokines used in the treatment of patients with severe surgical infections has been studied. methods: for two years patients were treated with cytokine mixture (ssp) obtained by arterio-venous perfusion of swine spleen and contained the following cytokines: il- , il- , il- , tnfa, ifny, gm-csf. results: ssp intravenous infusions were shown to accompany with mortality decrease from . % to . % in patients with abscessed pneumonia and lung abscesses and from % to % if disease course was complicated with sepsis. in patients with purulent peritonitis and sepsis efficiency of ssp was decreased due to endotoxieosis. thus, we used adoptive immunotherapy with mnc activated in vitro with ssp or recombinant il- . intravenous infusions of such cells resulted in transformation of a pathologic process from destructive into productive one. moreover, clinical manifestations of sepsis were controlled in % and mortality was decreased from % to %. conclusions: the use of eytokines themselves as well as cytokine-treated lymphoeytes permits to control the disease and leads to the mortnlity decrease owing to stimulation of host defence mechanisms. background: although red blood cell transfusions (rbct) are used to increase oxygen availability in septic patients, several lines of evidence suggest that rbct may actually worsen tissue hypoxia. thus, rbct may negatively influence outcome of septic patients. objectives: to determine the association of ) rbct ; ) number of units transfused; and ) mean age of the units transfused on the first day of transfusion with mortality of critically ill septic patients. methods: we prospectively identified patients who met strict criteria for sepsis syndrome (ss) seen in the icu of st. paul's hospital from to and excluded patients who died in the first days after the onset of sepsis. we recorded clinical characteristics, multiple system organ failure score, and apache ii at onset of sepsis. then, we retrospectively recorded the total number and age of rbc units transfused during the first days after onset of sepsis. overall -day mortality was %. results: the main results are shown in the table. the mortality of patients who received rbct was nearly double the mortality of those who did not receive rbct even after adjusting for severity of illness using apache ii. objectives: gastric mucosal acidosis is frequently observed in patients with sepsis. the aim of this study was to determine whether volume infusion using pentaspan| decreases abnormal gastric mucosal pco (pico ) in patients who have sepsis syndrome (ss) who have already been resuscitated using clinical endpoints. methods: we prospectively identified patients who met strict criteria for ss, had a pulmonary artery catheter and a gastric tonometer in place, and pico > mmhg. pentaspan| ( ml) was infused in rain. measurements of hemodynamics, hemoglobin, arterial lactate, blood gas analysis, and pico were performed before and repeated miff and hr after pentaspun| infusion. we calculated the pico -arterial pco' difference (pico -paco ) and phi (using henderson-hasselbach equation). anova was used to assess statistical significance. results: all patients werereceiving adrenergie drugs. map was : : mmhg and lactate . : : . mmol/l. pentaspan| increased ci by % (p< . ) but did not change pico ( and increase m oxygen o* wery were simimny achieved in both groups. nevertheless, epinephrine was associated with a lactic acidosis and increased laetate/pyruvatemia ratio (l/p) that evoke a dysoxia rather than a metabolic effect. an higher gastric mucosal pco in the ep group compared to nor-rob suggests the hypothesis of an anaerobic production of co in favor of a splanchnic hypoxia. in both group, arterial ketone body ratio that reflects hepatic mitochondrial redox state, compared to a control group without shock was decreased but increased between and hours after restoration of arterial pressure. the association norepinephrine-dobutamine seems to be better for splanehnic circulation than epinephrine and should be used for dopamine resistant septic shock. moreover, the increase in arterial pressure with nor-dob improved gastric mueosal ph and hepatic mitochondrial redox state and argue to reconsider arterial pressure as a significant goal for resuscitation in septic shock. conclusion: significantly higher malondialdehyde and ghitathione levels and glutathione-peroxidase activity in group ns at the end of icu stay were related to mortality these findings indicate an increased generation of free oxygen radicals together with increased anfioxidant activity in this group and sapport the employment of antioxidant interventions in critically ill patients. oblecfives: to determine the role of nitric oxide (no) in the mechanism of septic shock induced by isolated limb perfuslen with recombinant tnfcr methods: we have measured tnfr~ and metebo~ites of no in patients with signs ot septic shock following treatment with isolated limb perfusion for nonresectable soft tissue tumors and melanomas of a limb. perfuslen was carried out with melphalan (burroughs wellcome) and recombinant tnfcr (boehringer). tnfc~ was determined by specific radiometric assay (medgenix diagnostics), nitrate and nitrite were measured with a modification of the guess reaction ~. results: results are shown in the table. conclusions: during isolated limb pedusion with recombinant tnf~ very high levels of tnfcr were measured in arterial blood in patients. they all showed signs of severe sepsis syndrome with shock from vasodilafion, probably due to leak of recombinant tnft~ from the peduslen circuit to the systemic circulation. tnfc~-induced vasodilation was not accompanied by a rise in serum no-metsbolites. our findings do not confirm the widely accepted theory, mainly based on animal experiments, that genera• of no is the key pathogenefic mechanism in septic vasodilafion , nor that tnfrt invariably induces forreafion of no. the precise mechanism of shock in these patients remains to be elucidated. references: . moshage h, kok b, huizenga jr, jansen plm nitrite and nitrate determinaiions in plasma: a critical evaluation. clin chem : / . . moncada s, higgs a. the l-argioine-nitrio oxide pathway. n engl j med ; : - ec is a commonly used for prolonged, stable animal anesthesia. noting that the hypotension after iv lps was attenuated by ec, we hypothesized ec also protects against lps toxicity. sprague-dawley rats received ip saline (s), thiobutabarbita mg/kg (tb), or varied doses of ec, followed hours later by bolus mg/kg iv lps. -day survival is shown below: group: s tb ec( . gmikgi ec( .sgm/kg) ec(i. gm/kg) alive (n) t ~ total (n) s s "signiflcant;y different from all other groups, p< . s / rats given lps followed hours later by ec ( . gm/kg) also died. additional rats were treated with s (n= ) or gm/kg ec (n= ) followed by mg/kg lps, then sacrificed at hours. blood glucose (bg, mg/dl),.hematocrit (hct), leukocyte count (wsc/mm~ platelet count (pltxl ~/mm ), bicarbonate (hco, mg/dl), gross bowel hemorrhage (bh, - scale) and lung myeioperoxidase activity (mpo, ~vmirvgm wet lung) are shown below ( we conclude that ec reduces the lethality and multiple organ toxit;~ty of lps. its diverse effects suggest asite of activity upstream from the cytokine cascade. these results are important for studies of lps which may use ec anesthesia and may have potential in the therapy of septic shock. [zo = hz impedance (z; {dyn.sec.cm " }); zl = first harmonic z; zc = characteristic z; z ph. = t'trst harmonic phase angle {radians}; f, #, * at least p < . between fio . and . , fio . and fio . &no - . _+ . - . _+ . # - . + . m - . + . * - . + . * - . + . * - . _+ . * in hyperoxia, compared to dogs at the same q, minipigs had a higher ppa ( + rnmhg versus + mmhg; p < . ). hypoxia increased (ppa-ppao) at all levels of q by an average of mmi-ig in minipigs and mmhg in dogs. inhaled no inhibited hypoxia-induced (ppao-ppa)/q changes in both species. conclusions: we conclude ~ that the minipig is an animal model of elevated pulmonary vascular resistance and impedance, and ~ that hypoxia-induced alterations in pvz spectrum are due to changes of resistance in small arteries. objectives: ) to determine the toxicity of ng-monomethyi-larginine (nma) administered by intravenous bolus to patients with refractory septic shock. ) to investigate the biologic activity of nitric oxide synthase inhibitors in septic shock. methods: from august to january , thirteen patients with vasopressor refractory septic shock received nma intravenously in escalating doses from to mg/kg. results: no hepatic, renal, gastrointestinal, or hematologic toxicity was observed at doses of nma as high as mg/kg. significant biological activity was observed at all dose levels consisting of increased blood pressure (systolic blood pressure from . mm hg + . to . _+ . s.e.m., p= . , systemic vascular resistance ( + to + dyne.sec/ cm s, p=. ), and a decrease in vasopressor requirements. the magnitude and duration of these effect were dose dependent. decreased cardiac output ( . _+ . to . _+ . i/min p=. ) and increased pulmonary artery pressure ( . _+ . to . _+ . mm hg; p=. ) were also observed. no significant effects on heart rate, pulmonary capillary wedge pressure, or central venous pressure were observed. four of patients survived for more than days, patients died of cancer complications (all patients had maintained blood pressure for h on nma) and patients died of complication attributable to septic shock (mods, ards, dic, refractory hypotension), and patient was unevaluable. conclusions: no adverse clinical effects have been observed in patients receiving bolus doses of nma as high as mg/kg. the increased pulmonary artery pressures observed in septic shock patients is further augmented by nma and may limit the dose which can be administered by intravenous bolus. other schedules of drug dosing may attenuate this effect. glucose-insulin-potassium (gik) solutions have been shown to improve cardiac contractility and increase oxygen availability in experimental and clinical settings of septic shock. several mechanisms have been proposed to explain these effects including a direct improvemeut of the energy balance by glucose, a direct influence of insulin on cardiac performance or an increase in intravascular volume due to the hyperosmolarity of the solution. to explore the role of hyperosmolapity, we compared the effects of gik to those of a isoosmolar hypertonic saliue solutiou in endotoxin shock in dogs. methods : the study included mongrel dogs ( • pentobarbitalanesthetized aud mechanically ventilated with air. thirty minutes after the intravenotls administration of mg/kg of e. coli endotoxin, the dogs were randomized to receive a ml/kg infusion in rain of a hypertonic ( mosm]l) solution iucludiug either a mixture of glucose % with u insulin and meq kcl/l (glk-group ) or hydroxyethyl starch . % in naci . % (hes-group ). in each dog, a . % saline infi~sion was continued to maintain the puhnonary arlery occluded pressure at baseline level. hemodynamic, blood gas aualysis and laboratory data were collecled at baseline and miu, rain, rain, and nunutes later.. results : eudotoxin administration was followed by a fall in mean arterial pressure (map) aud cardiac index (ci) and a rise in blood lactate levels. resuscitation with either gik or hes hypertoaic solutions resulted in similm increases in map, ci, oxygen delivery and left ventricular stroke index (table ) . we conclude that during resuscitation from endotoxic shock the use of gik solutions is not superior to hypertouic hes solutions. the higher blood lactate levels observed in the dogs receiving gik can be attributed to the glucose metabolism. , for group , for group ) were drawn and immediately analysed at ~ using the abl radiometer for po , pco and ph, and the osm radiometer for hbo %, hbco% and methb%. psost (i.e. the ps at ph= . , pco = mmhg and temperature at ~ c) was calculated automatically by the instruments on mixed venous blood, as was the ps "in vivo" (i.e. the ps at the patient's value of ph, pcoz and temperature), using siggaard-andersen's algorithm. the data were compared by the one-way anova test and by the t-test for paired and unpaired samples. results: the mean resulting values (in mmhg) with the statistical differences are shown in table i. in addition, the time series analysis shows the mean ps~st values as statistically below the psin vivo" in the septic patients while the opposite is shown for the cardiac patients. no differences in the time analysis are demonstrated for the second group. a possible clinical significance may be drawn from these different behaviours. objectives:toxemia degree and humoral immunity condition have been studied in patients aged from to with progressive course of sepsis and polyorganic insufficience. methods: such toxemia and humoral immunity findings as lencositlcindex of toxication (lii), level of oligopeptides of the middle molecular mass registered at the wave length of nm(mmi) & nm (mm ), distribution index (id), immunoglobulins a,m,g, concentration of circulating immunocomplexes (cici & cic ) and also some clinical and biochemical findings on the , , day after the operation serve as criteria for treatment effect. results: it was founded that in intensive therapy and detoxication, level of lii is successively decreased from . ~ . to . +. on the -th day after the operation. true decrease of the level mm from . ~. to . +. un & optimal density and increase of distribution index from . to . are argued. conclusions: in studlng the dynamics of the immunoglobulin's spectrum and the true increase of immunoglobulin g level from . +. g/i to i . +. g/i on the -th day after the operation simultaneously with the decrease of cic from . ~ to . ~ . (p . ) were founded. some stages of the investigation true increase of lymphocytes from . + . % to . + . % was noted and it appeared to be a favourable prognosis finding for disease outcome. high correlation dependence between bacillus-and segmentonuclear neutrophils and immunoglobullns g & m (r=. -. in p<. ) was discovered and it also showed positive dynamics of the course of the disease. a year old male patient was admitted to the icu with severe paraquat poisoning. treatment consisted of gastic lavage and oral administration of fullers earth. because of very high plasma levels hemodialysis together with charcoal hemoperfusion was started within one hour after admission. this treatment was further continued by continuous veno-venous hemofiltration in order to remove the circulating paraquat and also circulating cytokines. nevertheless patient s condition worsened necessitating artificial. ventilation and hemodynamic support. patient died hours after admission of acute multiple organ failure due to paraquat poisoning. serum levels of paraquat were determined by colorimetric method (table) . levels of interleukin (il ) and (il ), tumor necrosis factor (tnf-alpha), interleukin i receptor antagonist (il ra) were determined both in plasma and ultrafiltrate ( q~!ectives : evaluate in critically ill patients the effects of tow-dose dopamine on gastric mucosal blood flow (gmbf) using laser-doppler flowmetry, a continuous non invasive method of assessing microcirculation. methods : patients requiring both mechanical ventilation and pulmonary artery catheterization for multiple trauma (n= ), ards (n= ) and pancreatitis (n=l) were included. in each patient, the laser-doppler (ld) probe was inserted through a naso-gastric tube. the ld signal is proportional to the number of red blood cells moving in the measuring volume and the mean velocity of these cells. when the ld signal was satisfactory, an aspiration was created into a catheter which was fixed in parallel to the ld probe, to maintain the tip of the probe against the gastric wall at the site of measurement. data (systemic hemodynamic parameters and gmbf) were obtained at the end of a rain resting period (baseline), then min after dopamine ( mcg/kg/min) infusion, and finally rain after the end of dopamine infusion (recovery gmbf _+ (perfusion units) gmbf ~a% vs baseline) * p < . vs "baseline" and "recovery". conclusions : ) despite a slight increase in co (+ %), the dramatical increase in gmbf (+ %) with dopamine, strongly suggests a selective vasodilator effect of low-dose dopamine on gasaic mucosal perfusion. ) laser-doppler flowmetry appears a promising method to assess gastric microcircalation in critically ill patients. increasing evidence suggests that the activation of inos is the final common pathway for vasodilation in human sepsis associated with endotoxic shock. activation of the cellular immune system induces the excessive release of the pteridines neopterin (n) and , -dihydroneopterin (nh ) by human macrophages/monocytes. besides the well established diagnostic value of pteridines in several inflammatory diseases, it is speculated that these substances per se exhibit biochemical functions. thus we hypothesize that pteridines can modulate inos gene expression in vascular smooth muscle cells (vsmc) in vilro. cdtured rat aortic vsmc from female wistar kyoto rats were incubated with n ( pm), nh ( ilm), lipopolysaccharide (lps, ~g/ml), and interferone-~/(ifn-~/, u/ml) for h, respectively, inos gene expression was measured by competitive reverse transcription polymerase chain reaction. the results are summarized in the table. the present study demonstxates a neopterin induced increase in inos mrna expression at the transcriptional level in vsmc. while coincuhation of cells with n + lps resulted in an additive effect on inos gene expression, n + ifn- seem to have a more than additive effect nh did not alter inos mrna synthesis, but it suppresses the lps as well as the ifn-yinduced augmentation of inos gene expression. we speculate that this pteridine-mediated modulation of inos gene expression is involved in the regulation of the vascular tone in endotoxic septic shock. the relationship of sepsis and coagulation abnormalities is well known, mainly in severe sepsis and septic shock. still farther, the extreme expression of hemostasis abnormalities (disseminated intravascular coagulation) in sepsis, has been extensively described. we studied the changes in several coagulation and fibrinolysis markers in septic patients, trying to correlate them with the evolution of the sepsis phenomenon, with an emphasis in its early stages, where therapeutic intervention might be more drastic. in patients, with sepsis, with severe sepsis and with septic shock, as well as in healthy volunteers (control group) we measured : platelet (ptl), coagulation markers [fxii, fvii, fviii, fvw, fibrinogen (fibr) we conclude that all parts of the coagulation system are gradually changed during the evolution of sepsis phenomenon , even in the earliest stage of sepsis. the expression of an inducible nitric oxide (no) synthase (inos) plays a major role in the pathophysiology of septic shock (ss). inhibition of inos could therefore be of therapeutic value. however, such an inhibition has been shown to be detrimental, increasing tissue anoxia (and end-organ damage), possibly through the simultaneous blockade of constitutive nos (cnos). thus, selective inhibition of inos might be more suitable. we evaluated the effects of l-canavanine (can), a more potent inhibitor of inos than cnos, in an animal model of ss. method: in anesthetized rats, catheters were placed in the femoral vein and artery. rats were given an iv bolus of lipopolysaccharide (lps, mg/kg), at baseline (to). after h (t ), rats received at random an infusion of either can ( mg/kg/h; can group, n=l ) or an equivalent volume of . % naci ( cc/kg/h; nac group, n= ), giyen over h (t -t ). a third group (sham group, n= ) received . % nac in place of lps, and then was treated like the nac group. mean blood pressure (mbp), blood lactate and nitrates (no ) were measured each h. glucose, creatinine and asat were also measured in rats (n= in each group). the can _+ * + "t . + . "~ . +_ . "t + " + " *p< . can vs naci ?p< . vs sham can suppressed the hypotension, reduced the hypoglycemia and hyperlactatemia, and attenuated the biological signs of renal and hepatic dysfunction induced by endotoxemia. these effects were associated with a lesser elevation of blood no , confirming a partial inhibition of inos. conclusion: l-canavanine attenuates the hemodynamic and metabolic consequences of endotoxemia in the rat. these effects may be related to a partial inhibition of inos. they contrast with the deleterious effects described with non selective inhibitors of nos. l-canavanine could become a new tool for the treatment of septic shock. rocalc tonin :marker of sepsis, ii~flammaiiur% t~ boifi .cheval*~ jf.timsit*, m.assicot**, b.misset*,/.carlet*, c.bohuon** saint joseph heap, paris**biochemistry institut g roussy, villejuif, ce bi~)l~i~ttectives_: high serum levels of procalcitoaln (proct) have been shown to be ~ss-ocinted with bacterial infection. however, few data exist about the ability of proct to differenciate septic shock and shock from other origin in which an activation of intlmmamtory mediators has been also demonstrated. methods: thirteen patients with bacterial septic shock (ss), patients with non septic shock (nss), patients with bacterial infection without shock ( nf) and icu patients without shock and without infection (control) were compared for proct levels at dayl, , , , . patients were classified blindly and independently fi'om proct results. twelve patients were excluded because any classification was impossible due to mixed pathology. proct was measured with ebemoluminescenee (brahms diagnostica-berlin). results: dayl, proct levels are significantly different between the four groups. dayl proct levels are correlated with saps (p= . ), infection ( . +_ vs _+ ,p= . ), shock ( _+ vs +.- ,p= . ), death at day ( _+ vs _+ ,p= . ). when shock and infection are introduced in multifactor &nov& only infection remains correlated with day proct levels ( = . ) in patients with shock, dayl proct levels are correlated with saps, infection and death at day , but not with arterial lactate levels (p= . ), white blood calls (p= . ) or fever (p= . ). proct levels remain higher i~i septic shock patients at day , and ( figure) . i c edpsion: procalcitonin levels in the first three days of shock are differen[" between septic and non septic shock patients. in patients with diseases known to induce acute an inflammatory process, procaldtonin seems to be a marker o~ infection. obiectives-to evaluate the effect of endotoxic shock on the distribution of blood flow between the mucosal and the muscular layer of the intestinal wall. methods: in fasted pigs, mean aortic pressure (map, mm hg), cardiac output (co, ml/min-kg),superior mesenteric artery flow (q sma, ml/min.kg), and phi, where measured before (control) and after i.v. endotoxin ( gg/kg). the blood flow to the mucosal and the muscular layer was measured in regions (proximal jejunum (pj), mid-small intestine (mi) and terminal ileum (ti)) by colored microspheres, using adjacent samples in each region. the muscular layer was separated from the mucosa by blunt dissection, and the flow determined independently in each layer. results: endotoxin with fluid resuscitation induced the expected decrease in map ( . _+ . vs . -+ . , p< . ), and phi ( . !-_ . vs . _+ . , p< . ), with a constant co ( _+ vs _+ , p= . ) and qst, aa ( . _+ . vs . _+ . , p= . ). the results of regional pertusion are presented in the table. (flow in ml/rain g of tissue; mean _+ sem ; * p< . vs control by two-way anova) conclusions-these data indicate that the mucosal flow increased during septic shock. they suggest that a decrease in phi may be due to hypoper~usion of the muscular layer or to metabolic alterations within the mucosa, despite a % increase in flow. acute increase in wbc count (from a mean of lo.oo mm a to o /mm~), between the rd and the th day of therapy. there was a decline of the wbc count to an average of about . mm a after decreasing the daily dose of the medication to mcg there was no increase in tile absolute number of the eosinophils during the whole course of the medication. there was a slight decrease in the c complement between . to . g/i. normal values . to . g/i there was no change in c values. conclusions : an early increase in wbc count was observed ( rd day) without subsequent increase in the number of immature types from bone marrow, probably due to the mobilization of wbc from the periphery and this increase was dose dependent. there was a slight decrease in c fraction of complement, probably due to the consumption of this fraction in the process of opsonization. no adverse effects of the medication were observed, during the treatment with the above dose. these data sugest that cm csf may be a useful complement to tile main antimlcrobial treat,nent ~ of septic [cu patients. objectives: as part of a large multicentric, placebo-controlled, randomized clinical trial investigating the effects of interleukin- receptor antagonist (ii-lra) in the treatment of severe sepsis and septic shock, this substudy evaluated in dem.il the acute hemodynamic effects of ii-lra in patients who were invasively monitored. methods: in a total of evaluable patients in whom vasoactive support was little altered, hemodynamic measurements were performed at baseline (twice), and i hour, h, h, h, h, and h after the administration of mg/kg (n= ) or mg/kg (n= ) of i - ra or the corresponding placebo (n = ). / patients ( %) were treated with adrenergie agents and / ( %) with mechanical ventilation. data were analyzed by a kruskal-wallis test. results: during the study, there was no significant difference with time or between groups in arterial pressure, cardiac filling pressures, cardiac index or left ventricular stroke work (figure). burmester, "~ man and h. djonlagic medical university (internal medicine, "cardiology, *'microbiology) and "**southern city hospital, lfibeck, germany obiectives: evaluation of the incidence of bacteremia and sepsis in patients with nontyphoidal salmonella (s.) infections, specification of risk factors, need of icu treatment, clinical course, and mortality in the group of the patients who developed septic complications. methods: data of all patients with microbiologically proven s. infections hospitalized in the medical university of lobeck and in the southern city hospital of l beck from to . results: within the observation period s. was isolated from the stool cultures of patients. in patients (g m, f, median age yrs) s. could be detected in blood cultures ( s. enteritidis, s. typhimurium). in addition, in of these patients s. was also isolated from other specimens (urine, liquor, and tissue fluids derived from abscess punctures). in all patients with positive blood cultures the clinical course of s, infection was complicated: ? patients developed mof (acute renal failure, ards, hemodynamic instability, dic) and required icu treatment for at least up to days, of the patients died. the predisposing disorders in the patients with s. bacteremia were (n=): aids ( ), immunosuppressive drugs ( ), chronic alcoholism ( ), malignancies ( ), none ( ). septic complications in patients with nontyphoidal s, infections are relatively rare (in this study < % of all hospitalized patients with microbiologically proven salmonellosis) but severe (mortality of approx. %). patients at risk for a complicated clinical course are predominantly those with predisposing disorders but occasionally also patients without evidence for an underlying disease. age (yr) + + death (n) duration of shock (h) + + noradrenaline (rag/h) , _+ + temperature (~ , + , + pvr (dynxsecxcm - ) + + co (ljmin) , _+ , , + , lactate (mmol/l) + , , + interleukin- (pg/ml) _+ + interleukin- (pg/ml) , _+ , , + , tnf-alpha (pg/ml) , + , + neopterin (nmol/l) , + , + crp (rag/l) _+ +_ pro-ct (ng/ml) , + , , + there was no positive correlation between serum lactate levels, degree of shock, hypoxemia and pro-ct positivity. pts with septic shock of bacterial origin entirely developed hyperprocalcitoninemia, whereas pts with cardiogenic shock, who expired within h did not. however, in late cardiogenic shock (> h) all pts developed fever of unknown origin and consecutive hyperprocalcitoninemia. these data suggest bacterial inflammation and/or mucosal translocation of bacterial products in pts with prolonged cardiogenic shock. the use of a loading dose of quinine ( . mg/kg base in h) is recommended in previously untreated patients (pts) with sfm, particularly in multi-drug resistance areas. this protocol is difficult to validate, since the viability of microorganisms is not assessed routinely in parasitology laboratories. objectives: to examine the evolution of parasite viability during the early phase of therapy of sfm. methods: from / to / , pts with sfm (who ) treated with iv quinine for less than h were included prospectively. blood samples were collected at o, , , , , and h viability was assessed by culturing parasitized red blood cells in the presence of h-hypoxanthine, and radioactivity was determined at h by scintillation counting. viability was expressed as the percentage of radioactivity compared to the initial sample. plasma quinine was determined by liquid chromatography. tile ratio plasma quinine (pmol/ )xlo /icso for quinine (nmo]/]) was called the parasiticida/ index. results: pts were included, • saps . -+ . . the initial parasitemia was t. + . %. complications of malaria were coma ( pts), shock ( pts), renal failure ( pts) and acute lung injury ( pts). all strains were sensitive to quinine (icso -- nmol/ ). in pts who were not given a loading dose, parasite viability increased by and %, with concomitantly low quinine levels ( and #mow] at h); pt died. in pts that received a loading dose (serum quinine at h = . -- . ~mol/]) a marked decrease of parasite viability (by +_ % at h) was shown. viability was inversely correlated with plasma quinine (r=. , p-.o ) and parasiticidal index (r=. , p-.o ). conclusions: even with fully sensitive strains, the use of a loading dose of quinine seems warranted in severe falciparum malaria in order to reach rapidly adequate plasma quinine ]evels, necessary to inhibit significantly parasite viability. l nkka, e ruokonell j takala. critical care research program, department of intensive care, kuopio univ hospital, finland objective: to determine the incidence of positive blood cultures, their microbial subgroups and to evaluate the outcome of icu patients with different bacleremias. material and methods: we analysed all positive blood cultures in consecutive admission to a university hospital icu in - and the icu and hospital survival of the bacteremia patients. during these years patients had positive blood cultures that were considered as clinically relevant, excluding colonizations or contanfinations. results: patients with positive blood cultures had an icu survival of . % (vs. , % in all icu patients) and six month survival of . % (vs. . % in all icu patients). the most common bacteria were enterobacteriaceae ( , %), staphylococcus aureus ( , %) , coagulase negative staphylococci ( . %), pseudomonas ( . %) and slieptococci ( . %). obiectives: to evaluate prognostic factors and mortality in consecutive patients (pts) with hiv infection and septic shock. methods: from - to - , records of consecutivepts with septic shock (crit care med , : - ) admitted to the icu were reviewed retrospectively. results: among pts with septic shock admitted during the study period, had hiv infection- of whom had aids-(gr. i) and were hiv-negative (gr. ill. ten gr. ii pts ( %) were irnmunosuppressed because of neoplastic or immune dlsease. mechanica] ventilation was required in % gr. i and % gr. ii pts in gr . i pts ( %) a multivariate analysis demonstrated that hiv infection and sap i were independently predictive of death in pts with septic shock. ~onclusions: evidence of increased mortality, number of organ failures and higher severity scores (saps i does not take into account immunosuppression) is demonstrated in hi v-positive pts, infection with hiv appears to be an independent prognostic factor in pts with septic shock. the frequency of opportunistic infections (often responsible for delayed diagnosis and treatment) may contribute to the poor prognosis in this population. obiectives: to determine interleukin (il)-i levels in plasma of patients with sepsis and septic shock. to analyze the relationship between plasma il- and the proinflammatory mediators, tumor necrosis factor-aifa (tnf) and il- , the underlying severity of the disease and the evolution of patients with sepsis. methods: we studied critically ill patients ( men, women; - years old) in three diferents groups. group i: patients without evidence of infection, group i : patients with sepsis and with septic shock (group iii). we measured plasma il-lo, tnf and il- levels in the first hours of diagnosis. severity of illness was estimated with the acute physiology and chronic health evaluation (apache ii) scoring sytem. results: plasma levels of il- were higher in group iii (median, pg/ml; range, - pg/ml) than in group ii (median, pg/ml; range, - pg/ml; p <. ) and group i (median, pg/ml; range, - pg/ml; p <. ). median il- concentrations did not differ among patients who survived (median pg/ml; range, - pg/ml) and those who died during the overall follow-up period ( days) (median, ; range, - pg/ml); but patients who died in short-term (< hours) with catecholamine-refractory hypotension showed the highest concentrations of il-io (median, pg/ml; range, - pg/ml). in patients with bacteriemia ( %), levels of il- were higher (median, pg/ml; range, - pg/ml) than in those with negative blood culture (median, , pg/ml; range - . pg/ml; p< . ). there was a good correlation between plasma il-io concentration and levels of tnf (r= . ; p < . ) and il- (r= . ; p < . ). the correlation between levels of il- and the apache ii score was significant only in the septic shock group (r= . ; p <. ). conclusions: in septic shock, il-io and proinflammatory citokines are released in high concentrations. the significant correlation observed in patients with septic shock between il- levels and apache ii, short-term death and bacteriemia can possibly be explained by the massive inflammatory response in septic shock with fulminant course. intensive care department -calmette hospital - lille -france. in septic shock, inadequate splanchnic blood flow may play a prominent role in the pathogenesis of multiple organ failure. measurement of gastric phi has been propose to evaluate tissue oxygenation in splanchnic organs. objectives: to compare gastric phi values with hepatic icg clearance, an index of liver blood flow and function ; to determine if one of these two methods could be proposed to assess the entire splanctmic peffusion in septic shock. methods : patients (age : • years ; saps ii : • were prospectively investigated (septic shock : bone criteria). following parameters were collected during hours : systemic hemodynamic parameters (swan ganz catheter a h -ref computer -baxter lab.), calculated systemic oxygen transport (do ), oxygen consumption (vo ) by indirect calorimetry (deltatrac datex lab.), gastric intramucosal pco (pco ss) and phi (trip -ngs catheter -tonometrics lab.) and plasma disappearance rate of icg (pdr dye) (femoral artery fiberoptic/thermistor catheter , cold z computer -pulsian medizintechnik, germany). correlations were performed using a linear regression. elevated in all days with the highest value in second and third days of treatment. nonsurvivors had higher values of these parameters than survivors but differences did not reach statistical significance. another trend of changes were observed in selectin p (gmp- ) concentration. in all patients concentrations measured were elevated but in survivors after not significant decrease this parameter in second day another one had simmilar values. in patients who died we noted significant decrease in third day (p < . ) whereafter prominent increase, significant after seventh day, in comparison to third day value and value in survivors group. icam- concentrations in all patients reached high levels and in nonsurvivors after four day of treatment significant increase in comparison to survivors we found. conclusions: multiple trauma complicated with sepsis induce rapid elevation of concentrations of il- , il- and increased expressior of adhession molecules (selectin e, p, icam- ) measure of icam- and selectin p concentration determine lung injury severity and prognosis as to health and life. (clp) .pathophysiology of cip is unclear, but changes in regional bloodflow may be a ~ignificant factor. nerve blood flow (nbf)is reduced in rat models of hemorrhagic shock (g),but no information is available in sepsis. we studied the comparative effect of acute endotoxemic shock {etx)& h on perfusion of rat sciatic nerve. methods: male sprague-dawley rats were anesthetized with pentobarbital (ip), instrumented with a tracheostomy, carotid arterial & venous catheters and mechanically ventilated (fi = . ). the left sciatic nerve was surgically exposed. monitored variables included: a) mean arterial pressure (map,mmhg) ,b) nbf (ml/ o g/min) by laser doppler flow meter,c) nerve internal arterial diameter (id ~ m) by video image shearing and splitting method. after stable baseline measurements were obtained, acute hypotension was induced by randomly assigning the rats to etx ( . b , difco) in saline at mg/kg or h. both interventions produced % reduction in map within min., which recovered to baseline values spontaneously in etx group, & by reinfusion of heparinized withdrawn blood in m. data were analyzed by linear regression, two-way repeated measures analysis of variance followed by bonferroni-t method. experimental stages were:( )baseline, ( ) mid-point of map reduction; ( ) nadir of hypotension, ( )midpoint of map recovery, & ( ) after stable recovery of map. both etx & h induced shock result in similar reduction in nbf consistent with lack of autoregulation in peripheral nerve vessels independent of etiology. since cip is primarily associated with sepsis, it is not likely that acute reduction in nbf alone causes cip. direct & indirect neurotoxic effects of mediators of sepsis need to be evaluated. .':_.~::::o o:oc ., objectives : evaluate the relationship between il- , a cytokine which inhibits tnf, production and protects mice from endotoxin toxicity, and the other proinflammatory cylokines, tnf~, il and ils in severe sepsis and septic shock. methods : twenty-eight icu patients ( m, f, mean age + y) were studied as soon as they developped a severe sepsis (n = ) or a septic shock episode (n= ) as defined by a conference consensus in ( ). tnf~, il , il s and il- plasma levels were measured by immuno-radiometrie assays from medgenix (fleurus, belgium). lc mean and range. results : the comparisons between cytokine levels in severe sepsis versus septic shock were made using the logarithm of the value in order to normalize the distribution of data, and student test. il- plasma levels were higher in patients with septic shock than in patients in severe sepsis. there was a significant correlation (p < . ) between il- and tnf a (r= . ), il- and il~ (r = . ) and il- and il s (r = . ) as well as between il- and apache n score (r= . ). patients who died (n = ) had il- levels higher than patients who survived but this difference was not statistically significant ( pg/ml vs . pg/ml; p> . ). conclusions : during severe sepsis and sepsis shock, il- seems at least to follow the same evolution (increase in plasmatic level) with the severity of sepsis as the other cytokines. reference : ( ) crit care med ; : - . objectives: to evaluate the effects of steroids on hemodynamics and mortality in septic patients with konwn levels of cortisol concentration. methods: retrospectively we analyzed data ofpatients with documented septic shock who received steroids after assessment of adrenal function. in all patients hemodynamic parameters as well as the necessary vasoactive medication were assessed, before and hours after corticosteroid medication. immediately before administration of corticosteroids adrenal function was evaluated with cortisol levels before and after synthetic corticotropin ( . mg). finally we studied mortality. we defined a positive respons on corticosteroids as an elevation of map of at least mmhg and/or a decrease in the necessary vasoactive medication of at least % within hours. adrenal insufficiency was defined as a cortisol level after stimulation of less than nmol/l. results: of patients were found to respond to steroid medication, did not. mean cortisol levels before and after corticotropin were • and • nmol/l in the responder group (rg) and • and • nmol/l in the non responder group (nrg). in the rg out of ( %) were found to have an adrenal insufficiency, in the nrg out of ( %). in the rg -weeks mortality was . % (l out of ), the overall mortality % ( out of ). mortality in the nrg was % ( out of ) (p < . ) and % ( out of ) (p < . ) respectively. conclusions: in patients in septic shock there is a beneficial effect of steroids in case of adrenal insufficiency, but also in a subgroup with normal adrenal f{unction. obiectives: intercellular adhesion is a critical step in the accumulation of leukocytes. postischemic cardiac lymph has the capacity to stimulate icam-i. in the coronary microcirculation neutrophils can be trapped and in many cases obstruct capillaries, previously we found that troponin t (s-tnt) a marker for myocardial iechemia, was increased in septic patients. the aim of the study was to follow slcam- and s-tnt levels continuously starting at the beginning of sepsis. methods: patients were ingluded in this institutionally approved study after relatives had given their informed consent. all patients were included within hrs following the beginning of sepsis. blood was drawn every hrs in the first ;~ hrs, after hrs, followed once per day for days. s-tnt, icam- , elam (elisa's, boehringer mannheim inc, r&d systems ltd.) arterial and venous blood gases were determined, an ecg and a complete hemedynamir measurement including cardiac output were obtained. all patients received adequate volume and catecholamine therapy (norepinephrine, dopamine, dobutamine; median (range) . ( . - . ), . ( . - ), . ( . - . ) pg/kg/min, respectively). statistical analysis: wileoxon signed rank-sum test. . ( . - . ) . patients had s-tnt levels > . pg/l. of these died, whereas only of patients died with s-tnt values < . pg/l (p= . ). all patients that died had elevated sjcam- levels ( ilg/l:cut-off ) whereas in the survivor group only % had elevated icam- levels (p= , ). conclusions: increased slcam- and s-tnt levels were found during early sepsis in the majority of patients, a high sicam- and s-tnt value was associated with a higher mortality. the research of the noninvasive haemodynamic monitoring accelerated recently all over the world. the aim of our study was to test whether the changes of the haemodynamk parameters measured by impedance cardiography (icg) were corresponded to clinical changes in septic patients. investigations were performed on critically ill postoperative septic patients (their multiple organ failure score was - /with icg monitor. in cases the investigation~ were performed in septic shock. the measured parameters were: heart rate (hr), mean arterial pressure (map), cardiac output (co), peripherial resistance (svr),preejection period (pep), and ventricular ejection time (vet). these parameters were measured during - hours in every minutes, depending on the patients cl~tnical condition. results: at the septic patients the hr and the co ]~reased. in septic shock the co was significantly higher the svr lower than in the septic group. in the hr there was no difference between the two groups. in septic shock noradrenalin influenced more effectively the measured parameters than dobutamin. conclusion: the trend of the measured icg parameters correlated with the clinical changes of septic patient's state. the noninvasive haemodynamic monitoring by impedance cardiography helps the planning and leading the adequate intensive therapy of these critically ill septic patients. to evaluate the development of sirs, sepsis and septic shock in hospitalized patients with fever, a prospective study was performed on patients using previously defined criteria. methods: normotensive patients with fever (temperature > . ~ axillary), admitted to the department of internal medicine were evaluated for the existence of sirs during the first three days of the study and sepsis at inclusion. during a follow-up period of days the patients were daily evaluated for the development of sepsis or septic shock. results: most patients ( %) had or developed sirs within the first three days, patients ( %) did not. sepsis was present in % at inclusion. in patients with sirs, % did not progress to sepsis or septic shock, % progressed to sepsis (mean interval . • . days), and patient (< %) directly progressed from sirs to septic shock. in patients with sepsis, % progressed to septic shock (mean interval . • . days). sepsis was preceded by sirs in %. septic shock was preceded by sepsis in % and by sirs in %. conclusions: % of patients with fever in an internal medicine department develop sirs, or sepsis. furthermore, progression from sirs to sepsis or septic shock is poorly predicted by fever or sirs. nevertheless, all patients with septic shock were preceded bysirs or sepsis. taken together, this may indicate a severity hierarchy of the syndromes. however, fever, sirs and sepsis are relatively poor indicators of development of septic shock. this supports further research on additional predictors of septic shock. b. m.manuylov, v.b.skobelsky (moscow) in recent years sodium hypochlorite (sh) has been successfully used to eliminate pyo-septic complications. moreover, the mechanism of the sh effect on the immune system has not been sufficiently studied. the aim of the present investigation was to study the mechanism of sh effect in inflammatory pulmonary diseases. patients with double pneumonia were subjected to the evaluation. sh in the concentration of mg/l in the volume of - m / hours was administered by drop infusion into the central vein. to evaluate one of the defence systems the leukocytes activity by the chemoluminescence technique was studied. in all the patients baseline secondary immunodeficiency which was indicated by the decrease in the luminescence level was established. even hour after the sh administration the leukocytes activation exp-ressed by the enhancement of their chemoluminescence . - times was observed. this supports the available findings that accumulation and liberation of the oxygen active forms (ol'oh, ' , h ) are accompanied by the increased phagocytosis, i,e. the signs of "the oxydation explosion" testify to the favourable sh effect on the course of inflammation processes. the use of sh permitted to decrease the percentage of lethality in double pneumonia by % in the intensive care unit over the year. at the same time, excessive activation of free radical oxygen may be a damaging factor. therefore, precise individual control over the choice of concentration, dosage and the preparation administration rate is required. prospective, double-blind, placebo-controlled, trial of atiii substitution in sepsis r. a. balk objective: pilot study to evaluate the efficacy and safety of atiii substimtion therapy in patients with sepsis. efficacy assessed using change in mortality or organ failure/dysfunction. adult patients meeting a definition of sepsis and cared for in a tertiary care academic medical center in chicago were identified and prospectively randomied to receive either atiii (kybernin p) or placebo in a double-blind treatment protocol. all other therapy and patient management were under the direction of the patient's attending physician. all patient's were followed for days and the organ dysfunction/failure were scored using published scoring systems (jordan et al crit. care med. , goris et al arch. surg. , kuaus et al ann. surg. colldusions:wha~ we met the shomaeker objectiv% the mortality and the pro~os[s were i~ttc*. those criteria were obtained with file tradititmal t~ctor likr doht~mme, hut c.~vh ~,as ca in~aertam measure. they ac~s smxergically in the optimizatic~l of the fell vmtrictdar work index, tad fimdameatally cavh seox~s to have an impo.aat role in the better respiratory ev-altmtioa, leaving yet the possibility to coltrol the flui& r althou~l eomproved it's not aec~pt~xl file importmlce h* the diminution, of the sepsis modiat~lrs llke fnt and il- with h~wmotiltrafi(al, stopphlg the evolution to nmltiorganic failure mid de~easethe mortality. with ours clhlicals results, we could saythat cavii in multiol~atlie disfut~oa septic patieats, se~r~ to be an c xilna] supoa or troatmeat maesure. of anaesthesia and intensive therapy, medical university of prcs, p~csf hungary. objectives: since some biological effects of bacterial endotoxin require an interaction between the lps molecule and a serum factor(s), we hypothesized that lps-induced no production and cgmp accumulation in vascular smooth muscle cells (vsmc), a mechanism ~thought to underlie cardiovascular collapse associated with septic shock, is modulated by serum factor(s). methods: cultured vsmc from rat aorta were challenged with e. coli lps for - hours either in the presence or absence of fetal calf serum (fbs), and no production was monitored by radioimmunoassay determination of cgmp content of hci extracts. results: in the absence of serum, o ng/ml lps was required to increase cgmp levels, whereas the presence of % fbs shifted the lps concentration curve i times to the left. similarly to fbs, human serum also potentiated lps-induced cgmp accumulation. in contrast to lps, serum had no effect on cgmp accumulation elicited by sodium nitroprusside, a no releasing agent, suggesting that the sensitivity of vsmc to generate cgmp in response to exogenous no is not modulated by serum. heat inactivation (> ~ min) but not removal of small molecules (< , d) from the serum by dialysis, reduced the potentiation of cgmp accumulation by serum. time course studied indicated that serum is required within the first min of lps exposure to increase cgmp levels. to investigate whether the effect of serum is specific for lps, we treated the cells with increasing concentration of interleukin -~ (il-i). % fbs shifted the il-iinduced cgmp responses five times to the left. conclusions: our study suggests that lower concentrations of e. cell lps and il-i require a heat labile macromolecule in the serum in order to elicit no production. this factor is present in the human serum and it may play a potentially important role during no synthesis induction in vsmc. objective: to evaluate the factors of acquisition and the outcome of methicillin resistant staphylococcus aureus (mrsa) bacteremia in an intensive care unit (icu). methods: all patients in which bacterermia due to staphylococcus aureus developed > hours following admission to our icu, during a year period ( january through january ) were reviewed. patients (pts) were included, mean age , y (sd , ), saps , (sd , ), mac cabe ( and ) %, mortality directly due to sepsis %. pts had mrsa bacteremia and methicillin susceptible staph. aureus (mssa) . both groups were compared using the chi square (with correction of yates), fisher's exact, student's t or wilcoxon test. results: there was no statistically significant difference between mrssa and mssa regarding at age ( , + , vs , + , ) , saps ( , + , vs , + , ), use of vancomycin ( % vs %), mechanical ventilation ( % vs %), number of days (d) before the drawing of the first positive blood culture (median d, range - d vs median d, range - d). more mrsa than mssa pts had previous use of nonsteroidal anti-inflammatory drugs (nsaid) ( % vs % p< , ), central venous catheter infection due to staph.aureus ( , % vs % p< , ), but previous use of antibiotics was not significantly different ( , % vs %). the outcome of the bacteremic pts was not statistically different: saps at the first day of bacteremia ( , +_. , vs , + , ), severe sepsis and septic shock ( % vs %), persistence of the bacteremia ( % vs %), mortality directly due to bacteremia ( % vs %). conclusion: previous use of nsaid, infection of venous central catheter are more frequently associated with mrsa bacteremia. thus, similar to others studies (hershow infect control hosp epidemio ; : - ) , these results do not indicate that mrsa is associated with increased virulence. objectives: to closer definition of mosf formation mechanismes in nosocomial sepsis (ns) the complex clinicobiochemical, microbiological, immunological, functional exaroination of cases with ns had been done. methods: examination of cellular and humoral immunity, nonspecific immunologic reactivity, systemic and hepatic circulation, microbiological examination of blood,electro-and echocardiography, sonography and computer tomography of chest and abdomen organs were obligatory. autopsy findings of dead cases had been analized. results: in cases ( , %) opportunistic pathogen microscopic flora ( staphylococcus anreus,staphylococcus epidermidis, staphylococcus saprophyticus) had been found out in blood inoculations. in cases ( %) side by side with destructive process in lungs the bacterial endo-and myocarditis with blood circulation failure had been determined.in cases ( %) simultanious lesion of three organs (heart,lungs,liver) had been found. morphologic examinations of dead cases ( %) internal revealed involvement of them in mosf-syndrome.hyperplasia of adenohypophysis;sclerosis of adrenal glands cortical layer;perivascular brain oedema,paralysis of brain capillaries and plasmorrhagia, cerebral thrombosis and cerebral abscess,necrobiosis of epithelium tubules of the kidney,pletora of hepar, fatty and granular degeneration of hepatocytes had been found.atrophy of white pulp and hyperplasia of red pulp, supress of lymphoid tissue, plethora and formation of infarctious had been found in spleen. mentioned changes in spleen were indispensable in ns. conclusion: in ns spleen can not secure it functions to support and appropriate detoxication potencial of organism,elimination of microbes,toxines,antoallergenes. insolvency of immunological link of antimicrobic defence is the starting mechanism of mosf developmentin ns. %neviere, jl. chagnon, b. vallet, d. mathieu, n lebleu, f. wattel ] ept of intensive care, hop calmette, lille, france ~everal studies have described tiypoperfusion of intestine during sepsis. owever, it is unknow whether the mesenteric blood flow is associated with nucosal hypoperfusion. additionally, the effects of resuscitation on the ntestinal microcirculation remain controversial. bjectives : to describe the effects of endotoxin in a porcine model during ~hock and resuscitation. ~ethods : ten pigs ( kg) were anesthetized and instrumented for "neasurement of cardiovascular variables. gastric and gut oxygenation vere assessed by intra-mucosal ph and microvascular laser doppler lowmetry. after baseline data collection, a minute intravenous infusion )f escherichia colt (serotype h , sigma, st. louis, mo) was begun ~t a rate of pg/kg. an infusion of either saline at . ml/kg/min (group ; n= ) or saline and dobutamine at a rate of pg/kg/min (group ii; n= ) vas begun mn after the end of the endotoxin infusion. tesults : to td t ~ fl w fluid ioadin,q alone sfyras d, k perreas, e douzinas, k spanou, m pitaridis and c roussos critical care dpt, evangelismos hosp., athens univ, school of medicine. obiectives: much controversy exists concerning the beneficial effects of cvvh on sepsis. we studied the effects of cvvh application on septic patients with reference to the following parameters: i) survival rate ii) cytokines' removal and iii) timing of cwh onset. methods: patients with sepsis (criteria according to accp/sccm, ) underwent cvvh as soon as they developed renal failure or dysfunction (urinary output< ml/ h, cr> . mg/dl and bun> mgd'dl ). specimens were collected: blood samples before cvvh and therafter both blood and ultrafiltrate (uf) samples on , and hours. cytokines tnfa, i - and ii- were measured by the immunoassay method in all specimens (uf and plasma -p) and sieving coefficient ([uf]/[p]) and h solute mass transfer of tnf and i - were calculated (v h x [uf] ). the apache ii score before cvvh onset, the duration of icu stay and the timing of cwh application related to the sepsis onset in days (ta) were recorded.with respect the mortality two groups were formed, i.e. group a (survivors) and group b (non-survivors) . the morbidity period in days of those septic patients who died in the past year and were not subjected to cwh (group c) was compared to that of group b. results: group a included pts and group b pts with mean+sd age ( _+ vs _+ , ns) and apache scores( _+ vs -+ . , ns). the mean ta-+ sd was . + vs -+ , p< . . the mean_+se morbidity period of group b vs group c was _+ vs _+ . p< . . the mean values of cytokines are presented in the following figures. the sieving coefficient for tnf was . and for i - was . . the solute mass tranfer was -fold the actual plasma content at a given time. . o conclusions: i) early application of cvvh seems to favourably affect the outcome of septic patients, ii) cytokine plasma levels do not decrease although cytokine removal is substantial, iii) it seems that cwh application in sepsis of any stage helps to buy time for further treatment. the most commonly monitored variables in shock stages idclude : arterial pressure, heart rate, central venous pressure, pulmonary artery wedge pressure and cardiac index. with vigorous therapy it is possible to bring these values back into the normal range in both survivors and nonsurvivors. therapeutic goal in septic shock stages is to maximize the values of cardiac index, delivery (do ) and consumption (c ). objectives: the main purpose of this article is to determine the relationship betwee~ delivery an consumption as a sign of hypoxia. fifteen patitents with septic shock were treated with intention to maximize the value of ci,d and v . we compared the levels of these parameters between the survivors and nonsurvivors and found no significant differences after hours. high levels of do and v may not guarantee against tissue hypoxia in early stage of septic shock. zjar~iic, dj janjic, lj. gvozdenovic, a.komareevic. t.petrovic, &marjanovic, institute of surgery, novi sad, yugoslavia objectives: evaluation and mutual comparison of clinical signs, laboratory data and microbiological monitoring in the patients with burn sepsis. method: retrospective analysis of the recorded data of all burn patients treated in our department between january and december . specially attentions were given to data considering wound infection, positive haemocultures, positive urinocultures and characteristics of septic state. results: out of patient there were ( , ~) adults and ( , ( ~) children. almost two thirds of the patients ( - , ~) were males. the predominantly cause ( , ~) of children's burns was scalding b~y hot liquids and flame burns ~ , ~) in adult patients. the most frequdntly species isolated from surface swat~ were pseudomonas aeruginosa ( " in adult patients) and staphyloccocus epidermidis ( , % in children). in only five patients ( , ~ the haenmcultures were positive -pseudomonas aeruginosa was isolated in three and staphyloccocus aureus in two patients. urine infection was diagnosed in , % of all patients. the treatment protocol included use of imipenem and polyvalent pseudomonas vaccine again~ pseudomonas aeruginosa and vancomycin and aminoglycosides against staphylococcus aureus. total mortality rate in this group of burned patients was , ~, but the mortality rate caused of sepsis was low (i %) . conclusions: early detection of any signs of wound infection and symptoms of septic state is a foundation for prevention and treatment of burn sepsis. the burn sepsis could be reliable detected by continuously monitoring the patient's status and by systematic microbacteriological monitoring of the burned patients. hyperdynamic vasoplegic septic shock p.f. laterre, p. goffette, j. roeseler, j.p, fauville, a. poncelet, p. lonneux, m.s. l~eynaert. dept. of intensive care, st. luc univ. hospital, brussels, belgium. splanchnic ischemia is described as a common feature of septic shock and could determine the development of msof. therapy such as noradrenaline (na) aiming at improving blood pressure is expected to worsen splanchnic ischemia by its vasoconstrictive effect and subsequent reduction in intestinal blood flow. ob[ective: evaluate the effect of na on splanchnic blood flow. material and method : in a patient admitted for variceal bleeding, ards and sepsis with positive blood culture, a fiberoptie catheter was positionned in the portal vein after recanalisation of its portosystemic stent shunt. blood pressure (bp-mmhg) , ci, svr, do (vigilance ~ baxter), v (indirect colorimetry), arterial, mixed venous and portal vein blood gases, phi were determined before (to) and during (t ) na infusion ( , to , hcg/kg/min.) . changes in splanchnic flow were assessed by changes in portal oxygen saturation (sp ) and arterio-portal oxygen saturation gradient (sao, -spoe laterre, ,lp. pedgrim, th. dugernier, v. delrue, ph. hantson, p. mahieu, m.s. reynaert. dept. of intensive care, st. luc univ. hospital, brussels, belgium. aim of the study : prospective determination of plasma levels of in patients with ss and their correlation with the type of microorganism and outcome. material and methods : in patients (pts) with ss and severe sepsis, plasma levels of tnfti, ill-b, il and il were determined every hours for days and on day after fulfilling the criteria of ss and severe sepsis. results : in pts, sepsis was caused by a gram (-) microorganism, in pts by a gram (+) and in pts no microorganism was identified. there were survivors ( %) (s) and non-survivors ( %) (ns) . cytokines profiles and levels were not different between gram (+) and gram (-) sepsis. ill-b levels were seldom elevated whatever the group studied. tnfot and il- were significantly higher in ns than in s ( objective: to evaluate the effects on the nitric oxide synthase inhibitor l-n~ hcl ( c ) on myocardial performance in human septic shock. method: septic shock was defined as severe sepsis with either persistent hypotension (mean arterial pressure; map< mmhg) or the requirement for a noradrenaline (na) infusion >_ .i ]tg/kg/min with a map _< mmhg. cardiovascular support was limited to na _+ dobutamine (db), c was administered for up to h at a fixed dose-rate of either , . , , or mg/kg/h iv. during c infusion, na was to be reduced and if possible withdrawn, whilst maintaining map above mmhg and the cardiac index (ci) as clinically appropriate. assessments were made at baseline (t = ); at i h from the start of treatment (t = ); and at the end of treatment (t = ) with c . conclusions: c can restore systemic vascular tone in patients with septic shock enabling na therapy to be reduced and/or removed. the ci tends to fall whilst lv performance is sustained over time. c is a novel vasoacfive agent for the treatment of septic shock, which is undergoing further clinical evaluation. laterre, f. thys, e. danse, j.p. pelgrim, e. florence, z roeseler, m.s. r eynaert. dept, of intensive care, st. luc univ, hospital, brussels, belgium. therapy aiming at improving blood pressure and cardiac index in septic shock (ss) might have deleterious effects on regional blood flow. objectives : compare the influence of volume loading (vl), dobutamine (dobu) and noradrenaline (na) on sushepatic oxygen saturation (shoe) and svoe-sho, gradient in treated ss. material and methods : in patients with ss, ci (thermodilution) , doe, svo,. sho,, svoe-sho e gradient and lactate (l) were determined before (to) and after (t ); vl, dobu and na. results: in patients with treated ss, tests were performed (vl n= ; dobu n= ; na n= method: septic shock was defined as severe sepsis with either persistent hypotension (mean arterial pressure; map< mmhg) or the requirement for a noradrenaline (na) infusion ~> . ~g/kg/min with a map _< mmhg. cardiovascular support was limited to na + dobutamine (db), c was administered for up to h at a fixed dose-rate of either i, . , , or mg/kg/h iv. during c infusion, na was to be reduced and if possible withdrawn, whilst maintaining map above mmhg and the cardiac index (ci) as clinically appropriate. assessments were made at baseline (t = ); at h from the start of treatment (t = ); and at the end of treatment (t - ) with c . conclusions: c is a novel vasoactive agent that can sustain map in patients with septic shock, enabling na support to he reduced and/or removed. there is a tendency for the ci to fall during treatment, which may be reflex in response to the increase in systemic vascular tone. c is a promising new therapy for septic shock, which will now be evaluated in a randomised, placebo-controlled safety and efficacy study. k. guntupalli objective: to evaluate the acute effects of the nitric oxide synthase inhibitor l-n~ hc ( c ) on selected indices of organ function in patients with septic shock. method: septic shock was defined as severe sepsis with either persistent hypotension (mean arterial pressure; map < mmhg) or the requirement for a noradrenaline (na) infusion --> . [xg/kg/ min with a map _< mmirlg. cardiovascular support was limited to na + dobutamine. c was given for up to h at a fixed dose-rate of either , . , , or mg/kg/h iv. during c infusion, na was to be reduced and if possible withdrawn, whilst maintaining map above mmhg and the cardiac index (ci) as clinically appropriate. indices of organ function were assessed at baseline (t = ); at the end of treatment (t = ); and h after treatment (t = ) with c . results. -median values (* assessment made at h or when c discontinued). conclusions: there was no appareut dose-dependent adverse effect on these indices of organ function either during or after exposure to c . the plmelet count tended to fall whilst creadnine appeared to increase over time in all dose cohorts. this novel and promising therapy for septic shock will now be evaluated in a randomised, placebo-controlled safety and efficacy sludy. pharmacokinetics of c in patients with septic shock preliminary results z. hussein, b. jordan, c. fook-sheung, k. guntupalli objective: to evaluate the pharmacokinetics of the nitric oxide synthase inhibitor l-n~ hc ( cg ) given by continuous infusion for h in patients with septic shock. method: septic shock was defined as severe sepsis with either persistent hypotension (mean arterial pressure; map < mmhg) or the requirement for a noradrenaline (na) infusion --> . ~tg/kg/min with a map _< mmhg. cardiovascular support was limited to na • dobutamine. c was administered for up to h at a fixed dose-rate of either , . , , or mg/kg/h iv. plasma was collected from each patient over a h period and analysed for c . pharmacokinetic parameters were derived from plasma concentration-time profiles using non-compartmental pharmacokinetic analysis. results: the (cm~ -maximum plasma concentration; auc -area under curve; cl -plasma clearance; v,, s -steady state volume of distribution; t'/ -plasma elimination halflife). conclusion: the pharmacokinetics of c in patients with septic shock are dose-independent at infusion rates up to . mg/kg/h. at higher rates, clearance of c decreases without any marked change in volume of distribution. c metabolism may be partially saturable at dose-rates above . mg/kg/h. obiectives: investigate the effect of the no synthase inhibitor, l-nt-methylarginine hc ( c ) on the haemodynamics and survival rate in a conscious mouse model of endotoxin shock. methods: female cd- mice ( - g) were instrumented under gaseous anaesthesia (isofluorane, %) and connected to a swivel tether system for continuous monitoring of blood pressure and drug administration. results: after h recovery, endotoxin administration (e. col• :b , - . mgkg - i.v.) elevated the plasma concentration of nitrite/nitrate (nox) and caused a progressive fall in mean arterial pressure (map) from + to + mmhg (n= , p< . ) at h, with a survival rate at h, h and h of %, % and % respectively. c administered as a h continuous infusion ( mgkg-th -t i.v., n= ), h after endotoxin, inhibited the elevation of plasma nox and attenuated the fall in map from + to + mmhg (n= ) at h, with an improved survival rate at h, h and h of %, % and % respectively. conclusions: this study suggests that overproduction of no is involved in the hypotension and mortality characteristic of septic shock. inhibition of no synthase using c represents a novel and promising treatment for septic shock. cultures of e.coli ( , %) and candida( , %) were olso received from autopsy material of children;p.aeruginosa,unspored anaerobes,proteus sp.,s.aureus,b.pneumonia were found in the few cases. in adults the spectrum of bacterioflora was mo~ re limited speaking about the number of species and cultures. in generalized forms of bacterial pyo-septic pathology a wider specific spectrum of causative agents was revealed usua fly with associations. e.coli and k.pneumonia played the leading role in children as well as in adults. in general,k.pneumonia ( , %cultures) and common e.coli( , %)prevailed according to the date of microbiological investigations of authopsy material in pyo-septfc pathology in . objectives: .in spite of all clinical exertion sepsis is still the reason for high clinica! lethality. this study is characterizing the group of patients which survived a septi~ shock. methods: during a period of months all surgical patients on icu were registrated prospectively, more than parameters for each of them were documented'daily in a paradox file. results (see table ): of patients fulfilled the criterion of a septic shock (r. bone, ) , of them died at the lth day, while the surviving group of patients stayed almost days at icu. obiectives: to compare the effects of and % pentastarch solutions to a human albumin solution on oxygen delivery (do ) in septic patients. methods: this stud}, included septic patients with fever (t > ~ tachycardia flqr > /rain), tachypnea (rr > /min) or mechanical ventilation, leukocytosis (wbc> /mm ) or leukopcnla (wbc< ()/mm ) and a clinical source of infection, who required a fluid challenge. in each patient the pulmonary arterial occlusion pressure (paop) was < mmhg. patients were randomized to receive ml of % albunun (n:i ), hydroxyethyl starch (hes -mw /d.s. . ) % (n: ) or t % (n=i ); patients were also treated with adrenergic agents. results cardiac index (c ) increased significantly only in % lies (table) hemoglobin (hb) decreased significantly at min in the same group. there was not significant change in oxygen delivery ( do ). baseline ci alb . :: . (l'min/m ) hes % . = . hes % . polyneuropathy of the critically ill (pci ) is a well recognized complication, acquired in the course of severe illness. we undertook a prospective study, to estimate the severity, extension and time of onset of pci in a selected group of patient with established septic shock ( bone's criteria ). all patients received inotropic circulatory support and were mechanically ventilated. none received relaxants or aminoglycosides. pci was diagnose<by clinical investigation and by emg, and repeated weekly ( axonal degeneration ). after days ( day = onset of septic shock ) in % of the patients pci was demonstrable. after days % of the patients had pci. there was a strong correlation between pci and other mof and between pci and encephalopathy ( eeg ). we conclude that i. pci is a common complication after septic shock. . pci develops early in the course of illness ( % after days ). . pci can be considered as a part of mof, suggesting a common pathogenesis. objectives: we addressed two questions: ) can serial measurements of vapco and avph also reflect the onset of tissue hypoxia during endotoxemia? ) are whole body changes in vapco and avph associated with parallel changes in regional circulation? methods: in anesthetized, mechanically ventilated dogs exhaled gases were sampled for determination of oxygen consumption (vo ). a catheter was positioned into the pericardial cavity to induce cardiac tamponade. ultrasonic flow probes were placed around superior mesenteric, left renal and left femoral arteries, and the corresponding veins were cannulated. group l served as control (n= ), group (n-- ) received mg/kg of endotoxin. thirty min later, tamponade was induced to gradually reduce do . results: systemic do crit was higher ( . _+ . vs . + . ml/kg.min, p < . ) and critical oxygen extraction ratio (o ererit) was lower ( . +_ . vs . +_ . %, p< . ) in the endotoxie than in the control group. meslenteric and femoral do crit were higher in the endotoxic than in the control group ( . _+ . vs . _+ . mlll g tissue.min and . _+ . vs . _+ . ml/min, respectively, both p< . ). regional o ercrit were lower in we endotoxic than in the control group (mesenteric: . _+ . vs .i_+ . %, renal: . + . vs . -+ . % and femorah . -+ . vs . _+ . %, all p< . ). vapco and avph followed a similar pattern in both groups, increasing slightly before do crit was reached, but dramatically wh,en do fell below do crit. in the presence like in the absence of endotoxin, systemic and regional do crit calculated from vo , from vapco , or from avph were similar; do crit was also significantly higher in the mesenteric and the femoral beds of the endotoxic than the control dogs. systemic and regional do crit could be calculated from the blood lactate levels only in the control group. conclusions: during an acute reduction in blood flow i) vapco and avph can reflect hypoxic threshold in the presence like in the absence of endotoxemia. ) alterations in organ oxygen extraction capabilities induced by endotoxin can be reflected by regional changes in vapco and avph. objectives: enhanced nitric oxide (no) release has been incriminated in the vasodilation and myocardial depression characterizing septic shock, but the administration of no blockers has yielded equivocal results. the present study explored the systemic and regional effects of a no donor linsidomine (sin-i) during endotoxic shock. methods: anesthetized dogs received endotoxin ( mg/kg iv), followed min later by a saline infusion to keep cardiac filling pressures constant. oxygen uptake (vo ) was derived from the expired gas analysis. regional blood flow was measured by an ultrasonic flowmeter (transonic). results: the control endotoxie group (n= ) maintained low arterial pressure and systemic vascular resistance. the dogs receiving a continuous infusion of , , ~ug/kg.min of sin- starting min following initial fluid resuscitation (each dose for h), had a higher cardiac index ( . -+ . vs . _+ . l/min.kg, p< . ) and lower systemic and pulmonary vascular resistance than the control group ( -+ vs -+ and -+ vs -+ dyne.sec/cm , respectively, both p< . ). arterial pressure and pulmonary artery pressure were not influenced. sin- significantly increased the mesenteric blood flow from -+ to + % of baseline levels and renal blood flow from -+ to -+ % (both p< . ) but did not influence femoral blood flow. the relative blood flow was increased in the mesenteric bed (at all doses), and reduced in the femoral bed (at highest dose). systemic oxygen delivery, vo , oxygen extraction and blood lactate levels had similar course in the two groups. conclusions: in fluid-resuscitated endotoxic shock dogs, the no donor sin- increased cardiac index without deleterious effects on arterial pressure, and increased splanchnic blood fl w. objectives: sepsis syndrome is associated with the release of a variety of inflammatory mediators, such as interleukins, tumor necrosis factor and platelet-activating factor (paf). administration of paf, a naturally occuring phospolipid, to laboratory animals has been demonstrated to resullt in pathological changes that closely resemble those found in sepsis. in addition, paf antagonists of various origins have been demonstrated to attenuate cardiovascular collaps and to protect against lethality in endotoxemia. in the present study the efficacy and safety of the paf-antagonist tcv- g was investigated in sepsis syndrome patients. in addition, the putative inflammatory parameters tumor necrosis factor (tnf), interleukin il) - il- , and soluble (s) e-selectin were measured in both tcv- and placebo treated patients. metho.~a randomized, double-biind, multi-center study was undertaken to compare the safety and efficacy of intravenously administered tcv versus placebo (takeda chemical industries ltd., osaka, japan). tcv- was used as add-on to conventional therapy in the treatment of patients with severe sepsis and septic shock ( . mg/kg body weight intravenously over hrs, twice a day for one week). day mortality was registered, as well as vital signs, laboratory parameters, hemodynamic parameters, apache ii score and mof score. plasma samples for the determination of inflammatory parameters were collected twice a day for one week. the study was approved by each institutional review board and written informed consent was obtained for each patient. results: a total of patients were included in the study. one patient had to be excluded as a result of protocol violation of the remaining patients patients were randomised to receive tcv- and to be treated with placebo. there were no significant differences between the treatment group with respect to age. gender, and apache i~ score on admission. however, admission levels of il- and il- , considered to reflect severity of disease, tended to be higher in tcv treated patients: . . vs . ng/ml for il- (p. ) and . vs . ng/ml for il- (p. ) in tcv- and placebo treated patients, respectively. a remarkable difference in survival, however not significant, was observed at day i.e. the end of tcv treatment (mortality: out of in the tcv group and out of patients in the placebo group). the inc;dence ui ~-,~verse events, 'n both treatment groups were comparable. plasma il- and plasma il- levels tended to decrease more rapidly in tcv treated patients compared to controls (p=. and p=. , for il- and /l- respectively) conclusions: tcv- is safe as add-on treatment in sepsis syndrome patients. the data presented indicate that tcv- may enhance survival of sepsis syndrome which will be assessed in a phase ill study. objective: oxidation of lipids by free oxygen radicals playes an important role in sepsis. an important source of oxygen radicals is the respiratory burst of phagocytic leukocytes, especially~ polymorphonuclear leukocytes. clinically the hallmark of sepsis is the capillary leak, which is mediated in part by oxyradicals. the adult respiratory distress syndrom (ands} represents the most studied capillary leak phenomenon in sepsis. methods: in ii patients (df,~m, aged - y.) with verified septicemia had been included in this study. beginning from day oxidisable lipidautoantibodies (club), ~opterin and crp were determined in the patients serum and compared against the apache ii score during the days - . patients, f and m died @u= ring their stay at the icu, of them in less than h~urs: after admission. at the begin surviving patients showed olab values between - %, non-survivors between - %, com= pared to neopterin values of - m~ol/l in survivors and - ~mol/l in non-survivors. apache ii score didn t show any difference. during the following days olab sbowed an in= crease in survivors, while in non-survivors olab stayed equal or showed a decrease. no difference between both groups was found in neopterin, crp and apache ii score. as result we conclude, tbat lipidperoxidation playes an important role during septicemia. olab as a marker of this process are predictive for the outcome of patients. obiectives : to evaluate the influence of cardiopulmonary bypass ongastrointestinal transit. meth~xls; gastrocoecal transit time (gtt) in consecutive children on day after uncomplicated open heart surgery was compared to gtt in children after closed heart thoracic surgery and to healthy controls. gtt was measured by hydrogen breath testing, using lactulose (o, g/kg in a % aequous solution) as nonabsorbable marker. exspiratory hydrogen content was measured using a electrochemical system (stimotron). patients in both groups had continuous infusion of morphine and midazolam and were comparable in respect to catecholamine dose. children with a history of toddler's diarrhoea and children with elevated central venous pressure were excluded. results: gastrocoecal transit was delayed in all patients after cpb, no transit was achieved within minutes in / . after closed heart surgery gtt was delayed (+- ) min vs. (+- ) compared to controls, but transit was achieved in all aptients within rain. mean dopamine dose was , mcg/kgmin after cpb and , meg after closed heart surgery.. conclusions: after uncomplicated cpb gastmcoecal transit is extremely delayed. the degree of delay is not explained neither by the use of analgetic and sedative drugs neither by the use of catecholamines. factors directly related to cpb (e.g. low flow perfusion, hypothe,mia) may be responsible. these findings are of limited clinical relevance in ancomplicated cases. in critically ill patients delayed enteral feeding may contribute to infectious complications. studies of therapentic measures to accelerate gastrointestinal transit therefore are warranted. neonatal ecmo -czech experience. nekvasil r., penkova z, vasek l., plier p., bobula a novotna e., pavlicek v., hnilicka m. nicu and ecmo center brno and dept. pediatric surge ', children's hospital brno, czech republic. introduction. in spite of the fact that neonatal ecmo has been gradually considered a standard method of solving uncontrollable respiratol t failure in newborns in most countries of the western europe, the introduction of this sophisticated method in countries of the former communist block meets a lot of problem. material methods. in the course of two years, only newborns, b.w. - g, were reffered to neonatal ecmo because of uncontrollable respiratow failure. at the admission the average values of oxygenation index were + , and aado , -+ , kpa. results. newborns ( %) died shortly alter" admission or during transport without possibity to further is newborns ( , %) were treated using high-frequency ventilation (hfo or hfjv), one for conspicuous airway resistance paradoxically with low-frequency ippb. all ventilated newborns survived. ecmo was applied in newborns ( , %) and of them survived the mortality rate of group mentioned was %. conclusion. the fact that in the czech republic ( mil. population with natality rate of about . newborns/year) only small part of patients was reffered to neonatal ecmo has a number of causes. the most important are: unacquaintance of prognostic and indication criteria at forwarding workplaces and the aversion of neonatologist to all new.therapeutic interventions. last but not least, a negative role is also played by current change of health service systems when, under the influence of health-service insurance companies a newborn in serious condition given only a standard intensive care in primary and secondary nicu is literally "the golden calf", and therefore these workplaces either do not send him or send him too iate ~o an ecmo center. stndy aim: urfactant, which is being applied into the lungs for treatment of respiratory distress syndrome, can reach the circulatory blood system by absorption. it refinances local fimctiun of macrophages and lymphocytes and increases the accumulation of nentrophils into the hmgs this eunld influence the local acute phase reactiml in the hmgs after surfactant application. "ilia aim of the stud), was to investigate the influence of oatural and artificial surfactaat onto the acute phase reaction and the blood elottiog system. material :and methods: a) measurement of tnf-a, - , ltb and thromboxane b release in whole blood: each of . ml hepariaised blood of healthy adult volunteers was iucubated lbr hears with eillier ill ug/ml lps, mg/ml b&,ine smfactant (alveulhct/themae), rag/nil artificial surfactant (exosurf/wellcome), og lps + i mg/ml bovine surfactant or i ug lps + i mghal artificial sorfactant, tleparinised bhx-,d without additions was used for controls. ' e concentration of e)lokines and eicosanoids ".van measured semiquantilatively by eijsa or eia (dimmva/hamburg) after ceatrifagalion, b) measurement of platelet aggregation: each of . ml haparinised hltxxl of healthy adult vohmteers was inculmled lbr hours with eilher aghnl i,ps, i mg/ml bovine surfactant (alveafact/thomae), i mg/ml artificial snrlhctant (exosurf/wellcotne), it} ug lps + i mghnl bovine snrfactant or ug lps + i mg/ml artificial surfaetant, tlepminised blood withont additions was nsed for controls. platelet aggregation in whole blood was measured by impedance in the aggregometer (g~nstaedt). c) measurement of the dotting activity: citrated bleed of healthy adult w~lunlccrs was mixed with difl'ereul couccalrations of imvine or artificial snrfactant fad then prolhrombiu tiaras and imrlial thrmnlx~plaslia filues were ineasnrcd. for measurement of the prokoagulatioo acfivily the reagent in the qnick test was replaced by surfaclant. results: dependmlt on the dose both surfactants stimulated the release of - but not or tnf-a io wlmle blood. "illere was no suppression of the lps-indaeed eytokine-release. both surfaclants did not directly influence the extrinsic blood coagulation system, while both activated the inniasie activity dependant on the dose. platelat aggregatiun was slightly stimulated by bovine snrfactunt and strongly inhibited by by artificial surthctant. in comhinafion with . ~ both factors showed no difft.'renca to lps ahme.'fhe reltu.tse of eiconasnid~." was stimolated more by artificial surfactant then by bovine surfilctant with regard to the cyehmxygelmse (thromboxsne b ) and the lipooxygenase (lencotrieae b ) pathways. conclusion: we cooclude: ) i:lovine as well as artificial surfaelant stimulate the cellular inmnn]e response. ) bovine as well as artificial surfnctant activate the intrinsic ctmgnlation cascade. ) bovine surlilctant stimulates platelet aggregation, artificial sar-fijctant inhibits platelet aggregation.. introduction: although the use of modem noninvasive methods like measurement of tope , tcpco or san leeds to an carly infonnation about pathologic alterations of the gas exdmage of ventilated patients, these methods cannot replace invasive bloodgas analysis. a new noninvasive method to monitor the capillary gas exchange is the reflection spectrophotometry. to record reflection spectra with a ldgh sensitivity on the surface of tissue we eonstnmted a special mierolightgnide photospectronmter (empito). this spectrometer enables the investigation of local helerogenities of llbo and lb content, capillary flow rate and oxygen uptake rate. aim of the study: ) to compare the sousitivity and specifity ofthe empilo with other non-invasive method~ of monitoring the gas exchange in neonatal intensive care patients. ) to investigate the possibility of obtainiug infonnations about the local nptake and the venous as well as arterial satnration. patients and methods:we investigaled non-selected patients of oar pediatric intensive care unit. for coulinnons monitoring we conslracted a special sappert to fix file end nf the microlightguide+ during the investigation tope , tcpco and san were measured by standard methods. "fo investigate the local tisslm oxygen uptake we recorded spectres within minutes by moving the lightgnide over an described area of the skin of the patient. 'lhe reselting i(gx) l ibo vahms were sorted by a software package. on the assumption that the low values correspond to the tibo value of the veaons ends of the capillaries and the high values to those of the arterial ends we ) it is possible to gel infonnations about the local o -uptake and arterial saturation ia the peripheral tissue by photoslg'ctroscopy. ) in combination with noninvasive pl i-measorement it seems possible to reduce withdrawal of blood for invasivn bhmd gas analysis. objectives:improve ventilation,tissue oxygenation and acid base balance among extremly low birth weightand premature infants. methods: fourteen cases were s~udied,their gestational age ranged from( - )ws.continnous positive air way pressure was applied to six cases at peep level from ( - )cm h o through nasal pronge,(group i),the other cases were managed as routine,(group ii).blood gases, tcpo ,tcco ,resp.rate,depth and pattern were monitored for assessment of tissue oxygenation and ventilation, results: our rasults showed that early application of cpap improve ventilation among ( . %)of cases,while ( . %)of cases need imv.the cases of group ii need imv among ( %)of the studied cases during the second or the third day of life. conclusions:-this preliminary study showed the import-............ ance of early application of cpap before the onset of respiratory distress syndrom to improve spontaneous ve ventilation,tissue oxygenation and to gecrease the risk of intubation. comparative assessment of pediatric intensive care; a national multicenfre siudy. reinoud j.b.j. gemkc, gouke j. bonsel , the dutch picasso (pediatric intensive care assessment of outcome) study group. twilbelmina children's hospital and utrecht university, utrecht department of clinical epidemiology, academic medical center, : amsterdam, the netherlands the performance of all ( ) pediatric intensive care units (icus) in the netherlands ( tertairy and non-tertiary) was analyzed in an open prospective muiticenter study. effectiveness of care was defined as the ratio of observed to prism-score derived expected mortality aenee, standardized mortality rate). efficiency was determined by objective criteria (mortality risk > % or administration of at least icu-dependent therapy)'. consecutive admissions aged < years old were included. overall, observed and expected mortality were in good agreement (p > . ). between hospitals, crude mortality showed wide variations (mean . %, range - %). however, in each center, observed and expected mortality were similar (mean ratio . , range . - . ). in tertiary care centres, severity of illness corrected mortality in high-risk patients was less than in non-tertiary care centres; paradoxically, in low-risk patients the opposite was found. probably the large proportion of low-risk tertiary care patients suffering from severe, incurable chronic disease, explains the higher mortality in this group. this indicates that simultaneous assessment of circumstances of dying and of long term morbidity in similar future studies is imperative. the average proportion of efficient icu days was %, however large variations between units were found (range: - %). in conclusion differences in mortality rates among pediatric icus were explained by differences in severity of illness. high efficiency rates in combination with adequate effectiveness, found in several centres suggest that admission and discharge decisions might be improved by a better selection of high risk patients requiring icu-dependent therapies, especially in less efficient centres. objectives: previously published studies showed that serum lactate levels correlated with outcome of severe ill adult, 'we hypothesized that critically ill newborns are often incurred hypopeffusion manifested by elevated lactate levels. these initial blood lactate levels should be related to nicu outcome. design: prospective study with ethical comfnittee approval. setting: the -bed neonatal intensive care unit of a university hospital material and method: a total of consecutive outbem newborns admitted to nlod from , . to ., . were enrolled to the study. babies who died or were discharged from the unit within hours of treatment were excluded from the study, mean birth weight was g (+/- r), mean gestatational age was weeks (+/- . wks), mean age at the admission was h (+/- hi. multiple (~_ j organ system failure occurred jn . % of babies at the admission./~tertal lactates were measure/at the admission, among - hour and - hour of n[c'lj therapy. outcome was defined as a mortality and length of nicu stay. results" survival rate was . %, mean length of nicu stay for survivors was . days (+/- . day). we found high lactate levels at the admission in . % babies (~ . % with levels above . retool/i). the mean arterial lactate concentrations for nonsurvivors were signiftcahtly higher than for survivors durin~ consecutive da~ as follows: objectives: the purpose of our research was to analyze the frequency of bronchial asthma (b.a.) exacerbations in pregnant women and health status of infants. methods: the research was based on the epidemiological investigation and prolonged observation of pregnant women with b.a. during the gestation period. remission of b.a. before the pregnancy in excess of years was recorded in patients ( . %), patients ( . %) reported a - year remission and patients ( . %) had a remission lasting less than months before they became pregnant. results: seven patients ( . %) developed medium attacks in the second half of pregnancy, four patients ( . %) experienced light attacks of b.a. asthma attacks were most frequently caused by acute respiratory diseases and stress factors. in two cases with grave manifestation of b.a., the pregnancy ended in abortion within the first - weeks due to the frequent and heavy choking attacks. to fight b.a. attacks, five patients used adrenomimetics (salbutamol, becotid) in sprays, six women were administered theophyllinum and salbutamol in the form of tablets during - weeks. a significant portion of pregnant women with b.a. ( %) exhibited frequent complications during pregnancy (toxemia, late gestosis, threat of miscarriage). our findings prove that babies born from women with b.a. of domestic and pollen origin had a low body weight ( - gr), functional immaturity and chronic antenatal and intranatal hypoxia twice as often as the infants born from healthy women without allergic background. conclusions: preventive treatment of women with b.a. prior to pregnancy is required to maintain a stable remission of the disease, which is a key to having healthy children delivered by mothers suffering from b.a. introduction. intracerebral hemorrhage (ich) is a common event in human prematudty, affecting about % of newborns weighing below g who are born before weeks of gestation, however, little is known about the pathogenesis of ich with exception of the prematurity of the brain itself, (birth) trauma, and asphyxia. the postischemic production of oxygen free radicals (ofr) dudng reoxygenation as a cause of brain damage has been demonstrated in animal research. since almost all preventive antioxidant activity of plasma is associated with ceruloplasmin and transferdn we investigated the association of such iron-oxidizing resp. iron-binding proteins and ich. we could demonstrate significantly reduced levels of both, iron-oxidizing and iron-binding proteins, in premature asphyxiated newboms pdor to development of ich. an increase of suparoxide after hypoxia in the presence of iron ions facilitates the formation ofthe highly reactive hydroxyl radicals. our data support the theory that ich may be caused by ofr, which can damage any sensitive tissue including growing endothelial cells. the estimation of transferrin-saturation and measurement of ceruleplesmin levels might help to identify an infant at dsk before the onset of ich. with the new medos | hia-vad | cardiac assist system the missing tool in the armamentarium of cardiac surgeons is available in two pediatric sizes: i -ml and -ml pump volume. the right sided pumps are % smaller for biventricular use. between february and may we implanted this assist system in children. the indications and demographics are indicated in the following table (left ventricular assist device-lvad, right vad-rvad univentricular vad-uvad, post cardiotomy cardiac failure-pcf, dilated cardiomyopathy-cmr bland white garland syndrome-bwg, tetralogy of fallot-tof, hypoplastic left heart syndrome-hlhs). objectives: evaluate tile effeci'of inhaled nitric oxide (no) as puhnona] t vasodilating agent ill tile posloperalivc period after correclion of congenital heart defects in infant. patient n.l: kg, lnonlhs, down syndrome undenvcnl rep~fir of atrioventricular septal defect (avsd). after surgery the puhnonary arlcry pressure (pap) slowly rose to tile syslemic dcspilc tnaximal eonvcnlional fllerapy (fentanyl mcg/kg/h, hypocapnia of mmhg and metabolic alcalinization). no was delivered into tile inspiratory branch of!be breathing circuit at ppm, and the gas aoalyser for no and no (polylron dmger) were situated at the espiratory branch, a rapid dccrcasc of pap io i/ of systemic was obtained with a dramalic improvement. no was continued at ppm for six days and the baby was exlnbated if! days after surgery and discharged from the icu days after. patient n. : . kg, monlhs, onderwen! repair of avsd. the day after surgery the systemic oxygen salnralion was % wilh a pap at % of systemic. two hours of c wenlional therapy failed o improve ihc patient and no administration was slarled at ppm. so dramatically incrcased to %, but the pap dropped only to % of syslemic. nevertheless ihe clinical conditions improved and the no administration could be reduced at ppm in the following days. she was extubaled days after surgery and discharged from the icu days after. patient n. : kg, 'ears. underwen| hearl tral~splantalion for congenital heart disease with moderate hypoplasia of pulmonary arlcrics. at the end of cardiopulmonary bypass the transpnlnlonary al~erio-venoas gradient yeas higher than mnfflg and we speculaled !hat w'ls due to a degree of puhnonary vasocostrictiont. the nsnal dose of no was otilised, however no significant modilicalion of pulmonary pressure or systemic oxygen saluralion was noled, and after h no was discontinned. tile palienl was carried io the icu with maximal inotropic support, extubated after d;b's and disclmrged from the icu after days. in all patient no major adverse effect relaled to no admilfistration ",','as holed. conclusion: in our experience no ms a pulmonary vasodilaling agent is effective and easily adjustable to tile palienls requiemenls, however its use remains limited ill those palienl ill whoin tile alnonll! of fixed inlllllojliify vascular resistance is predominanl. we report the use of ecmo support in two unusual cases of severe tracheal disruption in which it had become impossible to achieve adequate ventilation. case : severe tracheal laceration due to aspiration of a share forelan bodv: a previously healthy month old toddler was referred for ecmo following aspiration of a porcelain foreign body (with razor sharp edges) which had become embedded in the right mainstem bronchus with massive extrusion of air. this was removed on veno-arteda[ ecmo support, as the patient was unventilatable prior to bronchoscopy due to ongoing airieak. ecmg was continued after bronchoscopy to permit airway healing without the presence of an endotracheal tube. unfortunately, an extensive pulmonary haemorrhage on day of ecmo necessited re-exploration of the airway. this revealed a posterior tracheal tear from the cricoid to the middle of the right lower lobe. following repair the patient was left on ecmo support together with high frequency oscillation ventilation (hfov), the latter being used to minimise potential aideak and maximise alveoli recruitment. ecmo was weaned after days ( hours) -the patient was extubated weeks later. case : tracheal wound dehiscence due to seosls -tracheal transelant on ecmo: a month old infant with a c[inically significant congenital long segment tracheal stenosis and left pulmonary artery sling underwent resection of the stenosis, followed by primary reanastomosis. this was complicated, days later, by severe mediastinitis and complete dehiscence of the anastomosis. an autologous pericardial patch was used to repair this, however, the tracheal wound again dehisced days later making mechanical ventilation impossible. in view of ongoing sepsis and a severely disrupted trachea ecmo was the only possible form of support. following resolution of the local sepsis ( days) a definitive procedure in the form of a tracheal homograft (transplant) was undertaken on ecmo. the patient was managed on ecmo and hfov for a further days, the hfov being used to optimize rapid lung inflation. unfortunately this patient died months after weaning from ecmo due to complete disintegration of the homograft, which was not deemed reparable. conclusions: ) ecmo can be used in the acute management of oxygenation when there is major airway disruption making mechanical ventilation impossible. ) hfov was a useful adjunct in aiding recruitment of lung volume on ecmo in these two patients. backoreund: persistent pulmonary hypertension of the newborn (pphn) consists of a heterogenous group of diseases ranging from transient reversibte pulmonary hypertension to fixed primary malformations of the lung (primary pulmonary dyspfasia-ppd). inhaled nitric oxide (ino), a selective pulmonary vasodilator, has been proposed as a treatment for severe pphn. obiective and methods: ino was administered to near term neonates with severe persistent pphn, oxygenation index > and echocardiogrephic evidence of pulmonary hypertension, in order to further determine the clinical role of ino in the treatment of pphn. the response to ino was also analysed retrospectively to examine whether this could be of diagnostic value in differentiating at an early stage patients with reversible from fixed causes of pphn results: twenty one of the patients studied responded to the initial trial of no ( ppm x minutes), as defined by a greater than percent improvement in pad as well as a fall in the el to < . these patients were continued on ino therapy, with patterns of response emerging: pattern babies (n= ) continued to show a sustained response to ino and were successfully weaned from it within days -all survived. pattern babies (n= ) failed to sustain their response to ino over hours, as definded by a rise in the el > . six survived, five with ecmo. pattern babies (n= ) had a sustained dependence on ino for - weeks. all three died and lung histology revealed severe primary pulmonary dysplasia (ppd). patients with ppd (pattern ) not only required ino for longer periods of time than did the sustained responders (pattern ), but also required significantly higher doses of ino we report on the air transport of paediatric intensive care patients. these transports fall into three categories: ) retrieval of critically ill neonates and paediatdc patients referred for either ecmo or inhaled nitric oxide (ino) (n = ). one patient was transferred on ind. mean transfer time . hours (se + . hrs). ) long distance international transport using chartered aircraft (n = ). the indications for these transfers included both urgent retrievals for cardiac surgery and semi-elective transfer of stable patients back to their referring unit following treatment in tertiary centres. mean transfer time . hours (se + . hrs) ) long distance international transport using commercial aircraft (n = ). indications for transfer were either semi-elective retrieval for tertiary treatment or the return of stable chronically ventilated patients to their referring hospitals. mean transfer time hours (se _+ .fhrs, longest hrs). the transport team consisted of a paediatric intensive care doctor of at least registrar grade and a registered sick chidrens nurse with intensive care experience. the administrative components of the transfer (ambulances, airlines, customs) were managed in collaboration with companies specializing in air ambulance transfers. outcome: all the patients were safely transported to their destination without mortality or morbidity. complications durino transfer ir~lv~; ) patient complications -semielective endotracheal tube change and central access needed in the only patient brought to the commercial aircraft by the referring hospital (all others retrieved directly from referral hospital), seizure in patient with known encephalopathy, severe cyanotic spells in patient with fallots tetralogy who was retrieved for urgent surgery for this indication ) mechanical compfications -ventilator failure, incubator battery failure, oxygen regulator failure -all occurred with equipment sent from referral hospital, this was unfamiliar and unchecked by our transport team -it was not the decision of the transfer team to use this equipment on this single occassion. ) administrative complications -confiscation of incubator battery by airport security police, excessive delay by custom officials ( hours) in the airport. the incidence of such problems were felt to be low and unpredictable. in conclusion: mechanically ventilated paediatric patients can be safely transported on both chartered and commercial airlines. these transports are best accomplished by trained intensive care medical and nursing staff with the backing of an air ambulance organization competent in arranging the necessary administrative details. it is essential to use your own equipment and to retrieve the patient _directly from the referrin(] hospital to minimise ootential complications. our experience with anaesthesia for paediatric electromyography _w_._pla_ti_k_a_n_o_v, r.eousseff, k.pavlova, d.marinova dpts. of anaesthesiology and int. care and clinika] neurophysiology, med. university, pleven, bulgaria ~)_b_j#~ti_v~. to t~st a " heavv sedation " regimen of anaest-es~a for the purpose of paediatric electromyography d#s~gil~ non-randomized,non-blinded human trial in the seting of an uriiversity hospetal. _m_a_t_eri_a_is_a_nd_ m_e_th_od_s_. children,asa i-if,median age years,range - who undervent eleetrcmyography required anaesthesia. they recieved low-dose ketamine + i~iazepam or midazolam via musculary route( children,age - yrs,ketamine , mg/kg, diazepam - mg total dose ) or per os ( children,ketamine - mg/kg,diazepam , mg/kg or midazclam , - , mg/kg ) _resu_l_t_s. - minutes after medication a state of heavy sedation with weak spontaneos and stimuli-provoked movements was achieved in all children, that lasted - minutes and allowed adequate needle emg and nerve conduction investigation. children recieved additional , - , vol.% halothane during the placement of the needle. non -invasive blood pressure , breath and heart sounds and hb sad by pulse oxymetry were monitored.none of the older children disclosed memories of pain when asked after they regained adequate verbal contact.no complicationes were observed. antenatal maternal steroids reduce the risk of periventricular-intraventricular hemorrhage in very premature neonates treated with natural surfactants. i.apostolidou, c.papagaroufalis, g.touloumi, m.xanthou, n.kalpoyannis a' and b" neonatal icu "ag. sophia" children" s hosp. athens, greece. dept of hygiene and epidemiology, athens university, greece. obiectives: the aim of the study was to evaluate the association of periventricular-intraventricular hemorrhage (p-ivh) in surfactanl treated premature neonates with pre-and postnatal variables. methods: the population of the study was neonates admitted during the years to , with gestational age _< weeks and severe respiratory distress syndrome (rds) (mechanical ventilation and arterialalveolar oxygen tension ratio (ajapo ) < . ), who received rescue therapy of at least two doses of natural surfactants (alveofact or curosurf) and examined with ultrasound and/or autopsy for the presence of p-ivh (papile's classification). the examined factors in each neonate were the following: gestational age, birth weight, sex, multiple pregnancy, antenatal maternal steroids (complete and incomplete course of betamethasone), a/apo before the administration of the st dose of surfeclant, delivery, apgar score at min, type of surfactant, pneumothorax and patent ductus arteriosus. the statistical methods used were x and one-way analyses of variance followed by logistic regression medels, results: the incidence ot p-ivh was . %. three factors were found to have an independent relation to p-ivh (final logistic regression model): gestalional age, a/apo before surfactant administration, and antenatal administration of maternal steroids (complete and incomplete courses). for every weeks of lower gestational age the neonates had an almost doubled associated risk of p-ivh (or: . , % c : . , . ). for every . on average decrease of a/apo before surfactant administration the risk of p-ivh in the neonates was . times higher ( % ci: . , . ). the neonates whose mothers received antenatally steroids had only one tenth of the risk of p-ivh of the neonates whose mothers had not (or: . , % ci: . , . ). conclusions: our results suggest that the antenatal administration of maternal steroids, even less than hours before delivery, reduce the risk of pqvh in very premature neonates treated with natural surfactants, whereas the small gestational age and the lung immaturity still remain the main risk factors tor the development of p-ivh. we analysed retrospectively the management of ( boys, girls) accidental ingestions of foreign bodies in children (mean age : . years, range : months- years). no child had ingested more than foreign object. the majority of the ingested foreign bodies were : coins (n : ), toy parts (n : ), jewellery (n : ), batteries (n : ), "sharp" materials such as needles and pins (n : ), "large" amounts of food (n : ). impaction of food occurs more frequently in children after oesophageal reconstruction in cases of oesophageal atresia. although according to literature "coca-cola" is reported to be effective, this was not seen in our experience. / patients had minor transient symptoms at the moment of ingestion, such as retrosternal pain. only children experienced severe manifestations (cyanosis, dysphagia). in these children, endoscopy revealed oesophageal and gastric erosions. children were seen at the emergency ward within a few hours after the accident ( mean : hours, range min. - hours). chest and/or abdominal x-ray was performed as first-line investigation ( / objects were radio-opaque), and revealed an (unexpected) oeeophageal impaction in children. in / the foreign body was in the stomach. batteries, sharp objects and objects trapped in the oesophagus were removed, either by endoscopy or by magnet-extraction whenever possible. the outcome of the patients was excellent. no complications were observed. extraction is recommended in symptomatic patients, and whenever the foreign body is trapped in the oesophagus, or if the foreign object is "sharp" or a battery. objectives: two strategies were used for management of malignant diphtheria in children aged from . to years. methods: protocol n consisted of intravenous administration of diphtheria antitoxic serum, prednisolone ( mg/kg bw/day), plasmapheresis and supportive care. protocol n included the use of antitoxic serum against the background of high-dose dexasone ( - mg/kg bw/day), hemocarioperfusion and a preventive use (before the clinical manifestation of myocardial damage) of inotropic medications, inhibitors of angiotensin-converting enzyme and pentoxyphylline. each of protocols included the monitoring of serum toxin (diphtherin) levels. results: the group of patients treated according to the protocol n consisted of children with malignant diphtheria, of them with severe malignant diphtheria (grade and ). all patients exhibited the circulation of toxin during at least three days after the start of treatment. all patients with severe grade of disease demonstrated heavy cardiovascular disturbances associated with malignant diphtheria. of the children in the group died seven. the children of the second group were treated according to the protocol n . out of total of patients of this group. patients had severe malignant diphtheria. in all children a significant reduction in serum toxin level was revealed after hemocarboperfusion. in all but one case the satisfactory control of cardiovascular function on was achieved. of children admitted to the trial survived, one child with malignant diphtheria of grade and congenital filbroelastosys of the left ventriculum died. the severity of neurological complications was similar in each of groups. conclusions: the use of hemocarboperfusion, high-dose dexasone and early prevention of heart failure as a adjunct to the standart treatment has been shown to be of benefit in the management of malignant diphtheria. t. schaible, i. reiss, j. m er, l. gortner med. university of lqbeck, children's hospital, kahlhorststr. - , l~beck, germany surfactant therapy seems a promising approach for the treatment of the biochemical and biophysical abnormalities of the pulmonary surfactant system in severe ards. patients and methods: over a months period non-neonatal pediatric ards patients (age - months) in a "pre-ecmo"-situation (oi over h) were treated with bovine surfactant (alveofact| the underlying conditions-of ards were pneumonia ( ), sepsis ( ), immunosuppression ( ), near drowning ( ), neurogenous ards ( ). a total of - mg/kg b.w. was applied in several fractions. before surfactant therapy, we first tried different ventilation (best peep-finding, inversed i/e-ratio, hfo-ventilation) while monitoring the pulmonary mechanics. for hemodynamic stabilisation both norepinephrine and epoprostenol were used to optimize pulmonary perfusion for max. hrs. if there was no improvement of the oi by at least , further treatment with surfactant was initiated. in addition to surfactant all patients received a treatment with dexamethasone of mg/kg in doses. patients with no benefit (oi remained unchanged or increased within the max. - hrs) were taken on ecmo. results: nine patients improved within hours after surfactant therapy: the oi decreased from a level of (mean, range - ) before our treatment to a level of (mean, range - ) thereafter. in patients we were able to continue the positive effects of our treatment and they could be weaned of the respirator within - days. the other patients got worse despite respiratory improvement, they suffered of multiorgan failure of more than organ systems. the last patient did not benefit from surfactant, he had to be put on ecmo, but died because of a complication (hemopericard)after days. the autopsy of the ecmo-patient showed a pulmonary fibrosis, but the other death were not due to pulmonary failure. conclusion: a different sequential ards treatment integrating surfactant therapy can reduce the number of patients requiring ecmo. but ecmo as a therapeutic tool should be available in centers involved in ards treatment. l.blindl, t.p.le, h.weinzheimer, centre for paediatrics, university of bonn, germany selective reduction of elevated pulmonary vascular resistance by inhaled prostacycliu (pgi) has been reported in adults with acute lung injury, neonates with persistent pulmonary hypertension and in one infant with idiopathic pulmonary hypertension. we report on the effect of aerosolized prostacyclin in two children with secondary pulmonary hypertension. patient : in a boy with down's syndrome an avsd had been surgically corrected at month of age. at , yr of age a catheter examination revealed a pulmonary vascular resistance of % of systemic vascular resistance in room air and at an fin of . . prostacyclin ( . mcg/ml) was administered with a jet nebulizer at an fin of . . pvr declined to . systemic vascular resistance and returned to baseline after stopping pgi-inhalation. subsequent intravenous infusion ( ng/kg rain) had to be stopped after minutes because of systemic arterial hypotension. patient : a month old male infant with bronchopulmonary dysplasia developed suprasystemic right ventricular pressure inspire of therapy with oxygen and nifedipin. while he was spontaneously breathing % oxygen via face mask pao was mmhg, arterial ph was . . systolic arterial pressure was mmhg, a rv-ra gradient of mmhg was measured by cw-doppler. while fio was maintained aerosolized prostacyclin was administered over minutes. rv-ra gradient was mmhg, systemic blood pressure mmhg, pao mmhg. two hours later nitric oxide ( ppm) was inhaled at an fio of ( , . rv-ra gradient declined from to mmhg, systemic systolic blood pressure remained stable at mlnhg. discussion: sporadic experience shows that aerosolized prostacyclin selectively reduces elevated pulmonary vascular resistance in some patients. in patient the poor response to inhaled pgi compared to inhaled nitric oxide may be explained by the fact that the action of pgi is not independent from endothelial function, limiting it's effect in severe vascular disease. during the last two years ( - ), infants weighing less than gr. admitted to our referral unit. thirty four of them ( %) survived, ( % of infants weighing - g and % of infants weighing - gr survived) for the years - - the survival of these infants was % and for the years - - , % (p< . ). we analyzed the perinatal and neonatal factors influencing the outcome of these infants. the comparison among neonatal survivors ( ) to neonatal deaths ( ) shows: gestational age: . w ( ) to . w ( ) (s). birth weight: . g ( ) to . ( ) (s). apgar score: , ( ) to . ( ) (ns). presentation and mode of delivery: breech presentation is associated with higher incidence of neonatal deaths. i.v.h. (at the age of weeks): no one of the survival infants had evidence of i.v.h. respiratory problems: intubation, at the admittance of the infants . ",,( ) to % ( ) (s) use of surfactant: % ( ) to % ( ). bpd observed in % of the babies and only one was dependent on oxygen at home. antenatal betamethasone was given in % of the mothers. in conclusion: ) a great improvement in the survival rate observed in these infants the last years in our unit. ) factors with positive effect are increasing gestational age and birth weight, the absence of i.v.h. and the use of surfactant. the breech presentation and the severe respiratory problems increase the incidence of death. animal experiments demonstrated, that brain temperature determines the amount of neuronal damage caused by hypoxia and that mild hypothermia may have a protective effect. until now there is no method described and evaluated to measure brain temperature in neonatal intensive care units. we non-invasively measured brain temperature analogues, nasopharyngeal (tnasoph) and zero-heat-flux temperature (zht) at the temple whereby under zero heat flux surface temperature represents deep head and thus brain temperature. the aim of our study was to investigate the practicability of the method, the relationship of the two brain temperature analogues to rectal temperature (trect) and their dependence on insulation, thermal environment, body activity and time course. we investigated healthy preterms less then weeks postnatal age (gestational age +_ . wks; x + sd, weight +_ g) in an incubator. tnasoph was measured by a thermistor within a feeding tube, advanced to the nasopharynx, zht temple by a thermistor and a heat flux transducers both covered by an insulating pad, and trect thermal environment was characterised by operant temperature (tair . . + twall . ). body activity was video taped. measurements were performed during the following interventions: i/ insulation increased by turning the temple with sensors onto the mattress ( rain). ii) insulation increased by a cap ( min), iii) min after its removal, iiii) increased operant temperature by . + . ~ ( min). results: seven children with ea had a gasless abdomen, the endoscopic procedure excluded ( ) or diagnosticated an upper pouch fistula ( ). in patients who suspected "h" fistula ( ) broncoscopy has strong advocated method to make diagnosis and established cervical approach. from july newborns with ea and lower pouch tef received a selective transtracheal incannulation. we were not able to proceed just in case with congenital subglottie stenosis. in these patients we provided gastric drainage by radiopaque and flexible - french catheter. the knowledge of the precise anatomic position of tef consent to adjust the tip of the endotracheal tube in order to achieve best ventilation. the presence of the catheter through the fistula helps the surgeon to identify, it quickly. no complications were correlated to the procedure and no babies had early pneumonia. alimentary continuity was achieved in all patients ( primary anastomosis, resections of tef, oesophagocoloplasty and died with gastrooesofagostomy). the late mortality . % ( ) was only directly related to the severity of associated malformations. conclusion: the advantages of this technical approach are unquestionable for the anaesthesiologist and the surgeon. in our experienc e the procedure improves perioperative management of babies and appears to be safe. relation between cytokines, prethrombotic markers and endotelial injury markers in children with septic shock objectives: to establish the relationship between cytokines (tnf, il- , il- ) prethrombotic markers (d.d., pcam) and endothelial injury markers (tm, uwf) in pediatric patients with sepsis and bacteriemia without shock, and patients with septic shock. design and methods: prospective study, children ( months- years) were admitted in our picu in with the following diagnosis: bacteriemia ( ) sepsis ( ) and septic shock ( ) according to jacob's r f criteria. measurements: il- , il- , tnf, tm, vnf, d.d. pcam and routine laboratory data on admision, , , hours and on discharge. the prism (pediatric risk of mortality score) was also recorded. results and conclusions: two patients in the septic shock group died. significant differences were found between non-shock and septic shock patients in relation to tm, dd, pcam, il- , il- and tne high levels of tnf and il- are closely associated with the severity of septic shock with purpura in children. low levels of pcam on admission were associated with severe shock. who underwent open hea~nt surgery, hypervotaemia with or without oliguria was the most frequent reason to start pd ( %). in patients pd lasted less then one week and there were no complications; in patients it lasted - days (one child had a peritonitis). instillation of dialysis fluid into the peritoneal cavity was associated with a significant increase in central venous pressure. there were no significant changes in cardiac output or arterial oxygeu saturation. in all patients pd dhnjnished fluid overload or improved the metabolic status. patients ( %) survived the postoperative course and all had complete reintegration of renal function. conclusion: pd is a useful method to treat the fluid overload and acute renal failure in paediatric patients following open heart surgery with file effects of little importance on the cardiovascular fimction. obieetives: with the marketing of computerised systems for lung function testing in newborns, there has been an increasing interest in clinical approaches. percentile curves of pulmonary parameters permit an appropriate and clinically useful interpretation. however, the manual evaluation of the results using different curves is an impractical technique. therefoi'e a computer programme was developed. methods: the percentiles ( %, %, ~ %, %) of the most important pulmonary parameters were determined non-parametrically in weight-classes. for the calculation we have taken results of our own as well as other laboratories using a meta-analysis of reference studies. in all, individual data of - healthy newborns ageing between - days were collated. using these percentiles, for every parameter in relation to the body-weight the cumulative distribution was calculated approximately using piecewise linear and exponential functions. as shown in the figure the results of computing are represented numerically as well as graphically and can be included in the patient report. conelusions: clinic~d experiences with the programme have shown that representation of all measured parameters on standardised % scales allows an easy interpretation at first sight and improves the detection of pathologic patterns in the parameters. ")supported by bmft, fp "risikoneugeborene" prism (pediatric risk of mortality) score is a well known, already validated scoring system that quantifies severity of illness based on routinely clinical and laboratory variables measuring physiological instability. once computed the score by summing up the weights corresponding to the most abnormal value recorded during the first hours, the overall risk of mortality can be predicted by using the coefficients estimated by a logistic regression where prism score is the main independent variable. (pollack mm et al, -pediatric risk of mortality (prism) score. crit. care med. ; : - . to assess the applicability and validity of prism in the italian setting we launched out a prospective data collection in a sample of pediatric icus. measures of calibration (goodness of fit statistics) and discrimination (receiver operating characteristics and area under the roc curve) are planned to be adopted in the cohort of patients recruited during year period. as the validation study started on july , data collection is still on going and validation analyses will be carried out on july . up to now centers recruited cases. at present, characteristics of the sample recruited are the following: most of the patients were male ( %); the mean age is years with % of patiens having less than days; more than half were medical cases ( %) admitted from emergency room or from hospital floor ( %); % cases were admitted with an organ failure while % to be intensively monitored. icu-mortality was l %. the paper will present final results of calibration and discrimination analyses that will be carried out in the whole sample and across subgroups known to differ in terms of clinical relevance and prognosis. if calibration and discrimination assessment will produce not satisfactoty findings, a customization of the current coefficients will be made allowing a formal comparision of previous and new parameters. jf riera-faneao, m wells, j lipman. baragwanath intensive care unit, university of the witwatarsrand, south africa. [background the prism score is designed to assess the likelihood of death in ipaediatdc icu patients, using only acute physiological disturbances, age and [operative status to predict mortality. there is no evaluation of chronic health status, [including malnutrition. this may significantly affect its ability to accurately predict outcome in a population where malnutdtion is common. aim to determine the influence of nutritional insufficiency, as indicated by a low weight-for-age on outcome prediction by prism. patients & methods we analysed prism, weight and demographic data co ected prospectively from consecutive paediatdc icu admissions over a year pedod. a proportional weight (pwt) was calculated as a percentage from the th centile of the who weight-for-age growth charts. the pwt was compared for survivors and nonsurvivors, and mortality compared for pwt categodes nho wellcome classification). multivariate statistical techniques were used to identity associations with non-survival and to develop a modified logistic regression equation including a measure of i nutdtional status. receiver operating characteristic (roc) analysis was performed including and excluding patients with low pwt for the odginal and modified equations. results non-survivors had a lower weight than survivors ( . kg and . kg medians p = ) a lower pwt ( % and % medians p = . " . the incidence of malnutdtion , in our icu population was %. the mortality of manoudshed patients was' significantly increased (p = . ), with a good correlation with the degree of malnutrition. the accuracy of prism was significantly improved when malnourished patients were excluded from the analysis (roc value increased from . to . ). ! logistic regression and discriminant analysis identified a significant association between prism, pwt and outcome; age and operative status were not significantly related to mortality. the use of a modified equation including the raw prism score, pwt category and age can significantly improve the discriminatory power (az dm/elopmental sample . , az validation sample . ). the modified formula is: legit = - . + . *prism score - . *age + . *weight category, where the probability of mortality is exp(iog/t)/ + exp(iogio. discussion although we can improve the prediction of mortality by a modified or recelibrated formula, this still does not compare with the reference prism population. the need for validation of the score itself, in the association with outcome of the acute physiological variables themselves, is thus apparent. we conclude that while the odginal prism formula can be improved significantly, a modification of the basic variables in this and other third wodd populations may be essential. a high incidence of malnutrition is an independent risk factor of mortality, and an important cause of the poor discriminatory performance of prism. in order to improve the accuracy of prism, nutritional status should be taken into account. objectives: to assess the value of inhaled no to differentiate between pulmonary vascular constriction or fixed anatomical obstruction. methods: we assessed the response to ppm inhaled no in patients( m, f, median age . months, range day to years) with signs of increased pulmonary vascular resistance, there were pre and postoperative patients. patients were divided into responders(+) or non-responders(-). a positive response was defined as a % reduction in pulmonary arterial pressure and pulmonary vascular resistance(pvr) or in the presence of a left to right shunt, a fall in pvr accompanied by increasing pulmonary blood flow. left atrioventricular valve atresia + mustard pat: pulmonary atresia vsd: ventricular septal defect asd: atrial septal defect pda: patent ductus arteriosus tapvc: total anomalous pulmonary venous connection the responders( / ) were characterised by left to right shunts or pulmonary venous hypertension( / ). patient# was weaned from ecmo with inhaled no. patient# , without congenital heart disease, underwent a lung biopsy which confirmed reversible pulmonary vascular changes. patient# had a pulmonary hypertensive crisis which responded to no. all non-responders( / ) had evidence of anatomic obstruction to pulmonary blood flow (# , , )or a low pvr(# ) on subsequent cardiac catheterisation. in patient # , lung biopsy confirmed severe obliterative vascular disease. conclusions: inhaled no appears to be an effective pulmonary vasodilator. a failed response may be evidence of either irreversible pulmonary vascular disease or a residual anatomical obstruction which may be surgically remediable in the postoperative cardiac patient. therefore, inhalation of no may be a useful diagnostic test to differentiate between fixed anatomical obstruction and reversible vasoconstriction. results: during these years, the incidence of sdra was . % of the total of admissions. the most common etiology was meningococcic septic shock. since , there is a decrease of its incidence. (from % to %) and an increase of pneumonia and immtmodeficiencies. mean age of our patients was , years ( % males, % females), total mortality by sdra was % and there is an increase up to % since mean time of stay of the dead was , days and , days those who survived. although during the late years we offer in the picu a better attendance quality to the patients with sdra and the mean stay is longer, both for those who die and for those who survive, mortality of patients with sdra have increased. the incidence of sdra secondary to the septic shock of a meningococcic etiology have decreased. on the contrary, the sdra secondary to infections by opportunistic germs in patients with congenital inmmunodeficiencies or acquired immuodeficiencies have a tendency to increase. in our series, this change of aetiology is the responsible for the increase in mortality. hospital infantil unlversitario "virgen de roclo". sevilla. espalqa aims:to assess the incidence, etiology, clinical course, sequelae and mortality of the patients admitted to a paedfiatic intensive care unit with the diagnosis of severe traumatism. material and method: cases of severe traumatism in children admitted to our icu in the period from january to june were reviewed. age of patient ranged from months to years, % were males. in our series, % of cases suffered traumatism due to a traffic collision and % had a fall from a considerable height. only in one case was traumatism due to violence to the child. we assessed the first assistance received in % of cases: where was it performed, interval of time since the accident, and steps taken. these data were also studied in relation to the latter evolution. results: % of our patients suffered cranioencephalic traumadsm (ct); in % it was an isolated picture and in % of cases was associated to other lesions. there was participation of thoracic and/or abdominal organs in % of cases. % of cases presented important maxillofacial involvement. only one case presented serious cervical medullar lesion. mortality in our series was . %. in . % important sequelae remained. all of these patients presented tepas on admission equal or lower than . % of those with traumatises had slight sequelae. . % of the total evolve towards healing. a polytraumatized child is a patient that benefits considerably of it admission in a paedriatic !cu. the rapidity in receiving first aid and its quality are essential to avoid sequelae and to make mortality decrease. after unilateral lungtransplantation % of the patients develop a lung failure with decrease of perfusion and increase of pulmonary blood pressure in the transplantated lung. the improvement of perfusion is an importent task in the postoperative period. case report: a year old girl with idiopathic pulmonary fibrosis received a left sided single lung transplantation. during the early postoperative period occured a higtter demand of oxygen and an increasment of the pulmonary vascular resistence in the left lung. the pulmonary ventilation and perfusion scintigraphy indicated in comparison with the right lung a reduced perfusion of only % in spite of a ventilation of % of the transplanted lung. to improve the perfusion of the transplant we administrated per inhalation prostacyclin in a maximal dose of ng/kg/min. the arterial blood pressure decreased but the perfusion continued nearly at the same level. during the following administration of ppm no in the respiratory air we achieved a significant reduction of the respiration pressure f~m to nun h and of the pulmonary arterial pressure. the perfusion in the transplanted lung increased to ca/of the total pulmonary perfusion. after days of administration with no we were able to withdraw the axtifical respiration without any following complications. conclusions: the perfusion of transplanted lungs is a major proble_r~ in the postoperative period. this case demonstrated the advantage of no towards the inhalativ application of prostacyclin. no showed a significant improvement of perfusion in the transplanted lung of a year old girl. results: a total of children with ards were treated with bovine surfactant (alveofact| cases were evalable. the median age was . years (range weeks to , years). in six cases ards was associated with pneumonia, in two cases with lung hemorrhage; in one case isolated ards followed hemihepatectomy. the first surfactant application was performed with a median latency of clays (range - days) after first symptoms of ards witha median doseof mg/ kg (range - mg/kg). in patients doses of surfactant were applied. during the hour before therapy, the median pao / fio -ratio was - . within min. after application of exogenous surfactant the pao / fio -ratio increased to with successive decrease over a period of hours to . accordingly, an increase in pao and oxygen saturation and (less significant) a decrease in ventilation parameters could be observed. analysis of broncho-alveolar lavage before surfactant application in children receiving repeated doses revealed in most examined cases either clear surfactant deficiency or pathological function. of treated patients survived ( of the , respectively). of the surfactant doses were applied in the surviving patients.conclusions: the application of exogenous surfactant in children with ards caused a significant increase in oxygenation, which declined over a period of - hours. the effect often could repeatedly reproduced, in one case after applications. the increase in oxygenation often allowed the reduction of fio and/or the inspiratory pressure. no side effects were observed after exogenous surfactant application.in many cases the application of surfactant wag too late after first symptoms of disease (median latency days). ards mostly due to pneumonia seemed to respond to surfactant therapy less well or not at all. permanent junctional reciprocating tachycardia (pjrt) is the most common incesant supraventricular tachycardia (svt) in children. it is usually drug resistant and its onset in early life has been associated with dilated eardiomyopathy. we report our clinical experience with patients detected antenatally and another diagnosed at months of age. method.diagnosis: negative p waves were detected in leads ii,iii and f, p'r > rp" and there was not warm-up at tachycardia onset.clinical records, ekg,x-rays, echo and holter were reviewed. ep studies were undertaken only with therapeutic purposes. results. in a year period patients under y of age fullfilled diagnostic criteria; were detected prenatally ( - weeks) and one was diagnosed at age mo. the fetuses had intermitent svt during gestation. all of them had pjrt in the first month of life at rates between and bpm. they were admitted to the icu but did not develop signs of heart failure. they were controlled with digoxine (d); d and quinidine; d and propafenone in to days. one was in sinus rhytm until age y; he then showed persistent pjrt over % of the day on repeated holters and underwent successful radiofrecuency catheter ablation (rfca).the other two patients showed initially a lowering of tachycardia rate followed by sinus rhytm for over % of the day (follow-up ran and y). the mo. old infant was admitted to the icu in severe cardiac failure. echocardiogram showed marked systolic dysfunction (shortening fraction %) treatment with digoxine, amiodarone and propafenone were unsuccessful despite lowering heart rate to ; rfca was performed at m. of age with restoration of sinus rhytm and rapid recovery of contractility. all patients were given atp at admission with transient ( to see) recovery of sinus rhytm. ff,s clinical course of pjrt is variable. atp is useful only as a diagnostic tool. initial treatment with digoxine + amiodarone or propafenone is adviced. rfca is a very useful therapeutic modality and can also be performed in young infants twelve patients ( %) died. these were meningitis, head injury, sub-arachnoid bleeds, status epileptieus, leukaemie, drowning, and multiple trauma. calculated from the a admission day p edialric risk of mortality score (prism), the probability of death (p) ranged from - %. of the deaths, i were predicted by prism analysis except for the leukaemie patient (p i%) who died from haematological complications following chemotherapy. two children predicted to die (p % & %) survived. the median length of stay was days (range - days). patlents( %) received ventilatn~ support and patienta( %) were transferred to specialist units ( neurosciences, liver, cardiac, bums). this data supports the view that many paediatric patients are being adequately treated in a dgh icu. meningitis and other neurological illness caused the majority of deaths and respiratory problems caused most admissions. most deaths ( of ) occurred within a few hours of admission. ectopic junctional tachycardia (ejt) is one of the most dangerous arrhythmias in the postoperative setting of congenital heart defects since it does not respond to antiarrhythmics or defibrilation. the object of this presentation is to report on two patients who presented f_jt in the early postoperative period and developed intense congestive heart failure which could be controlled after treatment with moderate topical hypothermia. two patients, m and y, diagnosed of atdoventficular septal defect and tetralogy of fallot developed intense heart failure in the early postoperative period. taehyeardia rate was and bpm. medical drug therapy included weaning from vasoactive drugs, iv digitalization and iv amiodarone treatment. there was not response. they were both surfaced cooled by placing plastic bags filled with cold water over the patient's chest and abdomen. temperature was monitored to obtain a central temperature of ~ there was a gradual decrease in heart rate in the following hours ( - bpm) paralel to the degree of surface cooling and clinical course estabilized.both recovered normal sinus rhytm in to hours. there were not significant arrhytmias after the procedure and postop, was uneventful. conclusions. moderate hypothermia is a very useful manuever for the treatment of drug resistant ejt. since it lacks side effects of other antiarrthymics we beleave it should be the treatment of choice for the treatment of ejt in the postoperative patient. present understanding of the pathogenesis of sepsis, based on the theory of systemic inflammatory reaction, has risen new interest in the more invasive methods of treatment, like plasmapheresis, leucapheresis and exchange transfusion (et). obiectives: evaluate the effect of et in the treatment of neonatal sepsis. material and methods: from september to december , a prospective study was carried out, where the severest cases of bacteriologically proven neonatal sepsis (n= ) were treated with et. in total newborns were treated for culture positive sepsis in the intensive care unit during this study period. diagnosis of sepsis was based on the clinical criteria of suspected neonatal sepsis, used by mc harris et al., laboratory data and positive blood culture. newborns with severe congenital malformations were excluded. et was carried out with fresh (less than hours old) adsol-conserved erythrocytes, from which buffy coat had been removed, and same donors plasma, using a slow continuous two-site technique. the mean volume of et was . ml/kg. the effect of et was assessed as a change in the score for acute neonatal physiology (snap), general treatment results were compared with a historical control group of newborns, treated for culture-positive sepsis in the same icu during the first eight months in . students ttest and chi-square test were used in statistical analysis of the data. results: with the use of el a significant decrease in mortality was achieved: death of cases during the study period, compared to deaths among the controls (p< . ). no baby, receiving et, died. the incidence of severe complications did not differ in the two groups. the snap-score showed quick improvement by the first post-transfusion day (p<o. ) and the effect persisted during the five following days. conclusions: we conclude, that the use of et in the treatment of critical cases may help to lower the mortality of neonatal sepsis. it provides a quick improvement in the clinical condition of septic neonates. the granulocyte-colony stimulating factor (g-csf) is largely employed in granulocytopenic patients. experimental studies have' demonstrated the effectiveness and safety of the therapeutic use, nevertheless the human employ is reported almost exclusively in neoplastic patients submitted to chemotherapy and in patients with fungal or pseudomona~ spp infections. the authors report the data concerning three non granulocytopenic children: in the first, o girl years old, affected by pseudomomm aerug/n~a pyeionephritis, the antibiotic therapy was not tolerated in consequence of renal and hepatic failure. the second patient, an infant months old, has been treated with salbactam-ampiciilin, ceftriaxone and chioramphenicol because of haemepldlw iafluem~ meningitis; a late neurological aggravation required the antibiotic therapy recommencement the last, a years old insulin dependent diabetic boy, was affected by cared/do spp systemic infection; the antifungnl therapy w~ not practicable in consequence of renal and hepatic involvement in all the g-csf was administred subcutaneously at the'dose of $ u for days. a significant increase of granulocytes was ever noted already after the second administration until hours after the last dose. clinical signs, fever and biochemical values are promptly influenced positively and all patients are restored to health. no side affects were noted. the granulocyte colony stimulating factor, at indicated doses, can prove of great help in critical patients, in who the antibiotic or antifangal therapies can result toxye or cannot be tolerated. the ~reptococcas p~, among the gram pmltlve~ represents the main causative organism of bacterial meningitis in children, with the majority of deaths occurring within hours of admission, the third generation cepohlosporim were indicated as antibiotic of first choice, and some literature reports have demonstrated no significant dlffereuces in dinical course of patients treated with ce~rlaxoae and of those treated with ampieibin and chlornmphenicol. the authors report the ease of on infant, male, months old, submitted to a neurosurgical interventiol after days the infant became febrile and lethargic and a bacterial meningitis by ~trepmcecctz ~ was diagnosed, in spite of he was given the eoftriaxone therapy.. an amelioration was found only when a systemic treatment with vaucomyein was added. after days an intrathecal administration of vancomyein m~day was started: the norbmlization of the spinal fluid profile was uoted two days later and the infant receve~d progressively. the follow-up after eight months doem't evidentiate motosensorini sequelae-the individuation' of ~cscc~ pmmmsm/ae strains heta-loctam antibiotics resistant requires alternative therapeutic regimens: the k~terity quead vitam and quoad valetudinem of these infections justifies an intrathecal therapy, especially failing o prompt answer to the systemic treatment objectives: to evaluate the incidence of the burnout (bo) of the staff in a picu in a university hospital. the bo is a syndrome charactedzed by many factors like emotional stress, lack of personality and reduced work ability. this syndrome represents a kind of abnormal response in the working habitat, above all, where psychological and personal characters are mostly involved. the symptoms of bo are represented by demoralisation, demotivation, incapability, boredom, isolation up to organic disturbances, like migraine, insomnia, dizziness and gastrointestinal disturbances. the major incidence of this syndrome was found in the hospital staff, especially among the nurses, working in an atmospher of high emotional dsk, just like in an intensive care unit. methods: from january ' till april ' , all the staff of the picu of the a. gemelli hospital of rome, underwent a test to evaluate the bo. the test used was taken from the book "bumout: from tedium to personal growth", by pines and aronson. twenty seven people were studied ( females, males)i out of which doctors, residents and nurses. it was considered sign!ficant as mild bo, a score between - and as severe, the score of bo >. . results: subjects ( %) resulted positive for bo, out of which were females ( %) and were males ( %). the subjects with mild bo were / : was a doctor, residents and nurses. the subjects with severe bo were / , out of which resident and nurses. conclusion: the results obtained show that bo is a condition well represented in the staff of our picu. the category most at dsk seem to be the nurses ( subjects), as well as residents ( subjects), as in literature, which shows a major incidence of the syndrome in younger subjects and having a limited partecipation of functional decision. the results obtained obliged us to start a programme of serial controls so that the subjects most exposed can have a necessary psychological support to react adequately to this condition. the term systemic inflammatory response syndrome (sirs) was adopted by the consensus conference to denote a type of systemic response to severe infection or otherinsults in critically ill patients. when sirs occurs from infection it is called sepsis. sepsis occurs more frequently in persons with perexisting illness or severe trauma. there has been tremendous advances in prophylaxis, diagnosis, and treatment of sepsis. a comprehensive model of the disease progression from sirs to mods should be developed giving priority to severity of illness scoring system and other predictive methods. some recommendations for future clinical trials include: trials should not start with humans. before proceeding to human trials, animal studies should indicate an acceptable risk/benefit ratio. appropriate patient populations must be defined and treatment protocols should be standardized. full and rapid reporting of all results should be mandatory and a central repository of published and unpublished study results could be helpful. accrual at each center should be of sufficient size, and should include the number of patients accrued, mortality rates, and patient characteristics. pivotal trial should be preceded by sufficient pilot or phase ii studies. correct drug dosage and usage should be delineated in pilot studies. large, multicenter, trials should be used to enhance the unversality of trial results. analyses should be planned a priori. definitions for the target population should be explicit, reproducible, and include illness severity scores. outcomes should be relevant reproducible and include both measures of benefit and harm. mods and its reversal should be considered as an endpoint. quality of life should also be considered as an endpoint. the estimators of overall treatment effects should be controlled for base-line prognostic factors and subgroup anaiysis should only be used for hypothesis generation and not to modify the conclusoin of the trial. economic analysis should be included as part of clinical design. evaluatin of source control should be a critical component of any study. standardized clinical mediator assays should be pursued. placebo patients in clinical trials should be studied for a better understanding of the pathogenesis and epidemiology of sirs, evidence based medicine should be used to evaluate the validity of clinical. introduction: use of inhaled nitric oxide (no) as a modulator for optimizing ventilation-perfusion or lowering pulmonary artery pressure is becoming increasingly common. no is a free radical but little toxicological research has been published. clearance of nebulized mtc-dtpa is known to be, a sensitive indicator for early function impaimaent of the alveolocapillary barrier. we investigated whether exposure to no increased clearance of ~tc-dtpa from the lung. methods: three groups of white sealand rabbits (bw . kg) were anesthetized, tracheotomized and paralyzed. groups were ventilated for six hours at pressure regulated volume control, set to deliver ml/kg with a frequency of /rain, i/e ratio = : and peep = cm hzo using a modified servo ventilator (siemens, solna, sweden) with computerized no delivery system. gas mixture per group was either / or / [no (ppm) / fioz]. after six hours of ventilation in these groups and immediately after anesthesia in group (control), ~tc-dtpa was nebulized into the inspiratory line of the breathing circuit and administered as a fine aerosol. gamma counting was measured for minutes, monoexponential curves were fitted to the data and the clearance half-time (t was calculated. the t~/ mean • sd of the different groups were: t~a (mean -sd) h"e,i witl~ arf : di.ff:erent kinds, aged .q-ore mon't.hes to [ gears o : (bodi weight .~rom ., to kg), is presen .... "ed ( i,,~u::trl:e i:ibstraclive d:lse~se... ~ .ards'- ; :~,;,,arf o~ ::entral genes:i s .- , ,~ :inc lud ing men ingeenceph it :is- ~ reye ' s ~yrtdro~e-..#~,bri~:ln pes~.re~nimatior~ disease.." ). int:lrl~]. pa-. "iiulle'i,~s ariel regymes o+ l;mv,l;i"t"v were cle'l'.ermllled by ba- 'i~ier was. about . tuber,, dopamin tiara-:. t.io; was ~.".,,'.r:~r~led. cmv,cppv d~.!"~tion raniled -~rom f to dayns.,~ < .-:in , "t -irl lo;and> davs'-in 'l~atierr~{s i'i"ai s:ltiol~ o ; patterers to imv, simv modee was per.r:)rmed, ~herl pif:' decrease.d to - ml~ar, fi ~ecreased to , . lind less with a = /,,. i:lesq.lts:{ in pat:i.ents e{ group :l, who were tre,~d.ed w&th f'f'v, teoph :i. : . l:i.r~ (is- .mg/kg/day), g lucecdr t icostei~oids ( .... :~;mg/kg/day), when r exceeded in , -.];, times normal va i tea the e aqes/,'!:l"oln ~j,, ite :i.~;::.!;, ~ml"lrj), it was possible 't'(' ce 'e~ e aad]t:..~rom ! . '.' i', to !..'; , - , mml-lg in ~}.. :~.[~ houi,!; ~d'l(:i to ru:}l",g'd!~l:i. e i::h,:~e,'~c['el';i.stil obieetives : this chapter will describe what is knovca of the psychlogical responses of infant and children to hospiuiisation and attendant procedures. the factors which may modify these responses will he discussed and important considemtiorts will be outlined for optimal anaesthetic management and postoperative period of infants and children which will minimised the rise of emotional upset. methods : in this paper the autors will discttssed the probl of: . health children (asa i, ii) facing single uncomplicated surgical elective procedures . various abnormal situations including neurotic children, children facing repeted operations, chronically ill, buaaes and tsaumatically impired ones . unfortunate young patient facing and often expoclting fatal outcome from le "ul'ukaemia, tumors, cystic fibroses or otheq" disease. : management of each child must vary greatly, ifi general the phases of emotional conditioning include home and preadmissiun preparation, admitiun preoperated and operative care and postoperative period. the authors would be happy if the child passes all stages without any trauma which could be prolonged in the future life. introduction ino is used to selectively reduce pulmonary vascular resistan(~e. we applied ino in the postoperative intensive care of patients with pulmonary hypertension and the risk of right ventricular failure after surgical correction of a congenital cardiac defect. methods - ppm no were added to the ventilatory gas mixture using a specially designed equipment (messer-griesheim, germany/austria). indications for application included pulmonary artery pressure > % systemic pressure, critically depressed right, ventricular function or an oxygenation index > . assessment of n oefficiacy consisted of on-off-on measurements according to the clinical stability of the patient including hemodynamic parameters, pulmonary gas exchange, continuous monitoring of ventitatory function and transesophageal echocardiography of the right heart. results in situations ( patients, age days- , years), ino was applied - h postoperatively. oxygenation was improved in situations from _+ to + mmhg pc ; pulmonary pressure was reduced in situations from -* % to _+ % of systemic pressure. in situations, no reduction of pulmonary pressure was present, but measurement of cardiac output or echocardiographic analysis indicated an improvement of right ventricular function (right ventricular stroke volume + -* %, cardiac output + -* %). in situations (immediately postoperativ with suprasystemic pulmonary artery pressures [n= ], multi-organ-failure [n= ]), no response to ino could be determined. conclusions for a special group of patients, the selective reduction of pulmonary vascular resistance by ino has become an important part of postoperative therapy. using this selective afterload reduction, postoperatively depressed right ventricular function can be improved. this effect of ino seems to be the most important one in the postoperative period. thus, ino appears justified to be appfleo when impaired right ventdcular function could be improved even when pulmonary artery pressure is not raised or remains unchanged. obiectives : premature infant are exposed to danger of apaea due to anaesthesia during their tirst months of life. it is yet unknown whether prematurity is corelated to any other kind of reslgratory disorder due to anaesthesia within the tirst year of life. methods : we theretbre researched retrospectively for respiratory disorders in all infants under months of life belonging to asa group . they all had been anaesthetised in . in our clinic for the following surgical reasons: ingvinal haemia, umbilical haemia, hydrocelae testis and phymosis. results : in cases we tbund: lafingospasm during induction in anaesthesia ( , %), bronchospasm during induction in anaesthesia ( , %), impaired intubation ( , ~ postanaesthetic laringospasm ( , %), supposed aspiration ( , %),postanaesthetic inspiratory stridor ( , %), postinductional inngoedema ( , %), death after months in consequative of infection pneumonie ( , %), none of these disorders was correlated the prematurity, infants suffered of post anaesthetic apnea, of them had premature medical history. concludions : prematurity does not enhance the risk of respiratory disorders due to anaesthesia within the first year of life, except the danger of postanaesthetic almea needs spetial cosideration. it could be demonstrated that aepgi lowers pulmonary vascular resistance and indirectly improves cardiac function. this effect seemed to be selective, and was comparable to ino in the doses we have examined. therefore, aepgi could represent a clinically useful alternate to inc. however, further research is necessary to work up the benefits of either therapeutic strategy. objectives: heat and moisture exchange filtem (hme) are used as artificial noses for intubated patients to prevent tracheo-bronchial or pulmonary damage resulting from dry and cold inspired gases. furthermore they are used for the prevention of bacterial contamination of the anesthetic apparatus by the patient's exspired air. so they are considered as a time-and money-saving device in anesthesia. filters are mounted directly on the tracheal tube, where they collect a large fraction of the heat and moisture of the exspired air, adding this to the subsequent inspired breath. the effective performance depends on the water-and bacteria-retention capacity of the filter. this study evaluates the efficiency of four different filters under clinical conditions. methods: four different types of filters ( dar hygrobac, gibeck humidvent, medisize hygrevent and pall bb ) were investigated dudng mechanical ventilation over a pedod of hours. minipigs with hemorrhagic shock were intubated and ventilated for days in an animal intensive care unit (icu). after hours of mechanical ventilation the filter was randomly replaced maintaining the individual ventilatory conditions. the weight of the filter was determined before use and after removal after hours. the airway pressure was monitored online to record changes during use. tracheal secretions and both sides of the filter were microbiolologically tested to see whether bacteria of the animal's respiratory system could be found on the patient's side of the filter or if they even would have penetrated the barrier. results and discussion: over a pedod of hours of types of filters showed an increase in weight of + % and airway pressure. bactedal celonisation ccured in nearly all fillers ( of ) on the patient's side, whereas only three of four types of filters showed identical bacterial colonisation on both sides. the only filter that did not show bacterial penetration, increase in weight or airway pressure was the pall-hme, a condensation humidifier without hygroscopic salts for moisture retention. with respect to our data one should use a condensation humidifier if airway conditions should remain stable dudng mechanical ventilation and desinfection of the anesthetic apparatus should be avoided after each patient. aim: to assess the clinical uses of, and experiences with, the hayek oscillator. this is a non-invasive device capable ef delivering not only continuous negative pressure (cnp) but also external oscillatory ventilation around a negative baseline (eov-nb) using an external cuirass. this type of ventilation avoids the need for intubation and intermittent positive pressure ventilation (ippv) and facilitates weaning in ventilator dependent patients. patients and methods: patients in respiratory failure, age range weeks to years in a total of patient episodes were treated using either cnp or eov-nb mode. duration of treatment varied from hours to days. indications for use ef the device were: ) to facilitate weaning from ippv ) prevent reintubation of patients following unsuccessful extubation, and ) avoid intubation and ippv altogether using the hayek oscillator as the on[y means of respiratory support. results: there was an increase in pao :fio ratio after cnp and eov-nb (p < . , and p= . respectively, wilcoxon signed rank test). patients who were in respiratory failure with hypercapnia showed a statistically significant reduction in paco both with eov-nb and cnp (p= . and p= . respectively) but the magnitude of change was individually greater in the patients who were treated with eov-nb. all patients, however, showed a fall in respiratory rate (p< . ) after the application of the cuirass in cnp mode. there was no physiological deterioration related to the application of external extrathoracic negative pressure in either cnp or eov-nb modes. conclusion: the improvement in pao :fio , the fall in paco and respiratory rate were indicators of an improvement in ventilation. the proposed mechanisms include improvement in frc, recruitment of additional alveolar units, and improvement in secretion clearance resulting in reduction in the work of breathing. meek to ~ month of the lifo,the bemodyuanicfacls were defined uitb the help of tetropolar reography method!. the excretion of !he catbocholauines fcfi] mith the urine gas detertend by taylor ll,laoorsy ~ iacg/dayl. hsaltl in the hypercuagulation stage of bic we deflorteeed the acliuutiun of the tbrubio and plasiin syaet~ mitb the increase of the inhihitnrs, in this case we registered in full uahe dot this process coabined uitb the dayl~ excreliou with lho urine epinopbr ne e], nor~pinopbr no tel and dophanine io], lbat shod the inlensificatiou of the s~nthosis prnoe-s~es and the release of ea in blood fron hissue deport the actffat on of the svnpathadrenui systen ]sfisl assisted to furl the b?perd~nanical rosins of the eircuidion and increase the ,icrocirculatinn, the klinicai sings of the insufissieutly of the circulalion have not defined,that has been associated the conpensatury character uf the ehan~es of ~ and heludy~enic status, t~e uun~u|p-lion ceugulupatby bus been donoustraled in the hypocougulatien stage ~bat man xauifosted b the exhaust of lhe confulalion nod oessel-platel heuostasis, the consuxptton of cnnpononts tbronbln ,plnstin, kallek~eiu-kinln s~slots and the forniration eat in fell canoe clot uas accoqaued bs docrea,e of fl,nfl,o, the products of the xotabolisx of c~ and the activation of xonoaninoxydasu. the decrease of the extoll'on g and the exhaust deport co indicahd about t!e ]ou fund/anal reserve of ~fl~. it was one of the lain reason of ~bo heiod~uanic disbroed iheat insnfissient]~] and the uicrncireulaflion lintestinal codeme with the low effectife periferal flow] and nul[iplay organ failure,the distrued deport of sos mitb throubocytupenin no; be one of the nechanisn the dislrood of uessej-plalol heioshasis, the correlation bolueeo changes of boiostosis c~ and circulation ore reguired aduinistration nedidns, thai reslore the love s of c~ in the blood, prevent uulliplay organ failure and hetorrnge in children with sepsis, ~b~ectives: multi-measured correlative analysis of the most number of non-invasive indices of the cardiorespiratory system function was made to determine the structure of their interrelation and the ways of their adequate and effective correction. hethods: spiremetry, capno~raphy, oxygenography, indirect fick method at recurrent respiration, plethysmography, integral rheography -in all indices were used. the received data were processed on a computer by a standard package of statistical bmdp programs. results: women with ~h-gestosis (i group) and somatically healthy pregnant women (ii group) were studied. cluster analysis has shown that the rate of the mean correlation connection between ventilation indices was % in the ist group and % in the iind group; gaseous metabolism - % and %, respectively; central hemodynamics was ~ in both groups. conclusion: cluster interpretation allowed to suggest that an increase of the rate of the mean correlation connection between the indices was characteristic of effective adaptation as the system was multi-component and well-regulated. on the contrary, the increase of the rate of strong correlation connection between the indices reveals the rigidity of the system and the tensity of adaptation mschaniams, i.e. the proximity to decompensation. it follows from this that in cases of eph-gestgsis, the reliability of regulating ventilation and gaseous metabolism decreases. seve/e hypoxemia in non intubated patients represents a major contraindicafion to fiberoptic bronehoscopy (fob) and bronehoalveolar levage (bal), but these procedures are often required for a correct diagnosis of the causative agent of pneumonia. aim of this investigation was to veaify the safety and efficacy of bronehoseopic procedures during pressure support ventilation administered through facial mask (fm-psv). five intensive care patients, all immunoeompromised, ( males and females; mean age . • were enrolled in the study. all patients presented criteria for pneumonia with pao /fio ratio ~ and were responders to fm-psv. fob and bal were performed afte~ topical anesthesia with fm-psv ( ps = em h ; peep = emh ; trigger = -lemh ) continuously admires" tered ( ' before fob fio = . ; during fob, fio = and for ' alter fob, fio = . ). pao /fio ratio as well as saturation (sat) did not show signifteative changes during the procodure (fig.l) . no complication was observed and hemodynamic conditions were stable for all patients. cmv, pnenmoeystiis ( ), legionella and mycobaetermm tuberculosis were identified from bal allowmg a prompt and targeted therapy. we concluded that mask psv can represent an excellea~ technique to pexform fob and bal in severely hypoxemic patients without deterioration of gas exchanges and avoiding endotraoheal intubation. intensive care unit, hospital general of albacete, albacet~ spain. objective: to analyze the current incidence and epidemiology of total parenteral nutrition (tpn) among critically ill patients placed on mechanical ventilation. design: prospective observational study. setting: medical intensive care unit in a tertiary hospital. patients: a total of consecutive l'ritically ill patients with non-coronary related disease needing mechanical ventilation admitted in our icu during a months period. measurements: data of sex, age, diagnosis, and outcome were recorded. severity of illness and therapeutic effort in the first hours were measured using acute physiology score and chronic health evaluation (apache ii) and therapeutic intervention scoring system (ties). r~ults: mechanically ventilated patients, male and female, were studied. only ten patients needed tpn and their main diagnoses were: five cases of multiple organ failure secondary to pneumonia ( ), ards ( ) and septic shock ( ); two eases of acute panereatitis; and one mesenteric throngmsis, one status epilepticas, and one ,prolonged cholinergic crisis b~ suicidal organophnsphate insecticide subcutaneous injection. no statistically significant differences between both tpn and non-tpn groups were found: objectives: evaluate the efficacy of prone position in ards and determine its importance in the therapeutic algorithm. methods: consecutive patients with severe ards (murray-score > , ; pao / fit < mmhg; male, female, mean age years) were conventionally ventilated (pcv, peep - mbar, i:e=i:i, ppeak < mbar). if after hours pulmonary function did not improve patients were placed in prone position. change from prone to supine position was done every hours. beside ultimate survival, parameters investigated were aado , pao /fio , and venous admixture (qs/qt). results: during the first hours in prone position of patients showed a significant decrease in qs/qt ( . % vs. . %) and aado ( vs. mmhg), and an increase in pao /fio ( vs. mmttg). changes were most pronounced in patients with high qs/qt, and in patients with an onset of ards less than hours before first application of prone position. after an average of position changes ( to ) of patients could be weaned from the ventilator. patient could leave tile hospital. i the later course letality was primarily determined by additional organ failures and by the severity of the underlying disease. negative side effects were minor, including slight cardio-vascular depression and increase in p~co , and never posed a limitation to continuation of prone position. especially in patients with septic shock skin lesions in exposed areas could not always be prevented, prone position could easily be combined with all ventilation modes and with all intensive care interventions. also immediately after major surgery and in patients with open packing prone position was possible. conclusions: in this investigation prone position proved to be an efficient and safe method in the treatment of severe ards. patients with a pronounced ventilation/ perfusion mismatch and patients in the early stages of ards appear to profit most from prone position. though the immediate effect on oxygenation is striking, still more the % of all patients die from multi organ failure and underlying diseases. a proposed therapeutic algorithm for ards is as follows: if under conservative ventilation (pcv, peep < mbar, ppeak < mbar) pulmonary function does not improve within - hours prone position should be applied. when after - position changes no lasting effect can be achieved further ventilation modes (e.g. pc-irv, aprv, no, etc.) should be used in addition to prone position. standard intensive care principles, such as fluid restriction and optimization of circulation, apply also to patients in prone position. objectives: nitric oxide reacts with superoxide to form peroxynitrite, an extremely reactive and toxic species. we quantified the presence nitrotyrosine, the stable product of the interaction ' of peroxynitrite with tyrosine residues in the lungs of pediatric patients that died with respiratory distress syndrome (rds). methods: paraffin embedded lung sections, obtained at autopsy, were incubated with a polyclonal antibody raised against nitretyrosine, followed by a secondary fluorescent antibody. alveolar structure-associated fluorescence was quantified using existing methods. results: tissue sections from patients who died with rds exhibited significant specific immunostaining which was uniformly distributed across the blood-gas barrier. in contrast only background levels of fluorescence were seen in the lungs of patients who died from non-pulmonary causes. intense staining was also seen in the lungs of rats that breathed % for h, a condition known to result in rds-type illness; no immunostaining was observed in air-breathing rats. conclusions: significant levels of peroxynitrite may be formed in the lungs of patients with acute lung injury. peroxynitrite may be contributing to the pathology of rds by damaging key components of the alveolar epithelium including the pulmonary surfactant system. mechanical ventilation time was prolonged ,g • days in patients with ardsvs , _+ l, days in control . mean staylcuwas lg _+ ,g days in the ards group vs , • , days in control group postoperative mortality rate was % in ards patients vs , % in those without respiratory failure. -ards incidence in liver transplantation is low ( , % in our sene) but it causes high mortality ( %) page, gas ventilation of the perfluorocarbon-f'dled lung, supports gas exchange and circulation in small animals (< kg) with lung disease. we hypothesized that large animals could be supported by page without adverse effects on bemodynamics. we first elucidated the determinants of gas exchange in normal sheep, and applied them to a model of adult respkatory distress syndrome (ards). methods: using the ventilator settings determined to be optimal in our pilot study (fio of . , peep of cm h , imv of bpm, it of %, and tv of ml/kg), sheep weighing . ~ . ) kg had lung injury induced by instilling ml/kg of . n hc into the trachea. ten minutes after injury, sheep with pao < ton" were randomized to continue gas ventilation (control, n= ) or to institute page (n= ). page was instituted by instilling . l of unoxygenated pefflubron into the trachea and resuming gas ventilation at the previous settings. abg's were drawn at baseline, minutes after injury, minutes after injury, and then every minutes for hours. objectives: inhaled nitric oxide (no) can improve oxygenation and decrease mean pulmonary artery pressure (papm) in hypoxemic patients with ards. in severe hypoxemic copd patients, it is not known whether inhaled no can exert a similar effect on hemodynamics and gas exchange. therefore, we investigated die response of inhaled no in hypoxemic copd patients and the results compared with those obtained in a group of ards patients. methods: ten copd patients (age _+ y;fev~ . _+ . l) and ards patients (age _+ ; lis . _+ . ) mechanically ventilated were studied. hemodynamic parameters were measured using a swan ganz catheter. arterial and mixed venous blood gas determinations, sao , svo , hb and methb were measured (abl ,osm ). mean intratracheal concentrations of no and no were continuously monitored using a chemiluminescence analyzer (nox ) . during the study the ventilatory pattern and fioz were kept constant. the protocol was for ards group: basalt, no loppm, basal~; copd group: basalz, no lo ppm, no ppm, no ppm and basal . after a steady state of rain hemodynamic and gas exchange measurements were performed. a positive noresponse was defined as a % increment in pao . results: papm was similar in both groups and decreased significantly after no (ards, basal . _+ . mmhg, no . + . mmhg, p < . ) (copd, basal . _+ . mmhg, no- . _+ . nrmhg, p< . ). all other hemodynamic variables remained unchanged after no. basal oxygenation was higher in copd group (paojfio _+ mmhg) vs ards group (paojfio _+ mmhg)(p< . ). after no- , pao increased ( _+ mmhg to _+ mmhg, p< . ) and qs/qt decreased ( + % to _+ %, p< . ) only in ards group. in both groups, significant correlations between basal papm and inhaled no-induced decrease in papm were found. inhaled no-induced increase in pao /fio was not correlated with basal paoflfio . no responders were / ( %) in ards group and / ( %) in copd group (p< . ). conclusions. in hypoxemic ards and copd patients, inhaled no decreased mean pulmonary artery pressure. however, oxygenation only ameliorated in ards group because die number of responders to inhaled no were higher in ards group and this effect seems not to be related to the basal hypoxemia. these results might be explained by the v/q abnormalities present in copd patients. grant fis / . objectives: it has been recently reported that expired con slope as a function of time is modulated by total respiratory system resistance (rrs) in critically ill patients (chest ; : - ) . in this study, we analyze the relative contribution of disease (dis), endotracheal tube resistance (rtube), airway resistance (rmin), additional resistance (~rrs), autopeep (peepi) and dylmmic/static elastance (ed/es) to the co elimination in different clinical conditions. methods: we have studied adult patients ( controls, acute respiratory failure, severe ards and copd) mechalfically ventilated (servo and c, siemens) without peep. we recorded tracheal pressure, airflow and capnograms. signals were analogic to digital converted for posterior data analysis. objectives: alveolar ejection volume (van) can be defined as the fraction of tidal volume (vt) with minimal dead space (vd) contamination. according to the classical paradigm: limvd_~ [vco /vt] =facoz, vco vs vt relationship tends asyntotically to a constant slope when approaches end-tidal volume. we have defined van as the volume that defines this relationship until a limit of % variation. methods: six subjects with normal respiratory mechanics were studied during anesthesia for minor surgery. two subjects, otherwise normals but having high values of total resistance and dynamic compliance, were also studied. capnograms were recorded in steady-state at levels of vt ( . , . and . l) and four levels of peep ( , , and cmh objectives: patients with ards presented lung abnormalities which originate an increase in airway resistance (rmin), in additional resistance (~rrs) and in static elastance (ers). application of peep further increases ~rrs. capnographic indexes reflect lung ventilation]per fusion inhomogeneities. in these conditions, the effects of peep on lung mechanics could be better understood by simultaneous measurement of capnographic indexes. methods: we studied groups of subjects. n: normal subjects scheduled for minor surgery; arf: critically ill patients with mild acute respiratory failure; ards: patients with early ards (< h). we recorded tracheal pressure, airflow and capnograms. signals were analogic to digital converted for posterior data analysis. respiratory system mechanics was assessed by constant end-inspiratory and end-expiratory occlusions technique. at equal tidal volmne ( . l) a peep level of , , and cmh was applied in all patients. we calculated ers (cmh /l), rmin, c~rrs (cmh /l/s) and autopeep. capnographic indexes were alveolar ejection volume (vae)/vt ratio and expired co slope beyond vae (sipco in contrast to synthetic surfactant natural suffactants (alveofact| are able to inhibit pmn-activation. after incubation of activated neutrophils with surfactant, l-selectin expression is decreased. these effects depends on which preparation is used. we conclude, that natural surfactant (aveofact| can perhaps influence early recruitment (,,rolling") of pmn in patients with respiratory failure like ards. with ards hormann cb, baum m, putensen c, knapp r, lingnau w, putz g . clinic for anesthesia and general lntensiv care medicine, university of lnnsbruck, anichstrabe , innsbruck objectives: in thoracic ct scans of patients with severe ards atelectasis and pleural effusion can be found in the dependent lung regions. by rotating these patients from left lateral position to right lateral position a redistribution of the ct densities, a recruitment of atelectasis and therefore an improvement of gasexchange is possible within a few days ( , ). the objective of this study was to find out the mechanism of alveolar recruitment during lateral positioning by ct scanning in left and right lateral position. methodes: after approvel by the local institutional reviewboard we investigated ventilated patients with severe ards (entry criterias: murray score > , ) in the ct scann of the university hospital. after a stabilisation period of minutes in supine position a thoracic ct scan slice cm above diaphragm was taken. then two different positions of the patients were studied in a randomized order: a) degree of left lateral position, b) degree of right lateral position. each lateral position was held for minutes. at the end of each of these periods a thoracic ct scan slice cm above diaphragm was taken. quantitative analysis of ct scan data was based on the frequency distribution of the ct numbers. to quantify the alveolar recruitment during lateral positioning by means of ct scan we defined compartments within the lungs: a) normaly inflated lung, b) poorly inflated lung, c) noninflated lung ( = atelectases) ( ). results: independant of the side of lateral positioning (l) in the non-dependent upper lung a significant increase of the normaly inflated compartment (s: %; l: %) as well as a significant decrease of the noninflated compartment (s: %, l: %) was observed in comparison to supine position (s). in the dependant lower lung the normaly inflated compartment decreased significantly (s: %, l: %) whereas the noninflated compartment increased significantly (s: %, l: %). throughout the whole studyperiode we did not observe any significant change regarding gasexchange and hemodynamic parameters. conclusions: in lateral position the non-dependent upper lung is decompressed. therefore a significant recruitment of atelectases is observed in the upper lung within minutes. on the other hand the dependent lung is compressed by the weight of the upper lung and the mediastinum. a great amount of the alveoli of the dependant lung collapse in this short time intervall. therefore the net effect of recruitment of one positioning maneuver is very small. when positioning patients one should be aware, that the patient is kept in each lateral position long enough to clean up the atelectases in the non-dependant lung and short enough to compress less lung tissue in the dependant lung. objective: to analyze effects of low-dose no inhalation ia patients with severe aeut~ respiratory distress syndrome (ards) over five days. methods: we prospectively studied patients ( men, woman) with severe ards admitted to our icu between may and may who required no inhalation with a dose of ppm for at least days. entry criteria for no injaalafioa were murray score >i . aud pat/fie < nun hg with peep >~ em i~o for at least hours. all patients were sedated, intubated and mechanicauy vantil~ed with volume assist-control ventilation, and had indwelling arterial catheters (pulmonary artery, and radial or femoral artery) to measure cardiac output (by thermodilufion) and relevant intravaseular pressures, and to calculate derived parameters. no was administered between y piece of the ventilator and endotraeheal tube and flow was adjusted to obtain ppm no in the inhaled gas. the no, no and no x concentrations were continuously measured at the distal end of the endouacheal tube by the chemiluminiscence method (nox , see-seres, france). metahemoglobinemia levels were mesured daily. no inhalation was manteined if paojfio ~ improved at least % and was stopped when the change in pao /fio ~ was below % or when the patient presented a paojf > mm hg a~er minutes without no inhalation. every day we made an on-off test to determine if no inhalation improved pao /fio ~. statistics: analysis of vmiance. data: mean + standard deviation. results: the mean age was . +_ . years and mean lung injury score was . • . . mortality was % ( / ), metahemoglobinemia . • . %, and no concentrations zero. paojf~o always improved significantly al~er ppm no inhalation (see :~ conclusions: reintubation in salf-extubated patients strongly depends on the type of meehamcal venfilatory support: the probability of needing a reintabation ff ese occurs during fult vontilatory support is higher than ff ese occurs during weaning. these data suggest that some patients may remain under weaning from mechanical ventilation for unnecessarily prolonged periods of time. objective: the aim of this study was to evaluate the acute effects on gas exehonge and hemodynamics due to positional changes from supine (sp) to prone (pp) in patients with severe acute respiratory distress syndrome (ards). methods: nine intubated, sedated, paralyzed and mechanically ventilated patients with severe ards were prospectively studied. all had a murray score > . , and a pao /f~o < with peep ~ cm h for at least h. all patients had indwelling arterial catheters in the pulmonary artery as well as in the radial or femoral artery in order to measure cardiac output (by thermodilution) mad relevont pressures, and to withdraw blood samples. arterial blood gases and hemodynamie parameters were measured first in sp, and then in pp after minutes of stabilization. vontilatoly parameters remaing unchanged during all the study. statistical analysis was done by the non parametric wdeoxon test. data are expressed as mean ~= sd. results: there were men and women with a mean age of . years ( - ) and mortality was % ( / ). main results are shown below: objective: to describe and compare a new method for obtaining p-v loops (p-vcv) by using a two-way collins valve (twv) with thosu obtained by the supersyringe method (p-vss). methodology: we prospectively studied patients who had an aeute lung injury and were intubated, sedated and paralyzed, and mechanieany ventilated. we performed the p-vev loops and p-vss loops in random order, and the static inflation pressure was limited to emh with both methods. pressure (p) was measured at the airway opening by means of a differential p transducer, and volume was obtained from flow (measured with a pneumotacograph) integration. the p-vse method has already been described (h~trf a,et al.bepr ; : - ) . the p-vev method consists in the following: the inlet of a twv is connected to the ventilator's y-piece, and both outlets are couneeted to the endotraeheal tube by means of an additional y-piece; one of this outlets has a one-way rudolph valve in order to allow inspiration but not expiration during the inflation maneuver. changing the twv tap position allows basal ventilation or progressiveinflation of the respiratory system. this maneuver is as follows: during an end-expiratory occlusion, the ventilatory settings are adjusted to deliver a ml v r with a respiratory rate of /min and i/e ratio : ; at the same time the twv tap is ehonged in order to divert flow through the one-way valve. inflation then begins alter releasing the expiratory oonlusion. pressure and flow signals were digitized and acquired by a computer for subsequent data analysis. we analyzed the following parameters: inflation compllonee ( objective: to analyze the variables which eventually may differentiate ards patients who do and do not respond to low doses of inhaled no. we prospectively studied patients ( men, woman) with severe ards admitted to our icu between may and may who were treated with no ( ppm). the onta'y criteria for no inhalation were murray score >/ . and paojfo z < mm fig and peep >/ cm i~o for at least hours. all patients were sedated, intubated and mechanically ventilated with volume assist-control ventilation. tidal volume was between and ml&g, with constant inspiratory flow, respiratory rate was - /rain, and i/e ratio between : to : . all patients had indwelling arterial catheters (pulmonary artery, and radial or femoral artery) in order to measure cardiac output (by thermodiintion) and relevant intravascular pressures, and to calculate derived parameters. no was administered between y piece of the ventilator and ondotracheal tube, and flow was adjusted to obi~a ppm no in the inhaled gas. the no, no and no x concentrations were continuously measured at the distal end of the endotracheal tube by the chemilumiinscenee method (nox , see-seres, france). metahemogtobinemia levels were measured daily. we considered a response to no inhalation when an improvement in paoz/fo above % was observed after the inhalation of ppm no (group r) . when the cha~age in paojfi z was below % it was considered a lack of response (group non-r small airways functional abnormalities have been recognized as a common feature of lung pathology. however peripheral airways contribute relatively little (~ %) resistance to flow and there disturbances can not be adequately estimated by conventional measurements of respiratory mechanics. the purpose of the study was to evaluate the relationship between raw and small airways conductance following weaning from ventilator methods. patients (age: - years; males) with no serious complications al~er mitral or multiple valves replacements and with more than hrs on mechanical ventilation have been enrolled in this study. the modified flow interrupter technique (ptg "gould" with fleish head # ; differential pressure transducer pm- -tc "statham" w amplifier "kistler ") and flow-volume recording of forced expiration (fleish head # ) have been applied before surgery and following operation on mechanical ventilation (my), after extubation (t:xtijb), on ( nay) and ( day) days. airways specific conductance (sg aw) has been calculated as a mean of - consequent measurements in each patient at each stage. the sac was estimated by max expiratory flow at and % of vc on - f-v curves (mef .~ , mef ) all the data were statistically analyzed with t-test introduction : noninvasive ventilation (niv) reduces the need for endotracheal intubation, the length of stay in icu and the mortality rate in acute exacerbation of copd. however, some patients failed to be ventilated with niv. .objectives...; to further delineate patients who failed to be ventilated with niv and to obtain predicted factors of failure. patients : a cohort of patients ( • years) presenting with acute exacerbation of copd (fevi: • ml, paco : • , ph: . • . ) and nonmvasively ventilated (pressure support through a full-face mask) between april and may twenty-seven ( %) were successfully ventilated with niv (discharged alive without the need for endotracheal intubation) while ( %) failed, requiring endotracheal intubation. .methods : patients successfully ventilated and those who failed were compared according to respiratory and nonrespiratory variables univariate analysis (wilcoxon rank-sum test and fisher-exact test) was performed to select variables included in a multivariate analysis by stepwise logistic regression. results : underlying disease assessed by the simplified acute physiologic score ( • vs • , p = . ), creatinine serum concentration ( • vs • gm/l, p = . ), blood urea nitrogen (bun : • vs mm/l, p = . ), age ( • vs • , p = . ) were higher and encephalopathy ( vs %, p = . ) more frequent in patients who failed. multivariate analysis showed that encephalopathic patients (or (odd ratio) = , p = . ) older than years (or = , p = . ) and presenting with bun >_ mmyl (or = , p = . ) failed to be ventilated with niv. variables related to the respiratory" status (i.e. paco , pao , fev ) were unable to predict tile failure of niv. conclusion : copd patients older than years, presenting with acute exacerbation, encephalopathy and bun > ram/l, should be carefully monitored because of high probability of failure with niv. methods:from february to december we studied pa_ timnts, males and females(mean age +/- ); of the se had emphysema,lo chronic bronchitis, dilatative car diomyopatia,with tracheostomy and emphysema.mean pac at admission in icu was +/- mmhg,while when weaningbegan, +/- .mean autopeep was cmh ( - ).all patients were ventilated in crpv as long as four hours to calculate st tic and dynamic cmpliance and autopeep.then the ventila tion was continued with psv+cpap(peep cmh objectives: analysis of the incidence of neurogenic pulmonary edema (npe) in a population of headtrauma patients with acute respiratory failure (arf). npe can occur after a central nervous system insult. differential diagnosis: cardiogenic pulmonary edema and other forms of non eardiogenic pulmonary edema. true incidence and pathophysiohigy remain poorly defined, however the role of catecholamines seems undeniable. early onset npe (within h after trauma) is characterised by hypoxemia, transient pulmonary hypertension and bilateral central fluffy infiltrates on chestx-ray. characteristics of cardiogenic edema or pneumonia are absent. late onset npe, (beyond hours after trauma), is more insidious. the clinical and radiographic picture has to clear within to hours. ( ) methods: all headtrauma patients admitted from january to december , in a nearotrauma icu setting were retrospectively analyzed for arf with as sole criterinm a pao -fio ratio < . results: neurotrauma patients were admitted during . patients ( %) presented with severe head injury (gcs< ), patients ( . %) with moderate (gcs - ) and patients ( . %) with minor head injury (gcs - ). overall mortulity was . % early (within h. after trauma) and delayed onset respiratory incidents were distinguished, counting for ( . %), respectively patients ( . %), patients ( . %) had early and late respiratory complications. early respiratory insufficiency was caused in patients ( . %) by aspiration, in patients ( . %) by lung contusion, in patient ( . %) by fat embolism and in patients ( %) by npe. in the late onset group patients ( . %) presented with pneumonia, ( . %) with fat embolism and ( . %) with npe. the npe group, patients, presented as follows: patients ( . %) developed early npe, and ( . %) delayed onset npe. patients ( %) died within the first days after admission, showing high mortality. gcs was less than in patients ( . %), indicating severity of head injuries. conclusions: high incidence of arf with various etiology ( , ~ was found in this population. in about % of all admitted hcadtrauma patients ( , % of arf) npe was causing attetial hypoxemia. occurrence of npe seems to be related to the severity of the brain injury and thus to outcome. these data call for extreme vigilance in respect of the insidious occurrence of npe. were included if recovering from respiratory failure and if in the opinion of the primary physician were ready for extubation. patients were excluded if undergoing compassionate withdrawal of support or had tracheostomies. the attending physicians were blinded to the measurements. included patients were placed on pressure support (ps) of em h with demand-flow continuous positive airway pressure (cpap) cm h . after a minimum of minutes on the above sehiogs: gastric intramucosai pc'o , abg, and a p . were measured. the padents were then disconnected from the ventilator for a period of one minute and the patients" respiratory rate and minute ventilation were measured using a wrights respirometer to calculate the frequency to tidal volume ratio (f/vt). patients were then extubated. extubafion failure was defined as the inability to maintain spontaneous ventilation for hours for any reason. results: twenty patients met criteria and were studied over one month period in october . six of the twenty patients ( %) failed weaning. the mean and standard deviation is outlined in failure . +/- . . +/- . . +/- . . +/- . comparison between roc areas shows phi and p . to each show a statistically significant difference from an area of . (p<o. ). the area at which the test has no discriminative value. this is not the case for either the integrative index or the f/vt ratio. the differences between areas for each curve is not statistically significant. the area's for each roc curve is shown in table #' . results. of the newborns referred to us for ecmo-therapy developed barotrauma, of the required ecmo and one of the three other patients who did not recieve ecmo-therapy died. to determine the correlation between the risk factor barotrauma and the severity of the pulmonary situation, we determined the oxygenation index of each patient referred to us for ecmo-therapy . the table demonstrates , d- giessen, frg. inhalation of nitric oxide (no) and aerosulizatiun of prostaglandin (pg) i: have been suggested to achieve selective pulmonary vasodilation and improvement of arterial oxygenation in patients with acute respiratory distress syndrome (ards). we directly compared these two modes of transbronchial vasodilator therapy in ards patients mechanically ventilated for - h (mean murray lung injury score [ ] . + . ). patients were randomized to receive either first no and then pgi (n= ), or vice versa (n= ), with an intermediate baseline period. each drug was titrated individually to find the lowest effective dose for maximum improvement of arterial oxygenation. gas exchange variables, including data from the multiple inert gas elimination technique (miget), and hemodymmaics under application of no/pgi were compared to pro-and past-challenge values. no (mean dose . + . ppm) increased the mean oxygenation index (pao /fio ) from + to + mmi-ig (p < . ) and reduced the shunt-flow from . + . to . : . % (p < . ). aernselized pgi (mean dose . + . " ng/kg rain) augmented pao /fio from + to + mmhg (p < . ), and decreased shunt from . + . to . + . % (p < . ). the miget analysis showed predominant redistribution of blood-flow from shunt areas to regions with normal ventilation-perfusion ratios in response to both agents. in patients, both no and pgi caused an increase in pao /fio by at least rnmhg. two further patients displayed a corresponding improvement of arterial oxygenation in response to either no or pgiz. the mean pulmonary artery pressure decreased from . : . to . : . mmhg in response to no, and from . : . to . : . mmhg (p < . ) in response to pgi . cardiac output, systemic arterial pressure, and right and left heart filling pressures were not affected by either agent. we r that individually titrated doses of inhaled no and aerosolized pgi~ effect selective pulmonary vasodilation and redistribute blood-flow from shunt-areas to weu-ventilated regions with nearly identical efficacy profiles. obieetives: the use of extraeorporeal bypass systems for oxygenation and co -removal is performed in patients with acute respiratory distress syndrome (ards) to reduce mortality and to improve restitution of pulmonary functions. high-dose heparin -commonly used in such patients to prevent thrombotic complications -might cause incurable bleadings as a major risk. this lead to the development of heparinized bypass systems; results of animal and first clinical studies proposed that such systems might run with low-dose heparin or even without any systemic anticoagulation. methods: since , patients with severe ards were treated with extracorporeal lung assist (ela) using heparin-coated systems (type maxima, medtronic, carmeda coating) in our icu. they received a moderate systemic heparinization. standard clotting assays (act, aptt, tt, ptt, at-ill, fbg, fsp) as well as special tests (tat, r~-tg, pf , pal-l, d-dimers, protein c, cl-inhibitor) were performed. results: covalent heparin-eoating of the ela-systems combined with lowdose heparinization could not prevent major changes in coagulation: ) in mean, platelets dropped from /nl to /nl within h after onset of ela. ) tat levels were already elevated before ela start ( ug/]) and demonstrated an additional peak h after onset of ela (up to ug/[). ) in parallel, there was a transient peak of pai-i levels (from to au/ml} h after onset of ela. ) in mean, fibrinogen levels dropped from to mg/ ml within h after onset of ela. ) in mean, cl-inhibitor levels dropped significantly from % to % after onset of ela and reached the initial levels within h. ) after membrane change, there were significant changes for c -inhibitor, fibrin-d-dimers and tt. ) global clotting tests such as aptt, ptt and tt were within normal range. conclusions: patients with severe ards develop a prethrombotic state already before they get connected to the ela bypass system. after onset of ela, they experience additional involvement of procoagulant, anticoagulant, fibrinolytic, and complement systems. in parallel to laboratory findings, clinical data suggest that the use of heparin-coated systems with accompanying systemic low-dose heparinization does not avoid thrombembolic and hemorrhagic complications in tong-term ela patients. thus, at the present, higher dosed individually adjusted heparin treatment has to be recommended. thereby, standard clotting monitoring is not sufficient. objective: poor muscle performance contributes to prolonged dependence on mechanical ventilation. longitudinal data on muscle function in icu patients is scarce. we evaluated changes in inspiratory and peripheral muscle performance during prolonged intensive care in patients with acute respiratory failure. methods: patients requiring intensive care for more than one week were studied. maximum inspiratory pressure (pimax) was measured twice a week for a maximum of three weeks. handgrip power (dynamometer) and subjective fatigue (keldet score) were obtained in survivors only. one measurement was done on the date of extubation. pimax pressure was obtained by occluding the airway for seconds or at least five inspiratory breath attempts. obiectives -measurement of peep-induced changes in lung volume helps to assess respiratory mechanics in acute lung injury (ali). we evaluated the accuracy and stability of a new respiratory inductive plethysmograph (rip), in the measurement of peep induced changes in end-expiratory lung volume (eelv) and tidal volume (vt) against volume reference, pneumotachograph (pnt), methods -two hours periods of basal ventilation and stepwise increases and reductions of peep ( - - - - cm h ) were recorded in patients with all. in addition, the new rip device (respitrace plus tm) was compared to an older rip (respigraph tm, nims, fl, usa) to determine its thermal stability using cold and warm devices and a ventilated lung model. results -the difference between the new rip and pnt in the measurement of vt was - _+ mi (x _+ se) or a - . _+ . % error. increasing peep from to cm h induced a + ml change in eelv. a difference of + ml ( . % of max. eelv change) (ns) was found between rip and pnt eelv readings. during controlled ventilation with constant settings the mean change in eelv was _+ ml/ min (fig.i) . validation of calibration showed a mean error of -+ . % after rain. the new rip had a higher drift when cold (cold + ml/ min vs warm _+ ml/ min), whereas the old rip was thermally stable (cold - + ml/ rain vs warm i + ml/ min). trauma icu, hospital traumatologia y rehabilitacidn. vall d'rebron. barcelona. spain. recent data suggest that the proportion of patients with relevant discrepancies between two jugular bulb (jb) oxygen saturation (sjo ) values is higher than suspected. objectives: determine the incidence, the significance and the pro nosis value of those differences,if they exist,in severe head injured-patients. material and methods: severe head-injured patients, coma glasgow scale (gcs) , with diffuse brain injury according to marshall criteria in the first ct scan, icp recorded, with an interval from accident-double jb monitoring < than hours, were studied. data from bilateral sjo were obtained simultaneously, every hours. discrepancies were considered significant, when differences in sjo were > %. no chan es in treatment protocol (hyperventilation, man• etc) were carried out due to this study. results: men and women were studied, aged • yrs. at arrival at hospital, gcs were < in and ) in to. the incidence of high icp() mmhg) were sz at the entry. the mean therapy index level required to control lop was ~l all patients required vasopressor therapy to maintain upp over ds mmhg. in patients a s.s f swan-ganz fiberoptic catheter was used to obtain a continuous recording of sjo . in the others , sj were intermittently controhed.the mean time of monitoring were d. • days. ten patients died within this period. a total of . blood samples were analized. at arrival, sjo discrepancies were found in patients, b %. at hours, the incidence were lower, / , . %. at th day, were h/ , z and at day , when the catheters were retired, ii[ , z showed discrepancies. the ct showed new injuries in g z of patients with differences > ~ in sd values throughout treatment period. none of those were considered for neurosurgical treatment. no correlation was found between iop and sjo values and sjo differences. conclusions: the incidence of discrepancies between sjo was higher than expected in severe head-injured patients. these situation could reflect disturbances between demands. when differences are known, and those lend to change, the ct scan, nearly always, will show new injuries. platelet-activating factor (paf) is an inflamatory mediator implicated in the pathogenesis of bronchial asthma and acute respiratory distress syndrome (ards). its inhalation in healthy subjects produces transient bronchoconstriction and mild ventilation-perfusion mismatch, together with peripheral leukopenia as a result of intrapulmonary neutrophil (pmn) sequestration. likewise our group has shown in healthy subjects and asthmatic patients that aaibutamol (s) inhibits both pulmonary and systemic effects of paf, suggesting that s may inhibit paf-induced venoconstriction in pulmonary microoirculation. the aim of the present study was to investigate if s inhalation decreases pmn by lung sequestration induced by paf. we studied healthy, non-atop• nonsmoking subjects ( m/ f, + yr), which were pre-treated with s ( ,ug) or placebo, with a randomized, double-blind, crossover, design, before paf ( ,ug) inhalation. we measured the respiratory system resistance (rrs) by forced oscillation, arterial btood gases and both total white cell and pmn count every min over a min. period. simultaneously, we recorded continuously the lung dynamics of inm-neutrophil and tc m-erythrocytes activity, with a gammacamara. after placebo, paf inhalation decreased white cells (from to x /l), and pmn(from to _+ x /l), and increased aapo (from . _+ . to . + . mmhg, p<o. ) and rrs(from . . to . . cmh .s.i q ) (p < . each). the fall in total white cell count correlated with the intrapulmonary pmn retention (r = . , p < . ). by contrast, after s, paf did not induce any change in white ceil count (from to + x /i), pmn count (from to x /i), aapo (from . to . + . mmhg), and rrs (from . . to . . cmh .s.i ). compared with placebo, after s there was a lower ~lln-pmn lung activity ( . . vs . . cts/mci/pixel, p<o. ), lower intrapulmonary pmn retention ( . . vs . . %, p=o, ), and a faster washout ( . . vs . . s ~, p= , ). our results indicate that s inhibits paf-induced intrapulmonary pmn sequestration, being consistent with the hypothesis that s may offset the venoconstriction of pulmonary microcirculadon caused by this mediator. supported inpiratory muscle fatigue with failure of force generation as def'med by a tensiontime index (tti)> . - . has been shown to occur in normal volunteers and in stable copd patients with a specific imposed breathing pattern. its role, however, in hypercapnic respiratory failure is less certain. we studied failed weaning trials in copd patients in which breathing pattern, tension-time index (tti) of inspimtory muscles, dynamic peepi, dynamic lung elastance, lung resistance, and arterial paco and ph were measured at the beginning and end of a t-piece weaning trial. in addition, the change in esophageal pressure during a mueller maneuver (apes max) was measured. a weaning trail has been prospectively defined to have failed if one of the following criteria was met: a rise in pco > mmhg from baseline accompanied by a fall in ph< . ; a respiratory frequency (f) > /min; excessive accessory inspiratory muscle recruitment; and a marked increase in dyspnea. values are expressed as mean • se. weaning failure was characterized by a more rapid, shallow breathing pattern, worsened mechanics, hypercapnia and respiratory acidemia despite an unchanged tri and pes max. we conclude that in this setting hypercapnic respiratory failure is not a consequence of inspiratory muscle fatigue. rather the adopted breathing strategy and resultant hypercapnia may represent an adaptation to forestall the onset of muscle fatigue. concerning the investigated elf-par~eters, no stadstically signhqcant differences were detected between the pgi and the control group. histopathologlcal changes occured in both groups and consisted in rare focal flaaaning f tracheal epithelium with loss of cilia and slight inflammatory cell infiltration, as well as slight swelling of alveolar typo pneumoeytes. sections of generation , and from bronchial tree were free of pathological changes. conclusion: alter h inhalation of p~ji no signs of respiratory-lract tissue damage caused by the aerosol could be detected. the minor pathological findings in the trachea are most likely due to mechanical irritation by bronchoscopy, changes of the alveolar epithelium are known for long-term mechanical ventilation . objectives: the aim of this study was to evaluate of efficiacy of ganglion stetlate blockade in patients with respiratory failure. methods: two groups of patients were investigated: group i (n = ) trauma patients with acute lung injury (ali), group if (n = ) patients with asthmatic status. in all cases continuous mandatory ventilation (cmv) was used with bennett ae. in both groups bilateral ganglion stellate blockade with antero-lateral approach was performed, using . % marcain. the following parameters were analysed: pao , sao , paco~, pip and c~t~t. results: in trauma patients with aij after bilateral ganglion stellate blockade short -lived and slight improvement of pao and sao , decrease of pacoz and pir and increase of static compliance of respiratory system were found. in second group bilateral ganglion stellate blockade interrupted the asthmatic status and significant statistical improvement of parameters of oxygenation, ventilation and respiratory system mechanics were observed. conclusions: we suggest that the bilateral ganglion stellate blockade is a very useful method in treatment of patients with obstructive respiratory insufficiency. the aim of the study was to analyse whether there exists serum and urine electrolyte disorder in patients(pts.) with acute respiratory insufficiency(ari). the study included t pts. with ari (pao : , @ , kpa. paco : , i- , kpa, ph: ~: , , hco : , :~ , mmol/ , sao : , ~- , %) who were hospitally treated due to pneumonia( pts.),emboly of the pulmonary artery( pts.) and severe attack of bronchial asthma ( pts). among tham there were ( , %) males and ( , %) females, average age , ~: , years, otherwise previously healthy. electrolyte concentracions were measured at the onset of the disease in serum and urine collected during hours (sodium-na,potassium-k, chlorine-c , calcium-ca,magnesium-mgand phosphorus-p). the measured serum and urine electrolyte concentrations were compared with respective referent values (rv). by serum electrolyte analysis, the following average velues were obtained: na:l o, the object of our investigation was a group of pts with massive pneumonias, males ( . %), females ( . %),mean age yrs.thirteen ( %) of them were smokers, ( %) nonsmokers. only pt ( . %) had pre-existing chronic respiratory disease, and ( . %) were admitted for the first lime,with no previous respiratory anamnesis. diagnose was based on anamnestic data of productive cough in pts( . %),physicaly ~onchial breathing in i~s ( . %),white cell count onder x /l in pts( . %). radiographicly, bilateral massive homogeneous shadows were found in pts ( . %), onilateral in pts( . %),pleural effusion in pts ( . %). abnormal renal function was found in pts ( . %). sputum culture was positive in pts ( %): slr.pneumoniae, str.pyogenes, pse'udomonas aerug, in , , cases respectively. all patients had remarcable hypoxernia (pao range from , to , kpa) without hypercalmea. all patients needed oxygenotherapy together with antibiotics and other .symptomatic therapy. nineteen pts had anaelioration of general condition and normalization of blood gas analyses, while pts with the lowest hypoxcmia died.in conclusion, massive pneumonias are frequently followed by respiratory insufficiency which is one of the markers of pneumonia severity. as existing hypoxemia complicates the course of the disease,prolonges the recovery, makes therapy more complexe and may be cause of death , frequent blood gas measurement is recomanded. we studied the effects of bosentan (bos), an eta and etb receptor antagonist, to examine if endogenous et mediates pulmonary hypertension in anesthetized and ventilated dogs with acute lung injury due to oleic acid (oa). the gradient between pulmonary artery pressure (ppa) and occluded ppa (ppao), and gas exchange (evaluated by arterial blood gases and sf intrapulmonary shunt) were measured at controlled flow. in dogs (treatment), data were collected at baseline, during long injury (obtained rain after intravenous administration of oa . ml/kg), and again after bos ( mg/kg intravenously). in dogs (pretreatment), data were obtained at baseline, after bos and then after oa. in treated dogs, oa increased (ppa-ppao, mmhg, table, means + sem, * p < . vs base) and deteriorated gas exchange. after oa, bos did not affect pulmonary vascular tone nor gas exchange. in pretreated dogs, bos had no effect on baseline pulmonary vascular tone but prevented the increase in (ppa-ppao) after oa. the deterioration in gas exchange after oa was not influenced by bos pretreatment. objectives: the alveolar tension is measured by the application of the alveolar air equation in which the arterial pco is used or by the simplified form of this equation in which the respiratory exchange ratio is taken at the value of . . the purpose of this study was to estimate the effective alveolar tension (pao eff) during spontaneous breathing with a new bedside technique which is simple non-invasive in normal subjects and patients with chronic bronchitis-emphysema. we also compared these values with the ideal alveolar po (pao (i)), measured from the alveolar air equation in which paco was substituted by the effective alveolar pco (paco eff) and with the alveolar po measured from the simplified alveolar air equation (pa ). this study is complemantary to previous work for the estimation of paco eff. methods: the subjects breathed quietly through the equipment assembly (mouthpiece monitoring ring, fleisch transducer head) connected to a pneumotachograph and a fast response and co analyzer. the method is a computerised calculation of the effective alveolar po quite similar to that of paco eff, obtained from the simultaneously recorded at the mouth expiratory flow, and co concentration versus time curves. results: the results showed a mean difference (pao eff-pa (i)) of - . kpa in normal subjects and - , in patients. the mean of the difference (pao eff-paq ) and (pad (i]-pao z) was much greater than . in all subjects. the limits of agreement for the difference (paozeff-pa (i))were - . to . kpa in normal subjects and - . to . in patients, while those for the differences (pao eff-pad ) and (pao (i)-pad ) were very large ( > - . to > . ) in all subjects. conclusions: the effective alveolar po is very close to the ideal one in normal subjects, tn patients pao eff may excessively deviate from pa (i) due to the observed significant difference between the alveolar/tidal volume ratio for o and that for co . the alveolar po measured from the simplified alveolar air equation (pao ) differed substantially from pao eff and pad (i) in all subjects. the essential role of glucoprotein hormone erythropoietin is to control red cell production. hypoxemia, reduced blood -carrying capacity and increased affinity of hemoglobin for are the primary stimuli for erythropoietin production. both anemia and hypoxemia induce rapidly erythropoietin secretion. kidney erythropoietin rna levels correlate inversely with hematocrit and directly with plasma erythropoietin level. similarly, hypoxemia increases kidney erythropoietin rna and plasma erythropoietin. the effect of hyperoxemia (pa >lo mmhg) on erythropoietin secretion isn't very well understood. the purpose of this study was first to evaluate the erythropoietin secretion in patients with acute respiratory failure and second to determine the effect of hyperoxemia on erythropoietin secretion in patients with and without anemia. sixteen patients with acute or acute on chronic respiratory failure needed mechanical ventilation were included in this study. these patient were divided in two groups. the patient who developed anemia were included in group i and the patients without anemia in group i . erythropoietin was estimated in venous blood in three stages. the first sample was taken during hypoxemia, the second during hyperoxemia and third during normoxemia. all the patients had high erythropoietin level during the hypoxemia period (mean value • mu/ml). during hyperoxemia etythropoietin levels were reduced in both groups ( mean value . + . mu/ml in group i, . • mu/ml in group ii). in normoxemia stage, erythropoietin increased again in anemic patients, and decreased more in the patients of group i . we conclude that hyperroxemia inhibit erythropoietin secretion in spite of anemia and tow arterial oxygen content. hyperoxemia may be a factor of the insisted anemia in with oxygen treated icu patients. the purpose of this study was to determine the relationship between clinical features of acute lung injury (all) and parameters like total proteins, total and individual phospholipids, the presence of paf, and acetylhydrolase activity in bal of mechanically ventillated patients. acetylhydrolase catalyses the cleavage of acetyl-group from the second position of the glycerylether backbone of paf, leading to its inactivation. mechanically ventillated patients were divided to three groups. group i includes patients without all; group ii, comprisespatients with moderate degree all, ( . <g-i < . ); and group iii comprises patients with severe ards (all score > . ). broncoalveolar lavage (bal) was obtained after infusion of normal saline at ~ to intubated patients and cooled immediately. cells were removed after mild centrifugation ( x g, min, oc). aliquots from the supernatant were used for total protein, phospholipid and paf analysis and determination. acetylhydrolase activity was assessed after incubation of bal with h-paf labelled on the acetyl group. released label was measured by liquid scintillation counter in the supernatant after trichloroacetic acid precipitation of the non-reacted substrate. kinetic characteristics of the enzymes were also studied. total phospholipids appear reduced in bal of patients with all, while total proteins increase. these factors appear to correlate with the severity of all. paf was not present in bal samples pretreatad with equal volume of % acetic acid to denaturate acetylhydrolase. detection limit for paf under our experimental conditions: pg paf/ml bal. instead, acetylhydrolase activity was detected in amounts increasing with the total protein content. background: intubated patients without lung injury or impaired breathing control normally display an inspiratory peak flow of below l/s. the aim of our study was to investigate the inspiratory peak flow generated by patients with acute respiratory insufficiency (ari). we had to take into account that both an inspiratory pressure support (ips) and the resistance of the endotracheal tube considerably influence the flow pattern generated by the patient. patients and methods: to investigate the non-influenced flow pattern we developed a new ventilatory mode which automatically compensates for the flow-dependent resistance of the endotracheal tube (automatic tube compensation, atc). furthermore, the mode maintains a constant tracheal pressure in inspiration and expiratio n . consequently, the measured flow pattern exactly corresponds to the flow pattern generated by the patient except that the ventilator modified for this mode (evita, driiger liibeck, germany) was not able to deliver a gas flow of more than l]s. we have investigated patients with ari arising from different reasons. results: the inspiratory peak flow measured in the atc-mode was . l/s _+ . l/s. the maximal deliverable flow of l/s was obtained in of patients. the figure shows the flow pattern under atc and ips in [~s] oi:) one of these patients. conclusions: patients with ari display a highly increased inspiratory peak flow. ventilators used for spontaneous breathing should therefore be able to deliver a gas flow of more than l/s. an overproduction of no and reactive oxygen species (ros) has been demonstratred in septic shock. ros and nitric oxide (.no) are free radicals which are known to react together leading to peroxynitrite anions that can decompose to form nitrogen dioxide (no ) and hydroxyl radical (oh~ thus, no has been reported to have a dual effect on lipid peroxidation (prooxydant via the peroxinitrite or antioxidant via the chelation of ros). in the present study we have investigated in different models the in vitro and in vivo action of no on lipid peroxidation. copper-induced ldl oxidation was used as an in vitro model of lipid peroxidation. ldl ( ~g apob/ml) was incubated with cu + ( , ~tm) in presence or absence of no donor (sodium nitroprussiate or glutathione-no) from to ~m. oxidation of ldl was monitored continuously with conjugated diene formation ( nm) and hydroxy nonenal accumulation (hne). exogenous no prevents in a dose dependent maner the progress of copperinduced oxidation. ischaemia-reperfusion injury (i/r), characterized by an overproduction of ros, is used as an in vivo model. anaesthetized rats were submitted to hour renal isehaemia following by hours of reperfusion. sham operated rats (sop) were used as control. lipid peroxidation was evaluated by measuring the hne accumulated in rat kidneys in presence or absence of l-arginine or d-arginine infusion. l-arginine, but not darginine, enhances hne accumulation in i/r but not in sop (< . nmol/g tissue in sop versus . nmol/g tissue in i/r), showing that in this experimental conditions, no produced from l-arginine, enhances the toxicity of ros. this study shows that the pro-or antioxydant effects of no are different in vivo and in vitro and could be driven by environemental conditions such as ph, relative concentration of no and ros, ferryl species...these conditions are impaired in circulatory shock. methods:" the diagnostic and therapeutic approach was standardized so that data collected over a -year period were comparable. a progressive deterioration of clinical conditions and/or pulmonary gas exchanges was considered as indication for my. variables potentially predicting the need for hv were derived from clinical and arterial gas data, extrapulmonary diseases, use of drugs, chest x-ray and ecg abnormalities. results: rv, performed with external and/or internal ventilators, was necessary in patients ( %). at the hospital admission, pac was higher and ph was lower in patients requiring rv ( pneumomediastinum, pneumothorax, ateleetasis and myocardial infarction are rarely seen in bronchial asthma. these complications occur as a result of the severe asthma.the aim of our retrospective study was to analyse the complications seen in acute asthma attacks. during the years through , patients were admitted to hospital in acute asthma episode. there were ( , %) pts with complications; mean age of yrs; females ( %). clinical history, ecg and chest radiogr~hs were analysed. the mean duration of bronchial asthma was yrs (range from months to yrs), all patients were atopics. there were four ex-smokem and one smoker. the worsening of asthma symptoms begun two days before the admission (range from to days). on ecg all patients had tschycardia. rightward shift of the qrs axis and st-t changes indicative of right ventrieutur strain were found in three pts. these were the transient fmdings that improved after curing the acute asthma attack. non-q myocardial infarction oeeured in one patlent and resulted from the hypoxaemia of asthma. hyperinfl~ion was the usual finding on the chest radiograpk pneumomediastinum and subcutaneous emphysema were apparent in five pts and required no additional treatment unilateral pneumothoraccs were present in two pts and needed eontimous intrapleural drainage; one of these patienst died in eardiorespiratory insufficiency. ateleetasis of right upper lobe was present in one patient. it oceured due to inspissated secretions and needed no additional treatment all these patients, except one who died, improved on lreaanent with oxygcr~ steroids, beta-two agonists, theophylline and antibiotics. in conclusion, complications occur in acute asthma episodes as a result of the severe asthma mediastir,*l emphysema and atelectasis are not serious complications. pneumothorax and myocardial infarction are very serious life-treatening complications and always have to i:m considered in taati~ts with sev~ asthma. acute bronchial asthmatic episodes represent one of the most common respiratory mnergendes, its maximmum expression "status asthmatiens" is one entity of low incidence, still it is a risk to the physical integrity of the patient. during a total of patients with diagnosis of status asthmabcas were hospitalized. out of these palients six had a near-fatsl asthma and they were subjected to a complex examination. near-fatal asthma was defined as either respiratory arrest or acute asttuua with paco greater than , kpa and/or an altered state of consciousness. mean age was , -d: , yrs, four male and two female sex. at presentation two patients suffered from coma, others were confused. they exh'bited severe dystmoes, diffieul~ speaking, used accessory muscles of respiration, increased whee~tg while two cases had silent chest on auscultation. cyanosis indicated a very severe asthma attack in all six patients. mean respiratory rate was ~ /min and puts rate .d: bts/imn. arterial blood gases revealed a pao of , ~ , kpa, paco of , • kpa and ph of , -+- , . area-careful evaluation they received conventional therapy (immediately continuous oxygen, impelled nebulization with high doses of betatwo agonists and ipmtropium bromide, intmvanous st~oids and theophylline). in two eases signs and symptoms of deteriorating airflow and respiratory muscle fatigue determined the need for mechanical ventilation. out of six near-fatal attacks aggressive lrealanent was suscessfull in four patients and fatal in two eases. one patient admittcxl in coma died in severe hypoxae~a upon one hour and one mechanicaly ventilated died from cardiac arrhythmia. life-threatening attacks in asthmatics in our group developed gradual worsening despite neatment which r symptoms in most other patients. one patient had "brittle asthma", other long-standing acute episodes ireated with systemic steroids. conclusions: idantitiechon of fatality prone subjects may lead to fttrther muetion of seveze episodes. respiratory affest and coma upon admission, severe dyspnoca with silent chest on ausouhation, oyanusis and use of accessory muscles of respiration constitute the basic cfinieal picture. hypoxasmia must be immediately eon'ected.the patients and physicians should be able to assess the severity of asthma, a major factor in near-fatal and fatal asthma attacks. objectives :our purpose was to asses if the evolution of patients with a adult respiratory distress syndrome (ards) ,shows any relation to the pulmonary or systemic origin of the disease and whether or not there were differences in the frequency of the syndrome in both groups. methods : randomized prospective study in multidisciplinary icu. one hundred and sixteen patients with a high risk developing ards were distributed into two groups. one was named systemic origin group(so) and the other pulmonary origth group (po).ai patients only showed one cause (pulmonary or systemic) with potential risk of ards.the patient's hemodynamic and respiratory status was evaluated every hours the first day and every hours the second and third day. at the end of hours the patients were diagnosed as ards or non-ards. measurements and main results : of the total patients, were finally included in the so group and in the po group.patients in so group and po group had comparable ages (p<. ).peep in both groups was comparable (=. ) at the mmnent of admission to the study. there were no statistically significant differences for cardiac index and systemic vascular resistances. the pulmonary vascular resistances (pvr) showed significant differences at h.(p<. ) and h. (p<. ).the oxygen comsumption (vo) in patients of the so group showed statistically significant differences at h. (p<. ) with respect to initial values.fifteen cases of ards ( . %) in the so group and twenty five cases ( . %) in the po group were identified. the time of onset of ards was _+ hours in the so group and + b hours in the po group.the final outcome was very similar th both groups : mortality of % in the so group versus % in the pc group. conclusions : the pathogenesis of ards depends on whether the lesion is originated at or outside the lung. the po group showed a sborter thne of onset of ards, a faster and more severe increase of pulmonary shunt and a higher percentage of patients developing ards compared with patients of the so group.the so group showed a higher and faster increase in puhnonary resitances tbat po group and a decrease th oxygen comsumption earlier and more severe than in the po group. these data thus seem to show that there could be two mechanisms involved in the genesis of ards depending on the cause. the fact that the ards genesis is shorter in the cases of pulmonary etiology with faster impairment of pulmonary shunt, and a slower increase in pulmonary resistances in this pulmonary group, would indicate that the underlying mechanisms responsible for the hypoxemia are different to those which thitiate the increase in pulmonary resistances. finally, the exclusive inapairinent of oxygen consumption, which appears earlier than the onset of ards in the systemic origth group, could show the generalized character of the process in this group. perfusion of prostacyclin (pgi ) to treat pulmonary hypertension in adult respiratory distress syndrome (ards) worse pulmonary gas exchange due to a marked impairement of ventilation/perfusion mismatch. recently has been shown that if prostacyclin is given by aerosol instead of intravenous the net effect is an improvement of arterial oxigenation due to a redistribution of blood flow to well ventilated areas. objectives: to asses the effects of inhaled proatacyclin on pulmonary haemodynamics and gas exchange in patients with severe ards. methods : two patients with severe ards (murray score > ) recived inhaled pgi at - ng.kg.min " using an ultrasonic nebulizer. haemodynamic measurements, arterial and mixed venous blood gas analysis were performed before and after rain of pgi inhalation. results: short-terro p~i inhalation improved pulmonary g-~ e-'~hange in both patients. arterial oxygen partial pressure (pao ) increased from to mmhg in patient and from to in patient , the ratio pao to the fraction of inspired oxygen increased from to (patient ) and from to (patient ). venous admixture decreased from % to % and from % to % in patient and respectively. mean pulmonary artery pressure decreased slightly from to mmhg in patient and from to mmhg in patient . no effects on systemic haemodynamics were observed in any patient. conclusions: pgi inhalation improves gas exchange and produces selective pulmonary vaaodilation, thus can be an alternative therapy for the treatment of pulmonary hypertension and hypexemia in patients with severe respiratory falllure. methods: we treated ards-patients (age yr ( - ) mean, range) during - . the lowest pao /fio -ratio was ( - ), the worst murray score . ( . - . ), icu-stay ( - ) days and hospital mortality %. the costs of intensive care were calculated according to intensivity of patient care as assessed by tiss-scoring (therapeutic intervention scoring system). the more intensive the care, the higher are the costs. costs per year of life saved (=life-year" in us $) were compaired by other medical treatments ( - ). it is assumed that the mean expected length of remaining life in ards-survivors after intensive care is years. treatment life-year ($) ' bone marrow transplantation (acute leukemia) lowering cholesterol using iovastatin treating hypertension using nifedipine heart transplantation intensive care of ards-patients conclusions: intensive care of patients with severe ards is highly more cost-effective as compared with many other routinely used medical treatment strategies, the usually good recovery and the reasonable quality of life in survivors justifies investments to care of these patients ( ). there is a close correlation between these two methods of measuring evlw. however there is an underestimation of . % in this kind of pulmonary edema ( oleie acid induced ) with the double dilution method. although the size of the sample is small, in normal lungs there appear not to be this underestimation. the effect of peep on evlw has been studied with contradictory results, probably as a consequence oft differences in methods of measuring evlw, variations in the type and severity of lung injury, and different timings of peep application. objective= ) to analyse the effect of different levels of peep ( , and omh ) on evlw during hpe; ) to establish whether increases in intrathoracic pressure due to high peep levels can obstruct lymphatic drainage. material and methodet hpe was provoked in groups of dogs by inflating a foley catheter in left auricular to a pressure of - r~uhg. peep levels of , i or m~hg were applied. resultst objective: to assess the effect on extravascular lung water (evlw) of the application of peep and the reduction of vt in an oleic acid pulmonary edema model in pigs, using three ventila~ary strategies. material and methods: twelve adolescent pigs (weighing over kg) were randomly divided in three gmups immediately alter infusing via a central vein . ml/kg of oleic acid to produce a permeability pulmonary edema. the ventilatory parameters for each group were as follows: group i (n= ) : vt: - ml/kg; zeep. group :(n= ) : vt: - ml/kg; peep: cm h . group :(n= ) : vt: - ml/kg; peep: emil . (resulting in permissive hypereapnla) after a four-hour period of ventilation the animals were killed and the lungs excised to calculate gravimetrically the extravascular lung water using a standardized procedure ( hemoglobin content method ). ill evlw (ml/kg) group obiective: in the postoperative period, maintenance of adeguate arterial oxygen tension is a major problem in morbidly obese patients probably because of a large reduction in functional residual capacity (frc). the aim of this study was to evaluate the effects of peep on respiratory mechamcs and gas exchange in this kind of patients. methods: in nine postoperative mechanically ventilated morbidly obese patients (bmi> kg/m ) we partitioned the total respiratory system mechanics into its lung ( ) and chest wall (w) components using the airway occlusion technique associated with the esophageal balloon, during constant flow inflation (jap ; : ) . at three different levels of peep ( , , cmh ) we measured: compliance (cst), airway (rim) and "additional" (dr) resistance, frc and gas exchange. obiectives. to describe the use of prone position in our icu we analyzed the clinical records of all patients admitted in - , selecting adult patients with arf defined as: intubation and pao /fio < mmhg plus an fio > . or peep> cm i . results. patients met the arf criteria: of them ( . %) underwent prone positioning (p+). prone position use began in the early phase of arf ( . • days from the beginning, range - , median ). out of p+ pts were treated with controlled ventilation (cppv or pcv), while were on assisted ventilation (simv+ps) and on spontaneous breathing (cpap). only pts were awake when turned prone, while pts required adjuncts of sedation to tolerate the change of position. the duration of prone positioning was variable (average lenght . • h, range . - h). only minor side effects were observed (eyelids and facial edema, chest and facial pressure bruises). we consider responders (r+) those patients presenting at least . mmhg increase in pao /fio : / patients ( . %.) were responders when first pruned. the pao /fio changes induced by prone position are reported in the figure. pao /fio increased when patients were pruned (*p< . ) and remained higher than baseline values when returning supine(*p< . ). paco remained unchanged. prone positioning was used at least twice in / ( conclusions. this retrospective analysis confirms that prone positioning improves oxtgenation in the majorib' of arf patients. altough we have no available criteria to discriminate in advance r+ from r-pts, we now routinely consider the use of prone position in the treatment of severe arf. palo a, otivei m*, galbusera c, veronesi r, sala gallini g, zanierato m, iotti g, braschi a.servizio anest. e rianim. i, *laboratorio biotecnologie e tecnologie biomediche irccs s. matteo, pavia, italy inhaled no can improve arterial oxygenation and reduce pulmonary hypertension in ards patients; little information is, however, available about the dose-response curves. methods seven ards patients (lis . +. ) submitted to mechanical ventilation randomly received inhaled no doses in increasing or decreasing sequence: . , , , , , and ppm. reference measurements were obtained before and after the entire period of no inhalation. hemodynamic parameters and blood gases were measured after min in each condition. cmv was administered under sedation and paralysis, with constant ventilation, peep (lol-_ cmh ) and fit (. +. ). the changes in vt and fit due to the no ( ppm in n ) injection in the ventilator external circuit were compensated for. results . the dose of . ppm, ineffective on papm, significantly improved oxygenation. the increase of pat and the decrease of q'va/q' and papm were nearly maximal at - ppm. no deterioration of arterial oxygenation was observed at no doses as high as ppm. co exchange was not influenced by no inhalation. systemic hemodynamic variables did not change throughout the study. these results suggest that a concentration around ppm is adequate for obtaining maximum effects on hypoxemia and pulmonary hypertension in patients with ards. low-dose inhaled nitric oxide (no) induces redistribution of pulmonary perfusion in patients with severe ards and causes improvement of oxygenation [ ] . however, addition of exogenous lowdose no in the inspiratory gas mixture might be only a replacement of missing atmospheric no ( - ppb) in hospital central-supplied medical air. [ ] we have realised nitric oxide measurements in ten healthy volunteers, ( smokers and non-smokers) breathing with a mouthpiece and occluded nostrils through a ventilator circuit, with separation of inhaled and exhaled gases by a valve. no concentration was measured with a double-chamber chemiluminometer (environnement sa, france) and with charcoal/silicate purified compressed air. there was no nitric oxide detectable in the inspirat ry limb of the ventilator. unfiltered central supply medical air contained : - ppb of no and - ppb of no , whereas central supplied oxygen was no/no free. samples were taken after equilibration periods of minutes, with increasing fit levels of . , . and . for subsequent minutes periods; paired values were recorded every s. the mean no value was . ppb (sd . ) and n o significant differences were found for different fit levels both in smokers and non-smokers. these data suggest that the no concentration of pulmonary origin in the exhaled air of' healthy volunteers is probably lower than that reported by other authors [ ] and that, previously reported, differences between smokers and non-smokers are not always striking [ ] . we suggest the use of activated charcoal/silicate filters for clinical trials in order to achieve standard conditions. [ objective: to compare efficacy and safety of two doses of salbutamol. methods: sixteen adults who had severe acute a~hma were randomly assigned to receive either rag (n= ) or rag (n= ) of nebulized sulbutamol. both groups were similar with respect to age, duration of a~hma, duration of attack before arrival at the hospital and severity of a~hma according to baseline measurements (table) . evaluation was performed , , and rain after the start of nebulization. results: compared with mg regimen, mg regimen resulted in the same improvement in peak-flow and fischl index (figure). the changes in heart rate, respiratory rate and pace did not differ significantly between both groups. the incidence of side effects, which included tremor, palpitations, cardiac arrythmlas and other symptoms, was not sj~ificanfly different in the two populations. conclusion:the results of this study suggest that nebulization of ng of salbutamol is not more effective than rag in the initial treatment of acute severe asthma in adult patients. the prognostic factors of neutropenic patients admitted to the icu remain poorly known. the aim of this study was to determine the respective weight of underlying malignancy and organ system failures on the outcome of these patients. patients and methods: the charts of neutropenic patients (wbc < /mm and/or pmn < /ram ), admitted to the icu between and , were retrospectively reviewed. the characteristics of the neoplastic disease (h~emopathy or solid tumor, tumoral evolution, duration of cancer disease and of neutropenia), the mac cabe's score, the organ system (respiratory, hemodynamic, renal, neurologic, hepatic) failures and the severity scores (saps, saps ii ,osf) were registred within the st day in the icu. when discharged from the icu, the patients were classified as alive or dead. results: fifty-seven patients ( . %) had a h~ematologic malignancy, and ( . %) a solid tumor. fifty-nine of the patients died ( . %); the mortality rate did not differ between both groups ( . and % respectively, p = . ). with univariate analysis, none of the tumoral features is linked to the prognosis; only the respiratory (p < - ) and cardiovascular (p < - ) failures, and the number of organ system failures (p < - ) are associated to the risk of death. the saps (p < - ) and saps ii scores (p < - ) were higher in patients who died. with multivariate analysis (logistic regression), only the respiratory failure is correlated to the risk of death (p = - ); neither the features of the underlying malignancy (p > . ), nor the duration of neutropenia before admission in icu (p = . ), nor the severity scores figs ii: p = . ) are linked to the outcome. conclusions: the tumoral characteristics do not modify the prognosis after admission to the icu. they should not influence the decision to admit or refuse a cancer patient in the icu. respiratory failure at icu admission has the predominent weight on the risk of death in the icu. patients with respiratory acidosis due to asthma occasionally require levels of mechanical ventilation that place them at risk for barotrauma. a few case reports have described the use of an extra-corporeal membrane oxygenator(ecmo) circuit as an alternative means of co removal. generally, this has been used for short periods of time (< h) without serious complications and with low blood flows through the extra-corporeal circuit. we report a case of refractory asthma who could not tolerate even small-volume breaths from a mechanical ventilator due to severe bilateral airleak. ecmo therapy was initiated at the referring hospital prior to helicoptor transport. high blood flows were used ( % of the patient's cardiac output), sufficient to achieve both co removal and oxygenation. satisfactory gas-exchanged was accomplished (pco = - mmhg) with nearly total lung rest for a prolonged period ( h). however, the long ecmo duration was associated with two severe complica-ti ns: ) bilateral hemothoraces due to anticoagu!ation in the extra-corporeal circuit, and ) prolonged weakness as a result of neuromuscular blockade for six days. the patient was discharged from the hospital in good condition. we present the respiratory and hemodynamic features of this case aw well as the potential complications of ecmo therapy in asthma. objectives: parameters derived from tidal expiratory flow ~e) and volume (vt) can be used to detect airflow obstruction in copd patients who might be unable to perform forced spirometry (e.g., icu). however, indices such as ave/v t and at/re are highly variable (thorax, : ; ) . methods: we investigated whether the standardized for v m effective time (teff~) of a tidal breath, which is derived by asimple mathematical procedure (teff,= j'vdt/vt ), is a more reproducible and sensitive detector of airways obstruction, we studied nine normal subjects ( male, -+ yr) and copd patients ( male, -+ yr) in the seated position, with a noseclip on. they breathed quietly, through a pneumotashograph to measure flow (v). volume was obtained by numerical integration of thellow signal. each subject had an initial - min trial run, in order to become accustomed to the apparatus and procedure. when regular breathing had been achieved, all breaths over a min time interval were recorded. the mean value of six consecutive breaths (ers criteria) for each subject was used for analysis under the condition that within session variation of tidal volume (vt) was < %. lung function tests were: in normals (mean-sd), fevl%pred = • fevl/fvc%= -+ % , and in copd patients, fev~%pred= __. and fevi/fvc%= --. %. results: values are shown as mean-..+-sd in the following a su~ve~ os literature sources p~oves that t~aditlona], i.e. medicinal medication and physiothe~apeutic methods os t~eatment often p~ove to be insufficientl~ effective both currently and in the ~emote future. the goal of this study was to investigate the efficacy os t~eatment of b~onchial asti~ma patients by means os speleo-and artificial sp~ay therapy. speleotherapy t~eatment was conducted in the conditions os mic~oclimate os salt mine in solotvino hospital. a~tis sp~ay the-~apy was conducted by means os a self-made device. ou~ method is based on the p~inci-~ le os using the majo~ facto~ of speleo-he~apy -highly dispe~sed sp~ay s sodium chloride. the obtained ~esults ~e~e analyzed in five g~adations. at the end os the speleothe~apy improvement and considerable improvement was observed in , ~ os patients; inconsiderable improvement -in , ~ os patients. having evaluated the e~s os t~eatment using a~tis sp~ay therapy the indices a~e , h and , ~ ~espectively. remote ~esults of t~eatment a~e an important index os t~eatment, the ~esult os ~hich ~e~e studied by means s a ~uestionnaive-method. patients ~ho had been t~eated by speleothe~apy mo~e f~eguently ~e-po~ted a ~elapse in disease ust afte~ the course o~ t~eatment ( , h). ho~eve~, in a ]ate~ phase the ~emission ~ould last ]on-~e~ (s months in , ~ os patients, till one yea~ in ~ ~). in , ~ os patients who passed the co~se os a~tificial sp~ay therapy a ~elapse was ~egiste~ed immediately as the co~se os t~eatment. then thei~ condition stabilized ~hile in , ~ os patients a period os ~emission lasted s ha]s a yea~. , ~ of patients dida't ~epo~t a ~elapse of the disease du~in~ one yea~. evangelismos hospital, critical care department, athens, greece method#: mechanically ventilated patients ( copd, ards, other pulmonary diseases) were studied in two phases: ) during the acute phase of respiratory failure; ) during recovery - days later. we measured mip and monitored the pattern of breathing while the patients were breathing spontaneously through the respirator (pressure support mode with - cmh ) until either the point they were unable to sustain spontaneous breathing (sb) any longer (phase ) or for two hours when they could sustain sb indefinitely (phase ). subsequently the patients were sedated, paralyzed and mechanically ventilated. then we simulated the pattern of sb at the end of the sb trial by manipulating the variables of the ventilator and assessed respiratory mechanics b y the end-inspiratory and end-expiratory occlusion technique. . during recovery, a combination of reduced inspiratory load and increased venfilatory capability makes a patient previously unable to sustain sb to breathe spontaneously. . inspiratory load is reduced during recovery, mainly because both intrinsic peep and breathing frequency are diminished. obiectives: although elevated concentrations of a few cytokines have been shown to be present in the bronchoalveolar lavage (bal) fluid (balf) of patients with the adult (acute) respiratory distress syndrome (ards), the pethogenesis of ards is largely unknown. leukemia inhibitory factor (lif), a growth factor recently recognised as a polyfunctional cytokine integrated in cytokine networks was measured in unconcentrated balf of patients from different patient groups. methods: lif was measured in balf by means of a specific and sensitive elisa (detection limit pg/ml)in balf (lavage of x ml in the right middle lobe). results: lif was not detected in the balf of healthy control patients and in only one ( pg/ml) out of patients at risk for ards (after cadiopulmonary bypass surgery) who underwent bal h after the end of the extracorporeal circulation. high and detectable levels were found in the unconcentrated balf of out of patients with full-blown ards ( + , mean + sem, range - pg/ml). there was a good correlation between the level of lif in the balf and a number of markers of inflammation: neutrophils/ml (r: . , p= . ), albumin ( r: . , p= . ) and protein level (r: . , p= . ). conclusions:the biological role of lif in these balfs is not readily explained by its currently known actions and it is unkwon whether lif contributes to or is a response to local tissue damage. our results indicate that this cytokine with lots of interesting _functions is a pert of the inflammatory cytokine cascade in ards. background and obiective : we recently demonstrated that cisapride -a new prokinetic drug -enhanced enteral feeding in a heter genoas group of ventilated icu patients by significantly accelerating their gastric clearance (crit care meal, ; : - ) . it remains unknown, however, whether certain subgroups of patients might benefit more from adding cisapfide to their enteral nutrition regimen than others. patients with chronic obstructive pulmonary disease (copd) might represent such a subgroup since their illness and its specific treatment put them at risk for gastric emptying disorders. design and setting : prospective, consecutive sample study in an adult medical intensive care unit in a university hospital. patients : mechanically ventilated and hemodynamically stable copd patients. interventions : gastric emptying was evaluated by bedside scintigraphy and expressed as the time at which % of a tcg~-labelled test meal was eliminated from the stomach (t / ). baseline data (do) were recorded after enteral nutrition reached to ml daily. scintigraphic measurements were repeated days after cisapride ( ml orally, q.i.d) had been added to this regimen (d ). patients were considered cisapride responders when gastric clearance improved by more than % from baseline. results : normal values for the test meal and for scintigraphic acquisitions obtained in the supine position were found to be + min. in healthy volunteers (crit care med, ; : - ) . five patients responded to cisapride (t / : + rain vs. + min at do and d , respectively) and five did not (t / : + min vs. _+ rain at do and d , respectively). in contrast with non-responders, all five responders had clinically significant maldigestion at baseline (excessive (> ml) gastric residues, vomiting (> times/day and abdominal distension) which disappeared in of them after the administration of cisapride. conclusion : copd patients who tolerate enteral nutrition well have basal gastric emptying times which are comparable with those of healthy volunteers and are not influenced by cisapride. however, cisapride treatment provides both scintigraphic and clinical improvement in those copd patients who exhibit clinically obvious gastric emptying disorders. cernv v., dostal p., zivny p., zabka l. dept. of anesth. and critical care, charles university, faculty hospital, i-irade~ kralove , czech republic objective: the aim of the study was to evaluate the effect of early entera nutrition started within hours of injury on the incidence of multiple orgar failure (mof) in trauma patients requiring vantilatory support. methods: after institutional approval patients were enrolled in the study enteral feeding was begun within hours of injury in trauma patients (en group) admitted to icu. nasuenteric tube was placed as soon as possible after admission into the distal duodenum under endoscopy. additional parenteral nutrition was used to meet patients energy and protein requirements. the control group (pn) consisted of patients fed during this period paretuerally. severity score apache ii, trauma score, cumulative balance of nitrogen (g), incidence of mof (three and more organs) and length of ventilatury support (days) were calculated. values are expressed as mean + sd. results: tab introduction : parenteral nutrition (pn) is an important aspect in the optimal treatment of patients on gastroenterology or intensive care. the aim of this bi-center study in patients has been to assess tolerence and efficacy of a new protein-lipid mixture for pn from a simple preparation. patients and m~hods : patients were selected in two hospitals (tenon and saint-lazare, paris) and were divided into two groups : group a (gastroenterology~ l short bowel syndrome) and group b (intensive care, surgical patients). all patients likely to require pig for a period of days (group a) or days (group b) were studied. the pn regimens administered were the following : combination with g of mct/lct fat emulsion end , g of nitrogen, in liter end glucose requirements were met by imfizsion of l liter of glucose - % via a "y " connection. lipid thus provided % of the non introgen calories. total daily calorie intake was to ] kced. this study monitored, before and at the end of infusions, the sennn albumin (alb), preaiburtun (prealb), triglycendes (tg), cholesterol (cs), and the serum ammotransferases (sgot and sgpt) end alkaline phosphatase (alp) activities. statistical significances were calculated using the wilcoxon-tost. introduction: many cu patients present a catabolic illness in response to inflammation and infection, characterized by a rapid loss in skeletal-muscle mass despite optimal nutritional support. growth hormone (gh) is responsible for a rise of lipolysis, enhancing the energetic balance, and of protein synthesis. recombinant human gh (rhgh) is nowaday available for clinical use, but its cost is very high. therefore, rhgh should only be prescribed to icu patients when its efficacy can reasonably be anticipated (ie. when the patients are catabolic or stressed, but in order to avoid overprescription for unstressed patients and for those who are overly catabolic). hence, we, as others, recently demonstrated that rhgh had no favorable effect in highly stressed icu patients. objective: to detect on a clinical basis, low (ls), mild (ms) and severe stress (ss) states in icu patients and validate this clinical judgement by objective metabolic mesurements, in order to select early those icu patients potentially able to benefit from rhgh therapy. methods: consecutive icu patients were prospectively stratified as ls, ms and ss by two experienced icu senior consultants (temperature; agitation; heart rate; arterial blood pressure; presence of an infection; respiratory rate; exogenous catecholamines). anabolic (insulin, igf- , gh) and catabolic (cortisol, ghicagon) hormones, and nitrogen balance were determined for each patient within hours after admission in the icu. metabolic and clinical data were then compared. the clinical stress states determined by icu physicians correlate with an objective metabolic assessment. therefore, the patients who will more likely benefit from adjuvant rhgh therapy can be detected simply and early. a prospective study on rhgh therapy in ms icu patients is in progress. berger mm md , chiolero r md , pannatier a phd , berger l , cayeux c , voirol p , hurni m md . surgical icu, pharmacy, and cardiac surgery, chu vaudois, ch-iotl lausanne, switzerland objective. nutrition of the compromised cardiac surgical patient is challenging. numerous factors influence the gastrointestinal (gi) absorption function, among which gut perfusion, which depends largely on the systemic hemodynamic status. patients in hemodynamic failure are prone to organ failure, and may benefit from an early jejunal feeding. the study was designed to assess the absorption function after cardiac surgery in patients with adequate and altered hemodynamic status, using paracetamol as tracer of gi absorption. methods. after cardiac surgery, patients, aged _+ years (mean_+sd) were assigned to groups (anaesthesia: fentanyl gg/kg + midazolam): group (n= ): reference group, with normal hemodynamic status, easy recovery. group ('n= ): patients in low output syndrome, cardiac index < . i/m on day (d ) after surgery, requiring prolonged intensive care, mechanical ventilation + nutritional support. paracetamol g, was given intragastrically on d + d : plasma levels measured (h.p.l.c), at administration (to), t - - - - - and rain. hemodynamic status assessed with pulmonary artery catheter. healthy subjects served as controls. results. compared to healthy controls, absorption was strongly reduced on d in all patients (no difference between groups). on d , peak paracetamol level was significantly lower in group (low cardiac output): in group the area under the curve on d and d were similar. there was a large inter-patient variability, reflecting the hemodynamic status. conclusion. gi absorption was decreased on d in all patients, and reverted to normal between d and d in case of normal cardiac function, but not in case of low output syndrome. the decrease on d can be attributed to fentanyl, known to slow down the gi transit. in patients with cardiac failure, correction of altered absorption was correlated with the hemodynamic status, suggesting that gi absorption is dependent on adequate splanchnic perfusion. the aim of the work was to define specific significance and evaluate efficiency of enteral component of infusion therapy in the intensive care of gastroenterotogic patients of surgical profile with pyo-septic complecations. there were used the methods of radial diagnostics and polyelectrography; the laboratory control on oxygen-transporting function, volumetric and hemodynamic state, changes in metabolic, hormonal and immunologic status was conducted. from january, [ till november, there was carried out the randomized study of patients with general purulent peritonitis; among them persons constituted the control group and -the main one. in the main g~oup the intestinal lavage, enterosorption, enteral introduction of nutrient solutions with gradual turn to enteral nutrition by equalized mixture "ovolaet" were started from the first hours after operation. the data obtained allowed to define the specifity of the program of artificial medical nutrition in the group of examined patients, based on necessity of individual selection of media for enteral introduction depending on the stages of intestinal insufficiency syndrome. it was shown that inclusion of enteral component into the program of infusion therapy during early periods stabilized circulation in the regime of moderate hyperdynamia, considerably decreases the deficiency of circulating blood volume, normalizes the values of oxygen transport, consumption an}d extraction, provides the optimal level of mycardial adaptive possibilities without tension of its compensatory functions and pulmonary circulation overload. due to combined application of parenteral and enteral nutrition the metabolic processes are shifted towards anabolism. this is supported by decrease to normal values in the contents of blood aggresive hormones (acth,hydrocortisone) and increase in somatotrophic hormone. the complete parenteral-andenteral nutrition influences positively on restoration of cellular and tumoral immunity, activates the factors of organism nonspecific protection and recovery from immunodepression, prevents the development of immunodeficiency. impact tm vs control. s atkinson, n maynard, r grover, e sieffert, r mason, m smithies, d bihari departments of surgery and intensive care, guy's hospital, london, u.k objectives: comparison of the effect of an immunonutrient enteral feed versus a control on the outcome of a mixed intensive care unit (icu) population. methods: admissions to this multidisciplinary adu)t icu thought likely to stay more than three days and with tube access to the gi tract ~r randomised to receive either impact tm, a feed with supplemental arginine, dietary nucleotides and omega- fatty acids, or an isocaloric and isonitrogenous control feed. study end points included mortality and icu stay. approval was obtained from the hospital ethics committee. rosults: patients were entered into the trial. the two groups were well matched for age, sex, and admission apache ii with an overall mean admission risk of death of . (std. dev. -+ . ). on an intention to treat basis, there was a no significant difference in icu mortality, icu stay or standardised mortality ratio (s.m.r.) between the two groups (see table) . similarly, there were no differences after stratification for patients receiving or more litres of feed. conclusion: there is no evidence of an effect of impact@, an enteral immunonutrient feed, on pre-determined end-points (icu mortality, icu stay or standardised mortality ratio) in a mixed intensive care unit population over that of an isocaloric, isonitrogenous control feed. objeeflves: evaluate changes of blood laatate levels according to patient medical status after cvvhd initj,~ion using dialysate solution containing lactate. method: review of medioal records of consecutive patients ~eated by cvvhd (dialysate solution hmnosol lg , hospal,uk, lactate concentration retool/l). date obtained hr before and - hrs at~er cvvhd initiation were analysed. results: all data are presented as mean + sem. in one patient, pre end post filter lactate levds were measured during standard cvvhd setting (blood flow ml/mlu, dialysate solution flow i /hr), and approximate daily lactate flux into the patient was calculated to be as high as mmol/d. lactate leveh measured after cvvhd initiation increased significenfly compared to baseline levels ( . + . axtd . + . ,respectively; p< . ,paired t-test). when patiente with increased basal lactete (~- ) were compared to paliente with normal basal values (n= ), no difference in laotete increase was fmmd (p= . , manova). patiente with severe liver dysfunction ( points in mop scomlg, n= ) had higher basal laotate levels than patiente with normal or slightly abnormal liver teste ( or point in mof scoring, n=ll), rite values being . + . and . + . , respectively (p< . , student t-test). increase in blood lactate did not differ between these two groups after cvvhd was stetted (p= . , manova). in pafiente with invasive hemedynamio mo~, no oorrelation batween changes in lactate levels and eitlm" changes in oxygen ddivery (t =o.ol; p--o. ) or oxygen consumption (reversed fie, k) (r -q).o ;p-- . ) were found after cvvhd initiation. conclusion: blood lactate increases on cvvhd with dialysate soh~on rich in lactate. this increase is predominantly caused by influx of lactate into the blood via the filter end does not seem to depend on the liver fimotion and/or oxygen metabolism changes. objectives: the study was designed in order to determine the effect on plasmatic proteins, of two types of aminoacids solutions of parenteral nutrition (pn) adapted to stress, having different concentration of branched chain aminoacids (bcaa), when applying to politraumatized critical patients. methods: a prospective study was performed using a randomized double blind design of polytraumafized patients, split in two groups of ten patients each, with mean ages of _+ an -+ years. due to their condition, all patients required p.n. for at least days. both groups were subjected to isocalorie and isonitrogenous solutions ( ci/kg/ day and . g of nitrogen/ks/day), varying only in the concentration of bcaa; solution a having a % concentration and solution b %. blood samples determinations during days , , , after the beginning of treatment with p.n. were total proteins., albumin, trandferrine, protein binding retinol; prealbumine and fibronectine. the anova test (one and two way) was used to compare the values between the two groups. results: the administration of solution a, showed statistically significant increases in the determinations of the values of protein binding retino] (p < . ) and prealbumin (p < . ). no significant increases were observed in the values of total protein, albumin, transferrine and fibronectin. solution b produced statistically significant increases only in the values of total proteins (p < . ). the remaining proteins did not changed from their control values during the whole period of pn administration. comparing both groups, no statistically significant differences were observed related to the type of diet. nevertheless, differences were found in total proteins, albumin, protein binding retinoi, fibronectin (p< . ) and prealbumin (p < . ) in relation to the time course of pn therapy. only the albumin values showed significant differences (p < . ) when considering the interaction of both the type of diet and the time course of pn. conclusions: . solutions of pn adapted to stress, can maintain the control values of slow turnover proteins and improve the values of rapid turnover proteins. . no significant differences on plasma proteins were found between the two solutions having % or % concentration of branched chain aminoaeids. &determination of rapid turnover proteins does not seems useful for discriminating different solutions of bcaa during pn. obiectives; the hormonal changes in the post-traumatic situation often leads to an elevated blood glucose and a negative nitrogen balance. to reduce the elevated glucose production by aminoacids the apprication of xylitol may be an alternative energy source. in a double-blind randomized study we investigated the effects of a xylitol/glucose solution (group a: aminoacids g/i; glucose/xylito g/ g/l) on metabolism and particularly on pancreatic and liver enzymes compared to a glucose based nutrition solution regimen (group b: aminoacids g/i; glucose g/i). methods: the clinical trial was carried out after the approval by the local ethical committee on patients with severe brain injury. there was no difference in body mass index bmi (group a: . +/- . kg/m and group b: . +/- . kg/m=), age, and sex. daily individual energy expenditure was measured by indirect calorimetry (deltetrac "~). nutrition was started - hours after trauma or surgery with carbohydrates and aminoacids. fat was added h after nutrition had started. to analyze the effects on pancreatic and liver enzymes we investigated the following parameters for days: blood gtucose, serum lipase, serum amylase, asat, alat, ~gt, ap, and serum cholinesterase (che). results: due to the daily indirect calorimetric measurements energy requirements were satisfied. there was no difference in blood glucose concentration and cumulative nitrogen balance between the two groups. neither were there any significant changes in asat, alat, ap, and che for days in both groups. serum tipase steadily rose to lull in group a and . lull in group b, respectively. conclusions: there was no measurable influence of either nutrition solution on liver enzymes. the xylitol/glucose nutrition regimen does not have any advantage over the glucose based nutrition solution concerning blood glucose level or nitrogen balance. the elevation of serum lipase to a -fold level in either group needs further investigation on trauma patients. the effects of fat emulsions in lung function, particularly in lungdamaged patients, have been attributed to alterations in pulmonary vascular tone caused by eicosanoid production modificatione. as the eicosanoid production may depend on the fatty acid profiles of the intravenous fat emulsion, haemodynamic, pulmonary gas exchange and plasma levels of prostanoids were investigated in acute respiratory distress syndrome (ards) patients, during different intravenous lipid emulsions (providing different prostanoid precursors). we studied in a randomized double-blind design groups (n= each) with ards. group i (lct) received a fat emulsion with long chain triglycerids (lct- %), group ii (mct) an emulsion containing a mixture of medium and long chain triglycerids (mct/lct / - %) and group iii placebo (control), during h ( mg/kg/min each). we measured before, at the end of h infusion, and h after the end of the infusion: lipaemia, arterial and venous blood gases, pulmonary and systemic haemodynamics, and plasmatic levels (arterial and in mixed venous sample) of eicosanoids (txb=, -keto pgf~,, and ltb ). at the end of the fat emulsion, groups (i and il) to , • to , • mmol/i), the paoz/fio z remained unchanged in the three groups; no changes in intrapulmonary shunt (qs/qt) were shown; neither in the mean pulmonary artery pressure. in contrast, only in the lct group: cardiac output and oxygen consumption increased significantly ( . % and %) (p< . ). eicosanoids were increased at baseline compared to reference values (p< , ). a decrease (p<o, l in the arterio-venous difference of the vasodilatador prostanoid ( -keto pgf~=) was shown in lct group. our results indicate that fat emulsions in ards patient do not influence pulmonary gas exchange, provided that the perfusion rate were keept at mg/kg/min. the present study was designed to evaluate the metabolic changes of a carbohydrate-based diet versus a fat-based diet on oxidation rate of substrates and energy muscle metabolism in patients with an acute exacerbation of chronic obstructive lung disease. patients were mechanically ventilated with a volume-cycled respirator. after an overnight fast, patients were randomnly treated with a continuous enteral feeding over a nasogastrie tube with a carbohydrate/fat ratio of / in group i (n= ) , and a carbohydrate/fat ratio of / in group i] (n = ) during consecutive days. indirect calorimetry was set at baseline and hours later the enteral feeding was started. muscular quadriceps biopsy was performed on baseline and on day of enteral feeding. urine was collected during hours and was used to measure h urea nitrogen production. the caloric intake was set according to the measured resting energy expenditure. respiratory gas exchange rate were measured using a computerized open-circuit indirect calorimeter. quadficeps femoral muscle samples were obtained by muscular biopsy after local anesthesia. analyses of glycogen, glucose -phosphate, lactate, phosphocreatine and adenosine triphosphate (atp) and enzymes (glycogen synthase and glycogen phosphorylase) were determined. after h of nutrition patients in group i showed an increase in carbohydrate oxidation (from . % to . %, p= . ), and in group ii a decrease in protein oxidation (from % to %, p= . ). after days of enteral feeding a tendency to increase in glycogen concentration (group i, p= . ; and group ii,p= . ) was observed. while patients in group i showed a tendency (p= . ) to decrease glycogen phosphorylase, patients in group ii maintained these enzymatic levels. the increased in glycogen was correlated in group li with degradation enzyme (p = . ). in group i, the pao /fio ratio increased after days of treatment (from . to . ; p= . ), but no changes in days of mechanically ventilation, length of stay in icu, or mortality rate was evidenced between groups. it is concluded that the oxidation rate of substrates used for energy production varies according to the caloric sources administered. with nutritional therapy muscle glycogen concentration increases in both diets, with different enzymatic influence. more studies are warranted to further assess the clinical implications of the different pathophysiologic behavior of the two diets. under certain experimental and human conditions (radiation and/or thermal injury) the gi mucosa is unable to perform this protective function and could play an important role in the pathophysiology of the syndrome of multiple organ failure (smof). the objective of the present multicenter study was to know the gi mucosal permeability in critically ill patients and the relationship with their outcome. the method used to assess the integrity of the gi mucosal barrier measured mucosal permeability to various hydrophilic compounds. specifically, we measured the urinary excretion ratio (uer) of two sugars (lactulose and mannitol) that had been previously administered through a nasogastric tube. urinary excretion rate was measured in healthy humans (control) and in patients admitted to the intensive care unit (icu) at days , and . the apache ii of the patients studied was _+ . there was an increase of uer in the critically ill patients compared with controls (o. _+ . vs . _+ . ) (p< . ). in contrast, no changes in uer between days , and were observed ( . -+ . . significant changes were also established in the different groups: major and minor surgery, general and epidural anaesthesia , transfused and non transfused patients except for iron and ferritin plasma levels which didn't change significantly in patients who had received blood. a positive significant correlation was established between crp and ferritin (r= . ) for patients under general anaesthesia, but not on epidural, regardless of the type of surgery. conclusions: iron and transferrin plasma levels fell acutely in surgical patients while ferritin and crp rose. transfusion implied an acute iron load.the established correlation between cpr and ferritin in patients on general anaesthesia suggests that ferritin behave as an acute phase reactant in that setting, while epidural anaesthesia may ameliorate the inflammatory response. objectives: to analize the effect of gastrointestinal complicacions (gic) related to the enteral nutrition (en) in the real volume of diet administered to icu adult patients. a prospective multicenter study was designed (comgine study) in wich en application, gic definition and his management were defined by collaborative consensus.patients treated with en in one month pedod were included. in each case, daily volume of diet prescribed (pv) and administered (av) was recorded and the volume ratio calculated (vr=pvxl /av). patients were divided for days of en according to the presence (group ) or absence (group ) of gic (abdominal distension, increase of gastdc residuals,vomits or regurgitation, diarrhea,constipation and bronchoaspiration). differences between groups were analized by anova and mann-whitney u test with p< . for statistical significance. (vr%) group (vr%) tolerance to the enteral nutrition (en) has been related to the severity of illness in icu patients. the aim of this study was to evaluate this relationship. methods. a prospective, multicenter, study was performed (comgine study) in wich en application and related gastrointestinal complications (gic) were defined by collaborative consensus. adult icu patients treated with en in one month pedod were included. gic were classified as: ) abdominal distension (abd), ) increase of gastdc residuals (igr), ) vomits (vom), ) diet regurgitation (reg), ) diarrhea (dia) and ) constipation (con). patients were divided in two groups: group a: patients with gic dudng their evolution. group b: patients without gic. differences between groups were analized for apache ii admission score or evolutive variables ( carbon dioxide production is a major determent of work of breathing. increase in co production during nutritional support may precipitate ventilatory failure and difficulty in weaning from mechanical ventilation. in the present study, co output was calculated while passive mechanically ventilated patient were fed with different amount of calories and different proportion of protein, carbohydrate and fat. four clinical stable and mechanically ventilated patients were studied. carbon dioxide was calculated while patients were paralyzed, sedated and mechanically ventilated. six nutritional regimens were used: a) no calories, b) t gr glucose/ h, c) calories equal to rest energy expenditure in special formula with low carbohydrates and high fat (pulmocare) d) calories equal to rest energy expenditure in standard formula (nutdson) e) calories double to rest energy expenditure in standard formula (nutrison) and f) calories equal to rest energy expenditure parenterel (tpn). carbon dioxide output was low while no calories and/or only gr/ h glucose was provided, the mean values of vco output were _+_ and !-_ ml/min respectively. there was no difference in vco output between with low carbohydrates and high fat regimen and standard regimen, mean values were t . -+ and _+ respectively.the high calories regimen and tpn resulted in higher vco output, mean values _+ and • respectively. we conclude that the special with low carbohydrates and high fat regimen did not reduce vco output, in the contrary, high calories intake as well as tpn increased vco output under tested conditions. klouche k., cristol j.p., vela c., canaud b., b raud j.j. m tabolic intensive care unit and nephrology department. lapeyronie hospital. montpeuier france. predictive factors for rhabdomyolysis (rh) -induced acute renal failure (arf) were studied in a retrospective study from january to january . patients (pts) (male: , female:lo; mean age: + y.o.) were admitted with rh defined as cpk> iu/ . etiologies were: traumatic and ischaemic , infectious , toxic , excess activity . factors studied were: simplified acute physiologic score (saps: . + . ), organ systemic failure (osf: . _-!- . ), diagnosis delay (d: +_ h), clinical parameters (sepsis, dehydration), blood chemistry data (cpk, bun, creatinine, potassium, phosphorus, calcium, proteins, hematocrit) and urinary ph. severity of rh was estimated by ward score determined according to phosphorus, albumin, potassium, cpk, dehydration and sepsis. urea appearance rate (uar) and creatinine index (ci*) were determined over a hours period. arf was observed in pts. in non-arf and arf groups respectively, saps ( . _+ . vs . + . ), deshydratation ( vs ), sepsis ( vs ), phosphorus ( . + . vs . -+ . ), calcium ( . + . vs . _+ . ), ward score ( _+ . vs . + . ) were significantly different. however, no significance was observed in uar ( -+ vs -+ ) and ci ( _+ vs _+ ). patients required hemodialysis (hd) ( : sessions) and remained dialysis free. only osf ( . _+ . vs . -+ . ), ward score ( . _-/- . vs . _+ . ) and ci ( +_ vs -+ ) appeared significantly higher in pts requiring hd. pts died from associated disease. all patients suffering from arf recovered a normal renal function. we confwmed that an elevated ward score (over ) is a good predictive index of arf. in addition we found that ci is a severity factor for arf requiring hd. thus, patients suffering for rh with elevated ward score and ci, have a fair chance of dialysis and should be treated more intensively. * ci (expressed in mg/kg) = (car + feces creatinine) / weight. where car: creatinine appearance rate; feces cr~t..= mean plasmatic creatinine x . . tr~er k., cetin t.e., tugtekin i., georgieff m., ensinger h. universit~tsklinik flir an~sthesiologie, uim, germany introduction: endogenous as well as exogenous adrenergic agonists have a profound effect on carbohydrate metabolism in human critical illness. in this study the effects of noradrenaline (nor) and dobutamine (dob) on carbohydrate metabolism during a hr infusion were investigated. methods: after approval by the local ethic committee healthy volunteers were studied. hepatic glucose production (hgp [mg/kg/min]), using , -d glucose as stable isotope tracer, as well as plasma concentrations of glucose (glc [mmol/i]) and lactate (lac [mmol/i]) were measured prior and during infusion of nor ( . pg/kg/min) and dob ( pg/kg/min). blood samples were drawn before and during the agonist infusion. results: no major changes in insulin and gtucagon plasma concentrations could be found during the study period. ::i:::: :iiiii~ ~ i ::i: ~:: : :: i:ii. mean-+sd are shown. # p< . , anova for repeated measurments. conclusions: the effect of nor on hgp and glc were smaller as compared to adrenaline (i) with a similar time course. in contrast to the effects of adrenaline and nor, dob had a different effect on carbohydrate metabolism: a decrease in hcp and glc, which is uncommon for a / -adrenoceptor agonist. since hgp is an energy consuming process that might deteriorate hepatic oxygen balance in critical illness, the differential effects of adrenergic agonists may be of importance and need further clarification. the nutritional insufficiency often accompanies post-operative hypercaloric states, inanition, serious infections and weakening chronic illnesses. that is why the early nutritional support, sufficient and appropriate for each individual base, is a fundamental component of intensive care unit as an indispensable factor for recovery. per this reason, our unit, developed a software for the implementation and nutritional control of t~e assisted patients. this software is incorporated is an expert system called ~i~su, designed and developed by the computational division of our unit. this system arrives to inferred diagnoses such as : respiratory, hepatic, renal(with and without dialysis) dysfunctions, pancreatitis, ards, decrease of consciousness, diabetes. according to these data objectives: to compare the effect of short term enteral feeding versus parenteral nutrition, when a isonitrogenous and isocaloric feeding solution is administered by either mute. methods: in a prospective controlled clinical trial patients were studied; all exhibited moderate degree of malnutrition, normal liver and kidneys, and a functi ning gastrointestinal tract. the patients were randomized to receive a free amino acid and small peptide diet ( patients) or an isonitrogenous isocaloric parenteral support (tpn) ( patients) (total energy: kcal, nitrogen: . g, carbohydrates: g, fat: g, n/non protein calories: / ) at least for days. results: there were no significant changes in anthropometric parameters within either group. nitrogen equilibrium was aqhieved by day in the tpn group and by day in the enteral group ( . % of the enterally fed patients and % of the tpn patients maintained in positive balance the day of the study). there were no significant changes in serum albumin within either group. serum level of transferrin reached a significant increase in both groups (p= . ). thyroxine-binding prealbnmin rose significantly in both groups as well (p= . and . respectively). statistically significant rises in lymphocyte counts (p= . and . respectively), in levels of c (p= . and . ) respectively), iga (p= . ), igg (p= . and . respectively) and igm (p= . ) occurred in either treatment group. there was a high incidence of negative skin tests at the start of the study in the enteral group ( . %) and the tpn group ( %). by the end of the study the incidence of negative responsiveness was . % and . % respectively. despite maintenance of similar glucose levels in both groups, tpn led to significantly higher serum insulin levels. the serum insulin increased almost linearly over the study period and eventually prevented fat mobilization and lipolysis, so that free fatty acid levels had fallen significantly. a significant elevation of the liver enzymes over the study period occurred in . % of the tpn group, but not in the enterany fed patients. conclusions: the present findings provide no evidence that enteral diets containing free amino acids and small peptides, as their nitrogen sources, are in any way inferior to isonitrogenous isoealoric regimes parenterally given. aim: the aim of this study is to describe and explore the expectations of the functions of the critical care nurse to enable the formulation of guidelines for the scope of practice for the critical care nurse with a south african context, methods: phase i was to determine the expectations of the critical care nurse, the nursing service managers and the doctors with regard to the functions of the critical care nurse. a focus group interview was held with a group of experts in the field of critical care. the results were used to compile a questionnaire. this questionnaire was sent to the critical care nurses, the nursing service managers and the doctors in south africa for completion. from these results the functions of the critical care nurse were determined. phase ii was to formulate guidelines for the scope of practice for the critical care nurse within a south african context. through usage of the date (phase i) the scope of practice was formulated. guidelines were formulated for the practise, education and research regarding the limitations of the professional-ethical authoration and the implementation of the scope of practice for the critical care nurse. objectives : high output gastric aspirates arc occasionally observed during fasting in critically ill paticnts, preventing any attempt of feeding via the enteral route. although these patients are often said to suffer from "gastroparesia", the motor correlates of this condition arc lurgcly unknown. in this stud?', wc recorded the gastrointestinal motility of critically ill patients with abundant (> ml/ hours) fasting gastric aspirates. methods : antral ( sites separated each other from . cm), duodenal ( site) and jejunal ( site) contractions were recorded simultaneously by ~eans of a multihimen tube assembly positioned trader fluoroscopic control (perfused catheter technique). tracings from prolonged recordings were obtained on a multichannel recorder ( a recorder, hewlett-packard) then anal) ,ed visually, with a special attention for the following abnormalities which are characteristic of intcstinal pseudoobstmctiou: l) absence or aberrant propagation of the migrating motor complex (mmc), ) presence of bursts (> min) of nonpropagated phasic pressure and ) presence of sustained (> min) uncnardinate pressure activity. patients with a volume of gastric aspirates of • (sd) [median ml/ hrs were investigated for - [median minutes. results : only one patient had no detectable motor abnormality. mmcs were either absent (n= ) or migrated abnormally (retrograde propagation : n= ; retrograde and stationnary : n= ) in pts. bursts of nonpropagated phasic pressure activity were present in the duodenum in pts and sustained uncoordinate pressure activity was found in pts. additional abnormalities included episodes of prominent pyloric activity. (n=l) and sustained antral pressure activity (n= }. conclusion : critically ill patients with large volume of gastric aspirates have manometric evidence of intestinal pseudoobstruction. prokinetic therapy in these patients should thus focus not only on enhancing gastric motility, but also on restoring a normal propagative contractile activity in the intestine. this prospective, open-label, randomized placebo-controlled study included patients with hypokalemia in whom rapid potassium replacement ( meq kci in h) was performed: patients received mg sulfate ( g in hours) and patients received a corresponding saline infusion. measurements were made at time , + , + and + hours results: k levels increased more in mg treated patients than in the patients who received saline infusion at time and h (p < . -students-newman-keuls). (table ). introduction. dual lumen uaso-gastrojcjunal tubes are a major ads'ance in nutritional therapy of mechanically ventilated critically ill patients since the " authorizc jejunal feeding with concurrent gastric decompression, there,, reducing the risk for aspiration. unfortunately, placcmem of these tubes in the jejunum regularly dictates to resort to endoscopy in order to facilitate pyloric intubation. recently, the remarkable gastrokinetic properties of the well known macrolide antibiotic er}lhromycin have been demonstrated in gastroparetic critically ill patients . aim. in the presem stu~,, we evaluated the feasibility of placing dual lumen naso-gastrojcjunal feeding tubes at the bedside without endoscopy, using edthromycin to help iranspy'loric migration of the tube under fluoroscopic control. methnd each patient admitted in our icu during a months period and requiring artificial ventilation and enteral nutrition for a period of at least days was included in the study.. after inserting the tube (stayput| sandoz, usa) in the gastric anmnn, e.rythromycin ( rag) was aduunistored intravenously, to help fluoroscopic positioning of the tube into the jejunum. the total duration of the procedure (from nasal intabatiun to jejunal placement), as well as the duration of ftuoroscopy were recorded in each patient. results. patients (male/female : / : mean age : . + . years; mean apacbell score : .t • . ) wore enrolled into the study.the procedure was performed within the dab,s following institution of mechanical ventilation. jejunal access was obtained in all patients without resort to enduscopy in , • . min.(total duration of the procedure). mean duration of fluoroscopy was . + . rain. conclusion. we conclude that placement of dual lmnen naso-gastrojejunal tubes can be obtained in mechanically ventilated critically ill patients without resort to endoscopy., provided that e rythromycin is used as gastrokinetic agent to help pyloric intubation. the following ad and dis parameters were considered in all patients: -mid arm circumference, triceps skinfold thickness, serum transferrin, albumine and lymphoeites and urinary creatinine/height index. patients whose results were bellow % of normal values in or more of the above criteria were considered undernourished (und).statistical analysis was performed using % analysis.statistical significance was established at p<o.o . results: a total of patients( female, male) -with a mortality rate (~) of , % -were studied, aged -+ years and a median ]enght of stay of days. -nourished (nou) at ad and at dis - ; ~ %; -nou at ad and und at dis - ; ) , % ; -und at ad and und at dis - ; ~ . %; -und at ad and nou at dis - ; ~ , %. we observed a p< . when we compared groups and (p= . ), and groups and (p= . ). variation in nutritional status and longht of stay: -nou at ad and nou at dis = > median lenght of stay days; und at ad and und at dis = > median lengbt of stay days; nutritional status and age at admission: -age > = years : nou ( ) , und ( ) -age < years: nou ( ), und ( ) nutritional status and age at discharge: -age > = years : nou ( ) , und ( ) -age < years: nou ( ), und ( ) we observed a p<o. when we compared groups and (p=o. ) and groups and (p = . ) conclusion: such study permits to conclude that most of our patients ( . %), staying in icu for more than days, were und, namely the elder. we observed that this kind ef patient had a longer median length of stay in icu, associated with higher mortality rate. in conclusion, the nutritional management is an essential element in suportive care of critical)y ill patients. ohiectives: sirs is associated with hypercataholisnl and resistance to nutritional support, despite raised circu&ting levels of growth hormone and insulin. serum igf- levels however are low; thercti~re rhigf-i administration might produce nsefnl anabolic effects. the pharmacokinetics of exogenously administration rhlge-i in such patients are likely to differ from that seen in the less seriously ill due to changes in circulating blood volume, increased capillary permeability, poor nutritional stares, and alterations in binding proteins. the ol jective of this study was to determine the clearance, apparent vohnne (if distribution (vd), time to peak concentration (tmax) and elimination half-like (tia) of a single subctkaneous il~ieetion of rhlgf-i in patients with sirs on the intensive care unit (icu). methods: icu patients with sirs were entered into the study, which had been approved by the ethics comjnittee. in each patient baseline hlood samples for circulating gf-i were obtained over a hour period, prior to il!iection of p.g/kg of rhlgf- subcutaneously. a further l samples were taken fi'om each subject over the next hours. circulating ige-i was measured in serum at each time point by radioimmtme assay, and the area under the curve tbllowing administration of rhlgf-i was derived tbr individual patients fixm~ baseline a~!justed igf-i levels. clearance values were caietdated by dividing the administered dose of rh[ge-[ by the total area under the etnve. vd and tv were calculated by regression analysis of the terminal part nf the log sertnn concentration time profiles. results: results are expressed as median (range). age ( - ) years, apache ii score ( - ), and length of icu stay ( - ) days. baseiine circulating igf- levels were low ( < - ) ng/ml reaching a peak of only ( < - ) ng/ml. of the patients had basal levels predmnhaantly below the inwel-limit of detection of the assay, and showed no appreciable rise in serum igf-[ levels tbllowing rhigf-i injection. parmacokinetic variables could not therefi)re be determined in these two patients. following rhlgf-i injection in the remaining patients tmax was at ( - ) hours, clearance was ( - ) ml/min, vd was . ( . - . ) l/kg, and tia . ( . - . ) honrs. conchlsions: there was marked patient wu-iability in response to rhlgf-i administration. vd was similar to that seen in poshq~erative maior surgery patients ( . vs . l/kg), but clearance was greater ( vs ml/min), tmax earlier ( vs hours) and t~a shorter ( . vs i . homts). if riaigp-i is to be administered to dtically ill patients the dose should he nlodified m the light of these findings. baekgronnd: acute bacterial endoearditis continues to be a condition with high morbidity and mortality. the majority of patients are treated with high dose antibiotics, but a patient group requires surgical treatment. methods and results: from january to march , patients underwent surgery for acute bacterial endocarditis: had native valve endocarditis and had prosthetic valve endocarditis. there were men and women. streptococcus viridans was the predominant infecting agent in native ~alve endocarditis and staphylococcus epidermidis in prosthetic valve endocarditis. progressive congestive failure, uncontrolled sepsis and peripheral emboli were the most common indication for surgery. although the mitral valve is more commonly involved in acute bacterial endocarditis, the aortic valve most often requires surgical inlervetion. the postoperative bleeding in the no-aprotinin group was + cc and in the aprotinin was :t: (p < , ). the mortality in icu was , %, the staphyloccocical endoearditis mortality was , % and the no-staphyloccocical endocarditis mortality was , % (p< , ). conclusions: the early surgery is indicated if endocarditis has no response with medical treatment. the mortality of staphyloceocical endoearditis involving left valves was higher than caused by other infecting agents. aprotinin treatment improved prosta#andin metabolism and preserved pletelet function during open heart surgery and could be useful in this type of interventions. urgent surgery had a high mortality ( %). selective digestive decontamination (sdd) has increasingly become the subject of critical discussion sine~ the development of multiresistant organisms has become an issue. moreover, a selection of gram-positive pathogens must also be taken into account when using the sdd regimen, which is primarily directed at the gramnegative bacterial specumn, with the methicillin-resistant staphylococcus aureus strains (mrsa) being of particular importance. the objective of our study was to determine the extent to which colonization with mrsa strains is promoted by sdd. method: patients (ventilation period > days) were randomized and allocated to the sdd group (n= ) or the control group (n= ). in their general intensive care theraw, there were no differences between the groups. the sdd regimen consisted of the four times daily administration of rag polymi~ mg tobramycin and mg amphotericin b in the nesc, mnoth and stomach. systemic prophylactic ~dmini~/rution of antibiotics was not part of the sdd regimen. smears were taken from the nose and the rectum twice wceldy and from the pharynx and trachea once wceldy, and tested for mrsa. further samples were taken as clinically reqnircr results: smears were examined in the sdd group. mrsa strains were detected in samples ( . %) from patients, and in patients they were detected for a period of up to weeks. the positive smears were districted as follows: tracheal / ( . %), nasal / ( . %), pharyngeal / ( . %) and rectal ( . %). severe mrsa-induced infections were observed in patients (infection rate . % of the colonized sdd patients). smears were examined in the control group. ivlrsa swains were r in samples ( . %) from patients, but only repeatedly over a period of up to days in patients. the po~tive snmars were distributed as follows: traclmal / ( . %), nasal / ( . %), pharyngeal / ( . %) and rectal / ( . %). there were no mrsa infections in the control group. conclusion: the data collected support the view that the use of sdd promotes a selection and persistence of mrsa strains. longer-term colonization with mrsa and sovere systemic inf~ons were only found in the sdd group. although the clinical and epidemiological impact of resistance develol~ng when sdd is applied ~maine unclear, this question should be given close scrutiny. tazobactam/piperacillin (taz/p p) is a new broad spectrum antibiotic, in which the acylaminopenicillin piperaeillin is protected by the betatactamase inhibitor tazobactam from hydrolization by bacterial enzymes. taz/pip has shown to possess a high antibacterial activity against almost all clinically relevant bacteria and is a registered drug in germany. obiectives: purpose of this investigation was to evaluate, whether faz/pip . g is suited for efficient antibacterial monotherapy of severe infections and what influence dosage frequency reveals on clinical efficacy. methods: hospitalized patients have been documented in this multicenter trial during a year period. as this investigation should reflect the usual clinical treatment, the only criteria for enrolment were the typical signs of infection as e.g. temperature > ~ leucocytosis or an isolated pathogen. exclusion criteria did not exist and the patients were treated in accordance to the severeness of infection, underlying diseases, risk factors etc. with taz/pip . g t.i.d, or b.i.d. results: patients suffered in most cases from infections of the lower respiratory tract (n= ), followed by intraabdominal (n= ) and skin and soft tissue infections (n= ). % of the lrtis wvre nosocomial acquired and in % the treatment was conducted as monotherapy. in % the lrti was treated with taz/pip b.i.d, and in % t.i.d. pseudomonas spp. (n= ) and staph..aureus (n= ) were the most isolated pathogens pretrcatment. the clinical response rates (cured/improved) after treatment with taz/pip . g b.i.d, and t.i.d, were % and % respectively. results for intraabdominal-and skin and soft tissue infections will be presented. conclusions: in hospitalized patients with severe infections successful treatment with taz/pip in monotherapy is possible. in this population a reduction of the dosage frequency to . g b.i.d, revealed equivalent clinical response rates. objectives. retrospective evaluation of cases of severe generalized tetanus (sgt), treated in our icu the last years. we review cases of sgt ( m, f), mean age . years. in eases the entry site of c.tetanus was a skin laceration, in case it proved to be the external genitalia, while in the rest no portal of entry could be determined. in the first cases incubation period was short ( - days) and so was the period of onset ( - days). all patients needed mechanical ventilation (range - days), initally through an orotracheal tube,and later through a tracheostomy, performed • days after admission. clinical manifestations of sgt included muscle rigidity and i generalized spasms, persisting for up to weeks in the most severe cases. significant autonomic nervous system dysfunction was present in cases occurring - days after the admission and following the time course of generalized spasm. besides general supportive measures, specific treatment included passive +active immunization, penicillin g, magnesium sulphate and sedation in a variety of regimens. neuromuscular blockade was required in cases. nosocomial infections occurred in eases, with sepsis and mof in one. average stay in the icu was - days. one patient died with severe septic complications and one was discharged with severe disability due to anoxaemie ancephalopathy, after a cardiac arrest on admission. ~ disinfectant in suspension test, without presence of organic load, disinfectants showed efficacy on lm. in the carrier test, in the presence of organic load, out of examined disinfectants did not exposed efficacy on lm. the results of examinations clearly showed that evaluation of disinfectant's efficacy partly depend on the used test method. antun basi , intensive care unit, kb firule split spin~ideva ! jugoslavia bacteremia and sepsis are frequent complications encouuntered in severe icu patients.microorganism identification with hemoculture presents the basis for adequate and successful antibiotic treatment.in many patients damage and vulnerability of the peripheral veins presents an obstacle for obtaining the blood culture from the central venous (cv) catheter sample could be also used. material and methods blood cultures were perfomed in lo patients on blood samples simultaneously obtained from the peripheral vein and cv catheter three times in a -hour period.criteria for the suspected bacteremia were body temperature above c and leucocytosis above ioooo leucocytes/dl. the site for venipuncture and the cv catheter stopcock port were cleansed with povidon iodine.after the initial ml of blood were discarded,lo ml were used for the blood culture.standard laboratory technique for blood cultures was used. results and discussion in ( %) patients hemocultures was negative at both sites,whereas in the remaining ( %) they were positive.for twentyone ( ~ of the positive patients the same results were obtained at both sites (peripheral vein and cv catheter),whereas in ( . %) patients the blood culture were positive only for the cv catheter samples.the cv catheters were in place for less than days in patients and for more than days in patients.from patients with positive blood culture from the cv catheter,one patient had the catheter for three days,whereas the other had the catheter from - o days. we neither found significant differences in hemodynamic dates : objectives: , to count and evaluate bacteria isolated from endotracheal (et) suctiori samples (with and without saline). . to establish the exogenous source(s) of pathogens isolated from carer's hands and the equipment involved in sampling in order to reduce the incidence of contamination and infection. method~: this prospective study included consecutive ventilated patients ( male and female, _ + yr; apache ii score -+ ) over a period of months. et aspirated samples with and without saline were taken daily from day of intubation until pathogen~ were presented in counts of _> per ml. at the same time, samples from both carer's hands were taken before and after et suction and a swab from the ventilator tube. results: the overall length of intubation varied between to days. bacterial transfer between staff and patients was noted in % of patients until day of intubation. there was no significant correlation between severity score and appearance of colonization. the incidence of pneumonia in studied patients was % with an overall mortality rate of %. acinetobacter anitratas (no ), staphylococcus aureus (no. ), klebsiella pna~moniae (no. ) and pscudomonas aeruginosa (no. ) isolates predominated in all our specimens. we noticed increased resistance to most antibiotics with the exception of imipenem for gram (-) bacteria and vancornycin for gram (+) bacteria. conclusions: i. tracheobronchial colonization appears directly in the maiority of intubated patients. . there is a close relationship between the microflora of personnel, patients and equipment. . bacteria transfer was noted both to and from patients. . strict hand disinfection policy remains an important measure for the proper care of mechanically ventilated patients to reduce respiratory infections. nnseeomial pneumonia is the most common nnsocomiai infection in the icu-settiag, reported in up to % of patients admitted to the icu following surgery. it is associated with significant mortality that ranges from ~ to %. enteric gram-negative bacilli have been implicated in % to % of ventilntor-associated pneumonias and pseudomonas aeruginosa accounts for % to % of these pneumonias. importantly, epidemics of/ - actamnse-pruducing enterobacter spp or klebsiella spp that are resistant to extended spectrum cephalosporins or penicillins, pose serious obstacles to effective antibiotic choices. carbapenems provide in ~tro activity against a wide range of enterobacteriaceaeand other gramnegative aerobic bacteria, except steaotrophomonns maltophilia. in vitro meropcnem is more active against pseudomonas spp than imipanem (especially p. aeruginosa and p. cepacia), imipenem and meropenem are effective against more than % of strains responsible for nnsocomial infections. all major pathogens associated with lrti are usually covered by the carbapenems, exceptions are pathogens involved in so-called atypical pneuomouia like mycoplasma, chlamydia and legionella. carbapenems are highly stable in the presence of most chromsomal and plasmid-mediated blactumases and usually offer a postantibiotie effect lasting for three hours against most of the enterubacteriaceae. reeent studies comparing imipenem/cilastatin with other ~-lactams and fluoroquinolones in severe lrti in icu patients resulted in favourable clinical cure rates and good tolerance, but development of resistance in p. aeruginosa and ;. aureus during treatment were of some concern. meropenem offers the advantage of greater stability against enzymatic degradation, so no concomitant administration of an enzyme inhibitor is necessary, and meropenem appears to be associated with a lower risk of seizures, particularly when used at high doses. results from studies with meropenem in lrti, especially in critically ill patients with acute exacerbations of chronic bronchitis, demonstrated excellent cure rates and better gastrointestinal tolerance of this new carbapenem. both earbapenems are effective candidates for use as empiric monotherapy in nosucominl infections of critically ill patients. qbl~ctives a favourable effect of iv immunoglobulins in septic surgical patients has been reported, but not sufficiently validated. we conducted this study on trauma patients to: i) investigate the effect of ivig on septic complications and il) quantify this effect by means of serum bactericidai activity (sba) assessment and iii) to explore the effect of temperature increase (from to ~ c) on the sba methods: twenty trauma patierits matched on admission for age, sex, inju~ severity score and glasgow coma scale, were allocated to receive either wig (ivig group; i patients) or equal volumes of human albumin % (control group; patients). wig (sandoglobulin) was administered in a total dose of g/kg divided in a four time regimen on days , , and post-admission. three blood collections were performe& before the first dose (day ) and hours after the third and the fourth dose (days and respectively). complement, lgg fractions, the sba at ~ and at o c and clinical parameters were recorded. results-similar lgg and igg] serum levels were found in groups ivig and control on day ( +_ vs • ns and + vs + , ns), whereas they were significantly higher (p< ) in the v g group on days ( _+_ vs + , p< ) and ( _+ vs +i , p< . ). the various complement-fractions increased in both groups without inter-group differences the mean (• sbas ( ~ c) at rain in ivig group vs control group were: - _+ vs - • ns for day , _+ vs - _+ p< for day and _+ vs - + p< for day . the mean (+sd) sbas ( ~ c) at rain presented a significant improvement over those of ~ c but for the control group remained negative a~d were respectively as following: -~ • vs - + , ns for day , +_ vs - _+ , p< . for day and _+ vs - _+ , p< . for day . the increase of temperature induced a -fold improvement of sba in iv g group and -fold ofcontrol-~oup positive blood cultures, and the product of the infectious episodes number multiplied by days of occurence, were significantly lower (p< ) in the ivig group than in the control ( vs , and vs , respectively). conclusions: our study shows a significantly favourable effect of ivig administration on septic complications and on sba of trauma patients. the increase of temperature results in a significant improvement of sba of patients that received ivig, which theoretically means a farther prevention of infection in the febrile state. pharmaceutical microbiology, university of bonn, meckanheimer aune , d- bonn, germany infectious diseases in intensive care patients are common in comparison to patients on other wards and out-patients. the main difference is that intensive care patients are much more sensitive even to less virulent bacteria. thus, the spectrum of infecting organisms is different. strains often regarded as pathogens with low virulence cause serious infections in these patients. strains such as serratia, however, have intrinsic resistance to most commonly used agents such as rd generation eephalosporins. furthermore, the common pathogens like staphylococci, psoudomonas aeruginosu, enterocneei and gram-negative bacteria, enterobacteriaeceae as well as the non-fermenters are less sensitive if isolated from intensive care patients. it is difficult to generalize on intensive care units as different patient groups are in different icus aud there are great changes from one hospital to another and from one country to another. if we take s. aurens strains from one study from the'overall resistance in intensive care units towards oftoxacin was %, whereas in other hospital wards the percentage of resistance was . %, in out-patients, however, only .$ %. the same trend was true for entercnecus faecnlis, coagulase-negntive staphylococci, and other bacteria as well as other drugs. one most striking difference was found with klebsialla pneumoniae and gantamycin resistance, which was $ times higher in intensive care units as compared with outpatients, whereas in the same species no difference was to be seen with the resistance towards carbapenems. however, differences between countries seem to be even more striking, as example gantamycin resistance and staph. anrens is given. the extreme difference is more than fold. thus, it is evident that there is a general trend towards higher resistance in intensive care units, but no generalizatiouis possible. therefore, surveillance studies in intensive care units are needed and the antibiotic policy has to be adapted to the specific needs of the unit. in the icu setting the most potent antimicrobial agents are required to address problem organisms including those resistant to penicillins, cephalosporins and aminoglycosides. carbapanems would appear to present a useful option in this setting. objectives of this study was the evaluation of systemic candid• in postoperative cardiac surgery patients (pts) with prolonged icu stay. methods: out of postoperative adults pts of mean age . + . years old, with a mean icu stay of . _+ . days, following an open heart surgery from july to april , pts ( %) remained in icu for more than days because of severe perioperative complications. patients were included in the protocol if they had clinical signs of infection or sepsis, and fungi isolated in blood culture or in culture from at least three different sites. the patients who developed systemic candidiasis received iv fluconazole ( mg/day) ( patients) or amphotericin-b for at least four weeks, and then they were closely monitored. results: out of postoperative pts with prolonged jcu stay, pts ( . %) developed systemic candid• usually after the th postoperative day. they were males and females of mean age +_ . years old. this group of pts had prolonged bypass and aortic cross-clamp time compared to control group ( min vs , and vs min). all these pts received inotropes per• (mean value= . ). during their icu stay, pts developed sepsis of bacterial origin, while the other two severe infection, and received antibiotic regimens for prolonged period. the patients were submitted to mechanical ventilation for a median period of days. the median icu and hospital stay was and days respectively. all pts have been improved and finally negative cultures were obtained. conclusions: . a significant percentage of patients who remained in the postoperative icu for more than days developed systemic candidiasis. . all patients who developed systemic candidiasis had received antibiotics because of sepsis or severe infection, for prolonged period. . fluconazole seems to be a very good alternative to amphotericin-b. . fluconazole is a safe antifungal agent with few side effects. botulism is the most severe and an odd food poisoning. although it is more commonly related to preserved meat derivatives, preserved fish and vegetables are also responsible for a number of cases. obiectives: to evaluate four familiar outbreaks of botulism . methods: we study the patients that were admitted in our hospital because of botulism from may to february . results: the thirteen pacients involved had a previous history of home preserved beans ingestion. after a -hours incubation period, gastrointestinal symptoms (abdominal pain, vomits, constipation) appeared and lead them to hospital consultation in the th to th day after ingestion. two patients died (acute respiratory failure before admission), seven were admitted in icu, two in ward and two of them were discharged from emergency room. clinical symptoms and the previous history of the ingestion established the diagnosis, that was emg confirmed. in all cases, symptoms were consistent with b-toxin botulism. b-toxin was isolated in serum and food proceeding from the third outbreak, and the serum was negative in the other ones. neurological symptoms were predominant: midriasis ( %), dry mouth ( %), dysfagia ( %), asthenia ( %), palpebral ptosis ( %), accomodation paralisis ( %) and urinary retention ( %). muscle weakness lead to acute respiratory failure in three patients (one of them required mechanical ventilation). four patiens developed infections (respiratory, urinary and phlebitis). both died patients and one another presented severe hypertension. all admitted patients were treated with polivalent anti-toxin. the two patients who underwent a more severe muscle weakness received also guanidine hydrochloride, with no answer in one case and provoquing a cholinergic crisis in the other one. icu length of stay was days. at hospital discharge, patients continued symptomatic, mainly with dry mouth, disfagia and impaired vision. conclusions: although botulism is a serious illness, the pronostic seems favorable if treatment and support measures are avaible. usually neurological symptoms we predominant and at discharge some of them could still persist. the arrow "hands-off" (aho) thermodilution catheter (tc) is completely shielded during balloon testing, preparation, and the insertion procedure. in order to assess the value of the aho thermodilution catheter in the prevention of systemic infections associated with pulmonary artery catheterization (siapa), we conducted a randomized prospective study over an -month period. methods : the patients (pts) were randomly assigned to two groups : group i for a standard tc customarily used in the department, versus group for the aho thermodilution catheter. the diagnosis of siapa was determined on the basis of a positive culture of tc and bacteremia with the same organism, with out any other nearby focus, in association with regression or disappearance of the clinical signs of infection after removal of the thermodilution catheter. results ( objectives: the mortality rate (mr) of tb requiring mechanical ventilation (mv) is high ( - %). the aim of the study was to evaluate mr, associated factors, and prognostic significance of mv and hemodynamic disorders from tb in icu in patients with tb. methods: clinical parameters on admission, and complications in icu were related by univariate analysis to icu, hospital, and month outcome. patients required mv; were immunocompromised (ic) including hiv. tb was pleuropulmonary in , disseminated in and meningeal in . results: mr was % in icu, % in hospital and % at month. / ( %) < . mortality was associated with a high saps score, initial shock, mv and nosocomial septicemia. the mr dramatically increased when ards occurred during illness, despite the lack of correlation between mr and initial po /fio ratio or initial murray score. the site of infection did not influence the mr. surprisingly, the mean therapy delay was shorter for non survivors. mr was not related to ic status, nor hivstatus, but was only related to previous steroid therapy. conclusion: mr of tb requiring icu is high ( % at month). need for mv increased mortality ( % vs %). general severity and respiratory dysfunction seem to be major prognostic factors in icu rather than tb per se or than therapy delay. in spite of the improvement in the prognosis of pneumococcal meningitis (pm) with third generation cephalosporins (tgc), this infection still presents a great mortality which could be increased with the appearance of antibiotic resistant streptococcus pneumoniae. objectives: to asses intensive care mortality and morbidity of pm and to define patients (pts) at risk of complicated evolution. patients and methods: a retrospective evaluation of pm cases (all diagnosed by csf culture) admitted in our icu from january tit march . in all pts we analized: demographic data, underlying disease, apache ii score, clinical symtomps, treatment, complications and outcome. statistical analysis was done using bmdp sofware package. results:a total f pts were studied, males; mean age , _+ ( - ); apache ii score , + , ; glasgow coma scale (gcs) at admission , _+ , ; ( %) pts suffer from cronic pathology; ( %) pts diabetes mellitus (dm), ( , %) pts had had a previous cranial traumatism. in cases the source of infection was otic and also in ( %) episodes of pm there were bacteriemia. in out of ( %) pts that ct was performed no radiologic abnormalities were shown, of them presented cerebral oedema and pts a cerebral abscess. twenty-eight percent presented seixures, % hemiparesia, , % respiratory failure, , % shock, i % renal failure, , % multiple organ failure (mof). as for treatment refers , % pts recieved only penicillin, , % pts only tcg, , % pts tcg followed by penicillin and , % pts tcg+vancomycin. seventy-five percelat of pts recieved corticosteroids and , % vasoaetive drugs. the mean icu stay was , : days ( - ). twelve ( , %) pts died, two of them presented pm relapse (resistant streptococcus pneumoniae) and another two pts developed neurological sequelae. factors associated statistically with bad prognosis were dm, the use of vasoactive drugs, shock, mof, the apache ii score at admission, the gcs at the and hours from admission in the icu but not the gcs at admission. didn't resulted statistiealy signifcative age, previous eronie pathology, seizures, baeteriemia, renal failure and coagulation disorders. conclusions: mortality was high and associated to apache ii score at admission, to gcs at and hours after admission, shock, vasoaetive drugs and mof. objectives:the aim of the study was to analyse some of significant immunologycai changes in surgical patients,requiring intensive health care,and to determinate the possibility for evaluation,dynamical examination and importance of immunologycal problems for treatment. methodes:the study concerns a number of patients with expanded surgical intervention or serious postoperative complications.the results has been carried out with fiowcytometryc analyses of lymphocytic suhpopulations and routins methods for investigation of humeral immunity.the"panel" for evaluation of (} immunologycal parameters has been offered:t-calls total/cd +/;t-helper/cd +/;t-supressor/cd +/ th/ts ratio;b-cells/cd +/;naturai kilier/nk/cells;skin test for cellular immune function;phagocytic and oxidative activity;serum levels of immunogiobulins-g ,a,m;protease inhibitors;c-reactive protein.all patients have been studied during suffering and after surgical procedures dynamicaly. results:there have been estimated significant changes in immunologycal parameters especially:decrease of t-cells: cd +mean= . %/ . %- . %/and cd +mean= . %/ % - . %/;inverted th/ts ratio ,mean=o. / . - , /;reduced or negative skin teste;reduced phagocytic and oxidative activity before septic complications. conclusions:dynamical examination of immunologycal parameters shows,that the prolonged t-total,t-helper lymphocytopenia with functional deficience of ceils-mediated immunity correlates with the stage of clinical condition of the patients and has prognostic importance.it's clear,that immunologycal monitoring gives a possibility for immunecorrection. patients (pts) with the human tmunodeficiency virus (hiv) infection have a decreased immune response and are particularly susceptible to infectious endocarditis (ie). the aim of our study was to analyze the prevalence of ie, its clinical and therapeutic implications in a hiv population we prospectively studied pts, . % ( / -group ie+) with ie during the clinical course of this disease. we analyzed the following parameters: age, gender, race, type of hiv, cdc classification, number of t and t type cell population and its ratio, therapeutic with azt, type and number of opportunist infections (inf, mycobacteriosis (mb), neoplasm's (nee) the echocardiographic parameters were lv internal diastolic and systolic diameters, lv percentage of fractional shortening, interventricular and posterior wall thickness, the degree of valvular regurgitations and the presence of pericardial effusion. el was located at the mv in . %, tv in . %, av in % and pv in . ~ and was multiple in . %. hiv el+ pts had larger lv diameters and more frequent significant valvular regurgitations ( % tr, pe %, mortality %). these two groups differed significantly in the following clinical parameters: the typical symptoms were watery diarrhea, high fever, tachycardia,luekocytopenia and oligouria within th postoperative days. the patients with mrsa enterocolitis had positive mrsa culture from the many materials except feces.mesa strains frequently had coagulase type ,enterotoxin a and toxic shock syndrome toxin- .eight of patients had postoperative organ failure.most of the mrsa strains in japan were similar in coagulase type to our hospital and our department.all of mesa strains were susceptible to vancomycin and arbekacin,tbough most of them showed resistant to many other antibiotics.we have employed guidelines for therapies such as oral or enteral administration of vancomycin and correction of the hemodynamics for dehydration and circulatory failure due to diarrhea from .futhermore we have placed colonized or infected patients in private room,worn gown and mask,and carefully washed our hands from . these countermeasures for prevention of nosocomial infections after significantly reduced the incidence of mrsa enterocolitis. conclusions:earlier diagnosis and treatment, and distric prophylactic measureres against mrsa infections are very important. -- cdo ivda leptespiresls affects all the organs with widespread hemorrhage that is more prominent in skin, mucosa, skeletat muscles, liver and kidneys. lung involvement is usually mild and less common. suli, it is very uncommon acute respiratory failure to be the pr sontirlg symptom. a case with leptosplrosl..,s which was presenting with acute respiratory failure is described. a year-old man admitted to icu becauso of fever, myaigla, aevere c~, hemopty~s. his blood gases showed: pao : mmhg with fio : . , pco : mmhg, ph: . , hco : mecl chest x-ray film demonstrated diffuse bilateral alveolar pattern occupying beth lung / ). trarmamlnase, bllllrubln, ~ and esr were elevated, wbc was . mm , platelet: . ram , hematesrlt: %, hemoglobin: .sgrldl=. there was no clinical or ecttlographlc evidence of left heart failure.patient fulfilled the criteria for diagnosis ards he was found to have an ~lutinatlon tlter for leptoq~lral antigens(indirect he~lutlnatlon atomy, ilia} very high ( / , negative<ill ) and iglm antibodies also very high (elisa, a= . , negative< . ) strongly suggesting leptospirosls. treatment with tetracycline and non-tnvaslve mechanical ventilation by nasal mask were started. pre~jre support mode and cpap was usod. the following days the clinical picture,the oxygenation and the chest x-ray of the patients were improved, and patient dl~herged from the icu. the control of leptosplremlc phase as well as his good cooperation during nipsv contributed to rapid recovery and excellent outcome in hiv infections, pneumonias (pnm), mainly due to pne~ mocystis carinii (pc) have an outstanding place in pu ! monary complications. the aim of this work was to compare two group> of patients admitted with pnm in our icu during the same period ( - ): group a, patients hiv+, and group b, patients hiv-. apache ii was identical in the groups (p=ns). group a required more often mechanical ventilation (p= ,o ), had a higher p(a-a)o (p= , ) and metabolic acidosis was more frequent (p= , ). regarding laboratorial parameters group a had a lower no. of linfocytes (p= , ), a higher ldh (p= , ) and a more marked hypoalbuminemia (p=o, ). mortality was higer in group a ( , %) than in group b ( , %), (p= , ). analysing the a group patients, we found no significant differences between alive and deceased patients, with exception for albuminemia, which was lower in the deceased patients (p= , ). in conclusion, the hiv+ patient's pnm have a more agres sive behavior when compared with community acquired hiv-patient's pnm. the prognosis was not influenced by the apache ii. perhaps other parameters such as p(a-a)o , metabolic acidosis, linfocytes, ldh and albumin shoud be more evaluated as possible predictive indices. some prognostic factors, usually accepted as predictive in the analysis of hiv+ patients do not seem to be worth in the late stages of aids, mainly when they reqquire intensive care. intensive care unit, onassis cardiac surgery center, athens, greece. objectives of this study was the comparison of two different antibiotic regimens as prophylaxis in cardiac surgery patients. methods: in a prospective randomised comparative study, two different forms of antibiotic regimens were investigated : a single dose of cefuroxime (zinacef, gr) (group a) given during the induction of anaesthesia, versus a four days combination of amoxiculine (amoxil, gr tid) plus netilmicin (netromycin, mg bid) (group b). a total of patients (pts) ( males and females, of mean age . + . years old) were included in the study over a period of one year; in group a and in the group b. patients were checked for the occurrence of infection during the first postoperative month. results: the total rate of infection in cardiac surgery pts was . %; . % in group a and . % in group b (p=ns). pts ( . %) developed infection following cabg, pts ( . %) following valve replacement and pts ( . %) after other cardiac surgery. they were males ( . %) and females ( . %). endocarditis has occurred . % in group a and . % in group b. severe wound infection was recorded in . % in group a and in . % in group b. one case of sepsis ( . %) in group a and in group b ( . %). respiratory infection occurred in pts of group a ( . %) and in pts of group b ( . %). two cases of urinary tract infection was in group a and one in group b. catheterrelated infection was occurred in ( . %) in group a and ( . %) pts in group b. pts ( . %) had fever of unclear aetiology in group b. conclusions: there was no statistically significant difference regarding the rate of infection in both groups. a single dose administration of cefuroxime is accordingly just as effective as a four days regimen of amoxicilline plus netiimicin. legionella pneumophila is a common bacteria of the environment, and it is an agent responsible for severe community acquired pneumonia (cap). we analyzed the patients with lpp admitted in our icu during the last years ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . they represented . % of cap. seven patients were males and female, with mean age . + . years. tiss was . + . and apache ii . + . . all, but patient, were under mechanical yen tilation (mv) during a mean period of . • (min-l, max- ) days. two pneumonias occurred beyond the season, while patients had an epidemiological history. only patient had no risk factor. in all the others tobacco smoking and alcohol abuse was quite frequent. diagnosis was based on serologic test and culture or direct fluorescent antibody staining of bronchial secretions. seven patients had a multisystemic disease with hepatic dysfunction in , renal failure in (due to rhabdomy~ lysis in ). one patient had a prosthetic valve endocarditis and another developped ards. nosocomial septicaemie occurred in patients. mortality rate was %. deceased patients had initially higher apache ii, (a-a) , and lower natriemia. comparing lpp with the other cap (n= ), both submitted to mv, mortality rate was similar ( , % versus . %). in conclusion lpp can occur all over the year. there was a high incidence of severe complications and outcome was similar to the other cap when requiring mv. prospective specimen brash (psb) with culture > cfu cfu/ml. broncho-alv~lat lavage (bal) ~= c'fu/rnl or positive blood culture. were excluded for rapture of treatment ; were analysed (shift with oral antibiotic : ; prohibited antibiotics associations : ; resistant germ : ). clinical data : age , • , ; saps • , ; mac cabe i : , % -ii : , % -iii : , . , % of the patients were intubated and under mechanical ventilation. the pneumoaiae were : primitive in ( , %), copd ( , %), aspiration pneumonia ( , %). germs were isolated (psb , bal , blood culture ) : s. pneumoniac ( , %), h. influeazae ( , %), sttep~:occns ( , %), saar ns ( , %), enterobaetdrindr ( , %), mosexella catarrhalis ( , %), othem . / ( , %) were sensitive to freatment. the ltentment was mg/kg/d of ampiclllin and mg/kg/d of sulbactam in continuous iv adminisu'ation during at least days. clinical eff~ienev : success ( %), failures ( %) with superinfeetion , worsening or relapse , dead , side effects . there was no difference between etiologies : primiti~;e~ , %, copd , %, aspiration pneamoniae , %. the bacteriological effieieacy was evaluated only for patients with eradication ( , %), eradication but super~ection ( , %) : with pseadomoaas a&ogiuosa , eater~ac~ ; beeteriological failure ( , %). in conclusion, the aasor ampicillin -sulbactam is effective for the i~eatment of severe acquired community pneumonise. objectives : to assess the efficacy of chlorhexidine (cl) gel or suspension applied in the nose and in the op for the prevention of the tmcheobronchial colonization. methods : thirty-seven patients expected to be intubated for > h were randomized to received topical application oga cl suspension ( %) qshrs, a cl gel ( %) q hrs or a placebo. in addition all vpts received a nasal and a op spray ( %) of either cl or placebo administrated according to the same schedule. semi-quantitative cultures of the anterior nares, the oropharynx (op) and the trachea were obtained on admission and once a day until extubation (just before the next application). the results were assessed according to the following criteria: success = no acquisition of gnb in the trachea ; failure = acquisition of gnb in the trachea. acquisition was defined by a follow-up culture positive for a gnb not present in the trachea on admission. results : success failure nosocomialpneumonia overall morality clsusp. placebo clgel placebo n= n= n= n= / / / * / / / / * / / / / / / / / / i *p = , byfisher'sexacttest conclusions : these results suggest that topical cl gel administered q hrs may prevent tracheal colonization by gnb. f. daumal*, m. daumal**, c. plot**, v. vurmmen ~ e.colpurt**, b. manonry** * hygiene hospitali&e, ** service de r enmmtion, * service des admissiens-urgeuces centre hospitalier g- ndral - saint-quentin -france obiectives: evaluate the nosocemial risk due to peripheral venous inserted short catheters, and the quality of care. patients-methods: the intensive tare unit (i.c.u.) is a beds unit. the prospective study includes all the patients comn~ in from / / to / / . the recruitemont uses an evaluation schedule of local clinical signs. the nurses aimed to create this evaluation data which includes the place of entry site, the duration of catheterization and the cause ot withdrawal. only patients staying longer than days in the i.c.u. are accounted for. the diagnosis of uosoenmial infection is assured by the physician taking care of the patient and by the hospital epidemiologist on the next signs: evident pus at the catheter entry site, positive culture of the strain, with or without the same pathogen in the blood sla'uam,the patient having no other distant source of infection. analyses were performed on epi/nfo. results: the occurrence of nosoeomjal inthrtions: i abcess and bacteremia during the first part of the study lent the medical staff to modify the protocol of insertion end survey of the device. so we analysed different periods: period ( / / to / / ) and period ( / / to / / ) for all .e peripheral catheters inserted in the i.c.u. period , % , % en infection due to peripheral venous device is a daily threat. the severity of some clinical situations requiring admission in icu proves it. the motivation of nurses for rigid adherence to established protocol, the daily survey of the entry site, the withdrawal of the peripheral catheter every hours aimed to reduce significantly the local signs of inflammation end infection of peripheral catheters inserted inside the i.c.u. objectives: to investigate the use of a new metabolic monitoring device for different ips levels by comparing oxygen consumption (vo ) to measurements of the mechanical work of breathing (web) and p . . methods: the study was approved by the institutiotml ethics committee. eight patients were investigated during weaning after prolonged mechanical ventilation ( - days) for various diagnoses when the clinical physician judged the patient to be ready fur weainag. ips was setto , , , mbar far rain periods each. all patients had a peep between - mbar.. respiratory frequency (f), tidal volume (tv), minute ventilation (ve) were read from the ventilator display ( ae, puritan bennett, carlsbad, usa). flow and airway pressure were measured at the endotracheal tube site. esophageal pressure was measured using an esophageal balloon catheter (fa. ruesch, frg). web was determined as the area subtended by the pleural-pressure-vohime curve. p . was determined by using standard occlusion technique and graphical analysis of the airway pressure tracing. vo and vco were measured using the pb metabolic monitor (puritan bennett, carlsbad, usa) connected to the pb ae ventilator. all data are given as mean• deviation for each ips level. comparison between the different ips levels was performed using anova for repeated measurements. significance was considered at p< . , compared to ips mbar. results: the values for breathing pattern, web, p . , vo and vco are given in the table for the different ips levels; significance is indicated by ~. objectives: fluidized beds are often used in the management of critically ill mechanically ventilated patients. critically ill patients are increasingly colonized with resistent pathogens [ie: p. aeruginosa, methicillinresistent s. aureus (mrsa), extended spectrum i~-iactamase producing enterobacteriaceae ] that can ultimately cause nosocomial infection. methods: we prospectively monitored bacterial colonization of mechanically ventilated patients and of the fluidized bed (clinitron) inwhich they were treated. multiple samples for quantitative bacterial cultures were taken from oropharynx, trachea, feces and bedsores. samples of ceramic beads from the bed were also taken both during and after patient stay (after bed operation in the absence of patient). re,~ults: episodes in consecutive patients (mean age: . years) were analyzed. all had bedsores and/or urinary catheters and fecal incontinence, patients had nosocomial pneumonia, had urinary tract infection [ with extended spectrum imactamase producing k/ebsie//a pneumoniae (ki~lse)], one had positive blood cultures with mrsa, and one patient had a ki~lse found in high concentrations ( - s cfu/ml) in occasions in feces. patients were heavily colonized: the , samples from ceramic beads showed no growth or became sterile without any sterilisation procedure (even in one case of presence of kf~lse) during the patient stay. conclusions: fluidized beds do not put patients at high risk of acquiring nosocomin pathogens, and cross-contamination between patients seems unlikely, even when multiple resistent organisms were initially present. the recommandation from some manufacturers to undergo extensive sterilization of fluidized beds after use does not seem warranted, at least with the bed used in this study. ant. koutsoukou, a, tahmitzi, p. kithreotis, m. koutonlidou, k. stavrakaki, kainis e, g. vlahogiorgos and e. eliopoulos icu-centre for respiratory failure -chest diseases hospital of athens. the cost-effectiveness issue is becoming vital in modern medicine and may lead to moral dilemmas since sometimes certain groups of patients may not have access to highly specialised modalifies. objective: our study compared the mean daily cost for antimicrobial medication in copd patients treated in icu versus all other patients in the context of relevant epidemiological, prognostic and outcome data. methods: age, sex apache ii score, length of icu stay (los) and in -icu fatality were retrieved from the files of all icu admissions over . mean daily cost for antimicrobial therapy per patient (dcat) was estimated. these variables were statistically compared between copd and non-copd patients. significance was assumed at p< . results: of the total admissions were fully evaluable. of them ( %) were copd patients. data (m---sd) results for statistical test are given in table i . copd patients were significantly older spent more time in the icu and presented with significantly higher apache ii scores. outcome and dcat were comparable in the two groups. objectives: the use of heat and moisture exchangers (hmes) during long term mechanical ventilation (mv) is increasing. in icu patients, they are routinely changed every day, according to the recommendations of the manufacturers, but the clinical basis for such a daily practice is lacking. we therefore prospectively assessed whether changing hmes (dar hygrobac, spa, mirandola, italy) every h only would affect their clinical and bacteriological efficiency. methods: two consecutive groups of patients requiring mv for > h were compared: group = hme replaced every day, n= episodes of mv in patients; group = hme changed every h, n= episodes in patients. tubings were not changed in the same patient during the whole length of ventilatory support. diagnosis of nosocomial pneumonia (np) was based on a positive quantitative culture (~ cfu/ml) of a protected specimen brush in patients with clinical signs of pneumonia. quantitative cultures of pharynx, trachea and y-cannector were performed every h. results: the groups were similar in terms of age, indication for and overall duration of mv ( +_ . vs +_ days, p= . ), and severity of illness (saps: --- . vs . +_ . , p= . ). the maximal values for peak airway pressure were identical in both groups ( . -+ . vs . • cmh , p= . ). obstruction of the tracheal tube was observed in only one instance in a group patient who had tracheal bleeding. circuit colonization was very rare, and of low grade in both groups. the level of patient colonization and the type of organisms were identical in both groups. more importantly, the incidence of np was the same ( / vs / , p= . ), as was duration of mv before the occurence of pneumonia ( • vs . +_ . , p= . ) and overall mortality rate ( vs , p= . ). conclusions: the clinical efficiency of this hme does not seem altered after days of use. indeed, replacing this hme every h only neither affect circuit and patient bacterial colonization nor the incidence of np. therefore, substantial savings could be obtained changing hmes every other day only. obiectives: to evaluate the usefulness of different paraclinical investigations for the diagnosis and prognosis of acute viral encephalitis in icu patients. methods: we reviewed patients (pts) admitted to our icu from july to december with the diagnosis of acute viral encephalitis. all were in coma and were initially treated as presumed herpes simplex virus (hsv) encephalitis. the causative agents were: hsv ( cases), herpes zoster varicellae ( ), measle ( ), rabies ( ), unidentified ( ). eleven pts survived and three presented neurologic sequelae. twelve pts were investigated by mri, and eleven also by spect and multi-modality eps. including brainstem auditory eps (baeps). these investigations were obtained as soon as possible following admission and were repeated during icu stay when possible. the clinical outcome was noted. results: six pts ( / ) had an abnormal mri. among them, pts made a complete recovery, in comparison with / pts with a normal mri. in one hsv infected patient, mri remained normal despite clinical deterioration and bad outcome. when repeated, mri became abnormal in cases (with poor outcome in one) and was improved in one. spect was found abnormal in / pts (among them, pts had thus a normal mr/). the correlation regarding the topography of brain lesions was poor between mri and spect. the findings of spect could not be correlated with a poor outcome. the baeps confmned in % of the pts the clinical diagnosis of brainstem involvement. changes in visual and somatosensory eps were mild in all the pts and were not helpful for the prognosis. eps were otherwise interesting for the follow-up of the coma in these sedated and ventilated pts. conclusions: the value of mri and eps for the diagnosis of acute viral encephalitis is of limited interest. spect seems to show early modifications, even in pts with a normal mri, but this test is poorly specific and does not correlate with mri changes when present. concerning the prognosis, larger studies should probably confmn that a normal mri could usually result in a good outcome. this serie illustrates also that hsv encephalitis could be demonstrated only in a small number of cases and that the prognosis of non hsv encephalitis is not easily assessed. objectives: to study the influence of gram (-) bacterial lung infections on liver function i~ mv icu pts. pts and methods: we studied pts, # ( , %), ( , %). hean age: , • years ( - ). mean stay in icu: , • days ( - ). they were divided in groups: a( pts) who did not suffer from pneumonia and b ( pts) who developed a gram(-) bacterial pneumonia. both groups were consisted of pts with same age, sex and disease distribution and same systemic failures. we measured sgot, sgpt, total bilirubin(tb), direct bilirubin (db), alk.phosphatase (al.ph.), v-gt and albumin (alb.) times: on days o, and of the pneumonia for group b and respectively for g~oup a. conclusions: ) in elderly intubated pts of an icu, kp is isolated more frequently than in icu pts< years (p<o, ). ) the frequency of isolation of kp in icu pts during - was - %, but now ( - ) kp becomes more and more rare ( - %) and is isolated especially in elderly pts. ) its sensibility to antibiotics remains always the same. ) the prognosis of np in our study was independent from age (p<o, ), sex, presence of bacteremia (p< , ) and type of invading microorganism. gram neg bacteria and renal failure (rf) from aminoglycoside toxicity are common and serious complications in critically ill patients. the aim of this prospective, pilot study was to compare the safety and adequancy of the endotrecheal (et) vs the intravenous (iv) administration of aminoglycosides. were measured in the plasma and the bronchial secretions at and hours after the iv (bolus) or the et (nebulized) administration of mg of gm in seven criticcally ill patients ( with established renal failure). safety was defined as peak and trough plasma gm levels _< than and ijg/ml respectively and adequancy as gm levels in bronchial secretions _> , ijg/ml. results: gentamicin was administered by the et and iv routes in and separate sessions respectively. a total of samples were assayed, in bronchial secretions (bs) and in serum. the et route resulted in higher gm levels in the bronchial secretions compared to the iv route ( , + , vs , _+ , pg/ml respectively, p = ns ). adequate bronchial gm levels were achieved in % of patients after et administration, compared to % after iv aaministretion. the blood levels of gm were significahtly lower after the et vs the iv route ( , + , vs , • , pg/ml respectively, p _< . ). the et administration resulted in toxic bronchia~ gm levels in % of the specimens. % of these samples were from patients with renal failure, however toxic blood levels were reached in only % of these. gentamicin seems to be a safe and adequate alternative route of treatment for the lrti. however, in patients with renal failure the et administration of the aminoglycosides should also be modified and continuously monitored. in order to evaluate the pathogenic role of anaerobes in nosocomial pneumonia (np), we investigated the systemic humoral response in patients who developed a np with anaerobic bacteria, especially prevotella species. methods: blood samples from groups of patients were tested. group i: patients with a np in which prevotella spp. was isolated from protected specimen brush (psb), group ih a control group of patients with a np without anaerobic bacteria, group ill: a control group of patients with dental stumps but without pulmonary infection, group iv: a control group of healthy voluntary people with prevotella spp. isolated from the dental plaque. an elisa was used to evaluate the total antibodies level against a mixture of four prevotella strains and a western-blot method was done to identify the antigenic proteins. results: data are expressed as means .+ sd. the antibody levels in patients of group i ( • was statistically higher (p=o.o ) than in the control groups (respectively: + , _+ , _+ ). using western-blot method, the intensity of the response was roughly superposable to levels obtained by elisa and the profiles were different according to the prevotella species. the occurence of a np with anaerobic bacteria (prevotella species) isolated from psb leads to an antibody response which seems specific of the prevotella species isolated. fever is common in the intensive care unit, but is not always related to an infection. we sought to define the epidemiology of febrile patients in a general medical/surgical icu. methods: we prospectively analysed the source of fever (t > . ~ c) in all adult patients admitted for >- hours in the icu during a two month period. these patients were studied for consecutive days. and werc classified in groups according to the evidence of infection (center for disease control criteria) after complete evaluation: documented infection: cdc criteria + isolation of pathogen (d); possible infectron: cdc criteria without isolation of pathogen (p); unlikely infection: patients who did nol meet the cdc criteria (u). results: of a total of patients studied, dec'eloped fever ( %). including (after complete evaluation) d, p and u palients. both the highest temperature in tile first day of fever and the maximal temperature were higher in d than in u ( . • versus . • and . -~ . ~ versus . - . , respectively p= . and p= . ). most common sources of infection in d were the lungs in patients ( %) and urina .ry tract in ( %). of these patients had positive blood cultures ( %). the overall mortality was % ( % in d, % in p and % in u. differences ns). antibiotics were given in % of d, % of p and % of u ( patients). in p there was a non significant lower mortality." in patients who received antibiotics ( / ( %) versus / ( %) patients, respectively). conclusions: in febrile icu patients both the highest first day" temperaturc and maximal temperature are significantly higher in infected than in non infected patients, but the differences are too small to be useful clinicall). mortality rate is not significantly influenced either by the presence of an infection or by the administration of antibiotics, obiective: retrospective study to determine the influence of candida infection on icu outcome. methods: patieet with a stay of more than days in inteaasive care were screened for candida infection. patients were treated with antifungal therapy due to either an increased antigen titre of -> : or clinical evidence of candida colonization. serological candida-antigens (ramco, pastorex) and antibody titres (hemagglutination, lgg-, igm-elisa) were examined routinely. seroconversion was defined as a threefold increase of antibody titre or a titre of : or higher. results: the median length of stay was (ranging from to ) days, the mean apache ii score on admission was (+_ . sd) points. of patients patients died ( . %). in the group treated with antifungnls ( patients) patients died ( . %). although of the patients only ( . %) developed a candida infection as defined above the mortality in the group that showed signs of infection was significantly higher ( . % vs. . %, p < . [chi-square-test]). in patients an antigen concentration-> : was measured. seroconversion was found in patients. the most common fungus was candida albicans ( . %). furtberm re, candida glabrata was found in . %. most of the patients were treated with x mg fluconazole ( patients). in patients therapy was changed to amphotericin b/flucytosine. in patients therapy was started with amphotericine b and flucytosine. in patients a threefold decrease of candida antigen titre was found. patients showed a decrease of candida antibody titre. conclusions: meticulous screening for eandida infection seems to be necessary since the number of patients with fatal outcome is significantly higher in the group with signs of fungal infections and thus requires immediate antifungal treatment. objective: early diagnosis of patients with ventilator-associated pneumonia (vap), and subsequent identification of causative microorganism, and selection of the appropriate therapy are critical important points that affect morbidity and mortality. the results of the quantitative bacterial cultures are not available for at least hours, while a two hours period, since the specimen are obtained is enough to know the gram stain results. the aim of this study is to determine the usefulness of gram stain in specimens obtained by bronchoaiveelar lavage (bal), through the bronchoscope. material and methods: we studied patients ( males and females, age + ) with suspected ventilator-associated pneumonia. the bal gram stain was considered positive when the specimen after a centrifugation at rpm for min revealed: i) more than leukocytes per optic field, ii) squamous epithelial cell less than percent and iii) one or more microorganisms per optic field on magnification. all patients had been receiving antibiotics, with no change during the last days, prior to bronchoscopy. results: patients had vap and patients did not. in cases the bal specimens (quantitative bacterial cultures) established the diagnosis of vap in the remaining three patients the vap diagnosis was established by other procedures (blood or pleural fluid culture, clinical outcome, autopsy). apache fl score in patients with vap was , -+ , , while in patients without vap was , + , . there was a significantly higher incidence of vap in patients who had i) coma (gcs < ) and ii) been receiving neuromuscular blockade (p< . ) . the sensitivity of the gram stain for vap diagnosis was %, the specificity , %, the positive predictive value %, and the negative predictive value , %. conclusion: our data indicate that the gram stain of bal specimens is useful for the early diagnosis of vap and the subsequent administration of the appropriate treatment. the role of anaerobes in mechanically ventilated patients with pneumonia (mvp) have been poorly investigated aim of the study : analyse the prevalence of anaerobic isolation in mvp. methods : between october and february all suspected mvp were investigated using protected specimen brush (psb) technique. brushes were rapidly transported in shaedler broth to laboratory. a special care was tooken for anaerobic isolation. results : among the psb performed for suspected mvp ( nosocomial and community-acquired pneumonia), yielded at least one micro-organism (positive psb : %). of positive psb demonstrated only aerobic bacteria and ( %) yielded with anaerobes. in out patients, anaerobes were associated with aerobic bacteria. anaerobes were mostly isolated in nosocomial pneumonia ( / positive psb). strains of anaerobes were isolated. prevotella species represent out these strains ( %) the most frequent anaerobic species were prevotella oralis ( ) p. intermedia ( ) and p. buccae ( ). comments:using adequate methods, anaerobic bacteria are frequently isolated in mvp. it could be off importance to take in account anaerobes in the choice of empirical antibiotic therapy in mvp. objectives: the majority of patients with multiple trauma are considered immunocompromised. the aim of this study was to identify risk factors of pneumonia in mechanically ventilated patients with multiple trauma or after surgery. methods: in this prospective study we studied multi-trauma patients (mean age + years, apache ii . + ), admitted to a general intensive care unit (icu). all patients were intubated and mechanically ventilated. we were considered that a patient had ventilator associated pneumonia (vap) when the specimens of bronchoalveolar lavage (bal) or protected specimen brush (psi?,), ebb'ned through the bronchoscope, had one or more microorganisms in concentrations greater than and cfu/ml respectively. all patients had been receiving antibiotics, with no change during the last days, prior to bronchoscopy. results: patients had vap, and patients didn't. in the bivariate analysis, the glasgow coma scale (gcs)< (x = . , p< . ), the administration of neuromuscular blockade (x = . , p< . ), the duration of mechanical ventilation to be greater than days (x = . , p< . ), the flail chest (x = . , p< . ), the parenteral nutrition (x = . , p< . ), the ards (x = . , p< . ), the abbreviated injury scale (ais) of more than for thorax (:,: = . , p< . ), the pneumothorax (x = . , p< . ) were statistically significant related to development of vap. in multivariate regression analysis, using the stepwise technique, three of the seventeen studied factors showed to have an indepantent association with the development of vap:the administration of neuromuscular blockade (f: . , p< . ), flail chest (f: . , p= . ), and gcs (< ) (f: . , p= . ). conclusions: in patients admitted to icu for multiple trauma or major surgery, the administration of neuromuscular blockade, the flail chest, and the gcs (< ), in the population under study, were the indepedent risk factors for vap. mof is a sereous complication of differem states: infection, sterile inflamation, extensive fissure injure, intoxication, ets. there is close correlation between extension of mof and death, developement of nasocomial infection. immunologic disfunction. in order to prgnose probability of risk of mof development among the patients with sepsis and septic shock, we achived an eqation, allowing to recive a coeficient, closely connected with this probabiliti. we have used retrospective analisis of cases of sepsis. diagnosis of sepsis was based according to bone's criterions of sepsis. mof was assessed as disfunction of or more systems according to bone's classification of mof. having used correlation analisis we have estimated factors which have had high correlation coeficient with the probability of development of mof. there were: apache-ii score points, evidenceof septic shock, endocrinopathy. with the help of multyple regression analisis we acheved next equation: y= , + , x~ + , x + , x , were x i-apache-ii score points, x -evidence of septic shock, x -endocrinopathy. the explanatory power of this quation was evidenced by roc of . , se (v - . introduction: the presence of liver dysfunction in the process of multiple organ failure is associated with an adverse outcome, particularly when it becomes progressive to liver failure. disturbances of liver function may occur early and their detection may be of significant importance for the further development of organ failure. routinely used liver function tests appear to be inconsistent indicators of hepatic damage. in this study, we used p_lasma disappearance rate (pdr) of indocyanin-green dye (icg) as an early estimate of liver function. methods: we serially evaluated pdr and routine liver function tests (serum bilirubin, sgot, sgpt), as well as acute phase and non-acute phase proteins (crp, transferrin) in patients during the first week after trauma or the onset of sepsis. patients: group : (n = ) multiple trauma iss > , group : (n = ): abdominal sepsis, acute necrotizing pancreatitis (anp) grade iii. patients were selected on the basis of clin cal estimates that these patients would require continued icu observation. pdr was determined by means of a fiberoptic catheter and a computerized system (cold z- , pulsion), which permits repeated bedside measurements. the initial values of pdr, serum bilirubin and transaminases were not significantly different in trauma, sepsis and anp. in trauma patients pdr improved during the first week. in patients with sepsis and anp pdr remained low and worsened with time. the decrease in pdr preceeded an increase in biochemical liver function tests in these patients. + . &-_ ( - ) discussion: routinely available blood tests of liver function are usually altered several days after injury. however, they are generally non-specific indicators and they are influenced by extrahepatic factors. pdr seems to be useful to evaluate impaired liver function early after the onset of sepsis and trauma. objectives: to study frequency of organ system failure (osf) and it's influence on outcome in granulocytopenic patients with hematological malignancies and septic shock(ss). materials and method: retrospective review of medical records of granulocytopenie(wbc< , xl ) patients with hematological malignancies and ss, who were admitted to the intensive care unit (icu). frequency of osf before and after ss was analysed. the patisnts were categorised on survival and non-survival. results: signs of osf were observed in . % of patients before ss and in all patients after ss. only patients presented with hypotension refractory to inotropic therapy. nevertheless there was a significant increase of frequency of acute respiratory failure (arf), acute renal failure (arenf) and liver injury (li) after ss occurred(showed on the figure). only frequency of organ failure before and after objectives: statusmetria allows to define the effective level of oxygen status and accordance to it means of carbon dioxide and elec-trolyte in critical care. the conception of syndrome int~ive care (sic) is exhausted itself and invariable outcomes of sic of multiergan system failure (mosf) confirms that. therefore, an alternative to sic should be advanced. methods: efficlenoy of treatment has been asscsaed in patients with mosf using value of metabolic rate and ability of an organism to cover it by oxygen and substrate supply. oxygen pulse (op) and index of efficacy of oxygen transport (ieto ) was monitored. ~lt~.lntenaive care is considered to be homeostasis-securing therapy (hst) if energostructure deficit is eliminated and necessary for recovery regeneration rate is .restored. op in patients with mosf was . mt-m " , and le,~ and ie'i~ w~ . units in sic. we managed to maintain op of . - . ml.m " and ieto of . - . units in hst. patients from with mosf survived in sic and patients from survived in hst. efficiency of hst appeared to be two times as much as efficiency of sic. cr of homeostasia-se-'uring therapy is advancing. the conception provides restoration of regeneration rate due to effective then in sic elimination of en=gostructure deficit. the conception may be a basis of new technology for treatment of mosf. helen f goode phd, nigel r webster phd. anaesthesia & intensive care, university of aberdeen, ab zd, uk. objectives: xanthine dehydmgenase is converted under conditions of ischemia, reperfusion and endothelial damage to xanthine oxidase, with superoxide anion as a co-product of its catalytic activity. multiorgan dysfunction syndrome is associated with splanchnic vasoconstriction resulting in significant and prolonged gut ischaemia. aggressive volume resuscitation with prompt restoration of blood flow results in reperfusion of the tissue and is likely to cause xanthine oxidase-mediated release of oxygen-derived radicals. this study investigates xanthine oxidase activation and oxygen-derived free radical-mediated damage in such patients. methods: fourteen consecutive patients on itu who met established criteria for septic shock and secondary organ dysfunction were studied. serum xanthine oxidase activity was measured using oxidation of a chromagen in a dual enzyme system and plasma malondialdehyde was measured using a specific spectrephctometdc assay. apache ii scores, blood pressure, svr, cardiac output and day survival were also recorded. biochemical data were compared with results from healthy subjects. results: xanthine oxidase activity was . + . units/i in patients (mean :t: sem) and . + . units/i in controls (p<o. , mann whitney u test), and was highest in the patients who survived (p< . ). malondialdehyde concentrations were elevated ( . • . prnol/i compared with . • . pmol/i in controls, p< . ). xanthine oxidase activity did not relate to apache score or any of the cardiovascular parameters recorded, and was not influenced by the degree of organ dysfunction. conclusions: patients with sepsis and secondary organ dysfunction have xanthine oxidase activation and evidence of free radical damage. the finding that activity was highest in those patients who survived suggests that reperfusion was incomplete in patients who died, with decreased tissue xanthine oxidase ~tash out' into the circulation. influence objective : evaluation of the feasibility of measuring the intragastrie partial pr~sure of carbon dioxide (pco ) using a chemilumin~nt optode and fibre~ptics originally developed for intrav~cular blood gas monitorlng(p ) methods the pco electrode w~ calibrated i~-vitro according to the m~ufaeturers instructions the sensor w~ then stripped of its outer c~ing and threaded down the pile tube of a g~ c enema e (gt) so ha he ens ng portion sat well within the balloon. the modified gt was inserted n~og~trically after induction of anesthesia in ten patients undergoing c~diopuimonary byp~s throughout results: according to the datas of integral rheography % of patients were revealed to have hypodynamic type of blood circulation (cardiac output was decreased on - % and general peripheral resistance increased on - %). it was effective to use complamin (xantinoli nicotinas) in the complex therapy of su~h patients. the dosage was - mg/kg during - days. as a result of such therapy was also the oliguric stage shortening (ac n ).in , % of the cases unsatisfactory datas of central hemodynamic,combined with high (more than cm h ) venous pressure,lung oedema.then nanipruss ( , - , mkg/kg/min) was succesfutly used in complex with routine therapy.in the patients with low cardiac output and low general peripheral resistance mesaton ( , - , mg/kg) and dopamine ( - mkg/kg/min) were effective.change for the worse in the hemodynamics datas, inspire of therapy during - days,was prognostically unfavourable. conclusions:we consider early diagnostic of hemodynamic disorders and adequate correction of them is one of the most important factors, determined the outcome and prognosis in arf patients. obiective: to evaluate the results of renal replacement therapy (rrt) in surgical icu patients with acute renal failure (arf). arf in icu patients is associated with high mortality rates. we investigated the relation between mortality and organ failure in different categories of surgical icu patients receiving renal replacement therapy (rrt) for arf. methods: the records of surgical icu patients with arf requiring rri-(haemodialysis or cavhd) were examined. five different patient categories were defined; .ruptured aneurysm of the abdominal aorta (raaa) n = .elective vascular surgery (evs) n = .abdominal sepsis n - .trauma n = .others ( e.g. malignancy, obstetric emergencies) n- seven organ systems (cns,circulatory,respiratory,hepatic,renal,digestive,haematologic) were scored for possible failure using the mof score. results: mortality was as follows: all patients % , group % , group % , group % , group % , group % . mortality increased with the number of failing organ systems; mortality for all patients with > failing organsysterns was % the only exception being the subgroup of trauma patients where mortality under these circumstances was o% conclusions: mortality in surgical icu patients receiving rrt for arf is high. no significant difference in mortality is found between raaa and evs. mortality increases with the number of failing organ systems. the subgroup trauma patients shows a lower mortality compared to the group as a whole, even with > failing organ systems. to look for the most accurate scoring system to measure the severity of the complications occuring in the early phase ( first day) of kidney transplantation and to asses their prognostic value. methods: in our retrospective study we applied the apache li and the goris scoring system for the kidney recipients who developed multiple organ failure (mof) as a consequence of their pulmonary and. cardiovascular complications following kidney transplantation. we evaluated the recipients the distribution of the women and men ( % ~ % ) was the same as in the kidney recipients. applying the apache ii system most of the patients had their score between and , and the function of , or organs were affected at the time of the onset of mof. the apache ii system gave adequeate information about the disturbance of the function of other organs beside the kidney failure even at the time of the transplantation. the scores and the number of the affected organs correlated with the condition of the patients in the goris scoring system but not as sensitively as in the apache ii scoring system. conclusions: both the goris and the apache ii scoring system can be applied to measure the severity of the multiple organ failure occuring during the early phase of kidney transplantation. however the apache ii system is more suitable to follow not only the stateof the patients at the time of the admission but also the changes occuring in their condition during the complication. v.v.erofeev, v.v.ivleva scientific research institute for general reanimatulogy russian amsci, moscow, russia objectives: the analysis of ssc and results of their treatment in patients following critical states showed the necessity of developing a combined antibacterial therapy. methods: according to the protocol patients ( - years old) with combined trauma and massive hemorrhagy following vast aml traumatic operations were examined. microflora's composition and resistence to up-to-date antibiotics was studied using the anaiyser iems reader by "labsisteme"(finland). general clinical, bacteriological, immunological indices, as weil as the duration of the treatment and recovering rate served as criteria of the combined antibacterial therapy effectiveness. results: it was proved expedient to administer antibiotics in staphylococcus infection in the following combinations: riphampizin with fluoroquinolones; i-ii degeneration, cephalosporins with aminoglycosides; cephalosporins with fluoroquinolones. in case of singling out the exciters of the euterobacteriaceae family, including the pseudomonas aereginosa, -fluoroquinolones combined with modern amynoglycosides; fluuroquinolones with ureidopenicillines; ureidopenicillines with amynoglycosides; amynoglycosides with the ii-iii generation cephalosporins; cephalosporins with fluoroquinolones. in severe ssc caused by combined infection (including anaerobes) clindamicin with modern amynoglycosides was prescribed. conclusion: the combined antibacterial therapy allows: ) to increase the effect on microbic agents and the efficacy of treatment in combined infections; ) to lessen the possibility of the exciters'resistence to antibiotics; ) to prevent the development of superinfection: ) to decrease the doses of medicine and its toxic effect. objectives: two methods of blood volume measurement in a group of critically ill patients were compared to investigate the practical possibilities of a new easy to use method based on carbon monoxide (co) uptake. methods: all patients had multi-organ failure and haemodynamic monitoring with a swan-ganz catheter. mean apache ii score was ( - ). when indicated, patients had blood volume measurements simultaneously based on the techniques of, i) dilution of ~cr labelled red cells, and ii) inhalation of carbon monoxide gas with measurement of the rise of carboxyhaemoglobin produced. the co was administered via a newly designed, ventilator driven, fully closed circle system ensuring co retention and co removal with automatic addition of oxygen to m}ttch patient uptake. a portable computer performed all necessary calculations. results: volumes obtained by co uptake were compared with the "gold standard" radiolabelling method. mean blood volume determined by the co method was ml ( - ml) compared with ml( - ml) with slcr labelled red cells (r= . ). regression analysis produced an intercept at ml. the slope of the regression line was . ( . - . , % confidence limits). discussion: the co method produces volumes in excess of the radiolabelling method. there appears to be a systematic error, and one possible explanation is co binding to substances other than haemoglobin. conclusion: the co method is easier to use than radiolabelling and of the lower cost, since cohb measurement only is required. aceuraey is sufficient for clinical use and our preliminary findings suggest this system will meet the requirements. objectives: this study was conducted to determine the role of nitric oxide (no) in the pathophysiologic alterations and multiple organ damage, and the possible effects of " " " (l-n -monomethyl-l-arglnlne nmma) on hemodynamics and mortality in rats caused by a prolonged hypovolemic insult. methods: a prolonged hemorrhagic shock ( - mmhg for rain) was induced in anesthetized rats followed by adequate resuscitation. l-nmma was administered intravenously at doses of . mg/kg or . mg/kg at the end of resuscitation. results: infusion of . mg/kg l-nmma diminished the fall in mean arterial pressure, significantly increased the cardiac index (ci) and stroke volume (sv), together with remarkable protection from multiple organ damage compared to the controls. the h survival rate was significantly improved from . % in the control group to . % in the treatment group (p< . ). in contrast, the high dose of . mg/kg l-nmma resulted in a strong blood pressure response but a marked reduction in ci and sv concomitant with an increased total peripheral resistance index within the observation period, and caused severe damage to various organs at h after treatment. in addition, marked elevation in both endotoxin and tnf levels were observed in animals subjected to shock insult. conclusions: these results suggest that no induced by hemorrhagic shock in rats is an important mediator for pathophysiologic alterations associating with cardiovascular abnormalities, multiple organ dysfunction, and even lethality. thus, regulation of no generation and use of no inhibitors might provide new aspects in the treatment of hemorrhage related disorders, and the use of l-nmma would be either deleterious or salutary in a dose dependent manner. (hebert, chest- ) . the purpose of this study was to assess the risk factors for hepatic dysfunction in mosf. methods: patients have been hospitalized in our icu from january to may . , ( %) with mosf. among mosf pati~ts, ( %) have had hepatic dysfunction defined according to hebert (bilirubin ~ ttmop , chest ). thirty six of these patients acquired hepatic dysfunction after admission in the icu. these patients were compared with mosf patients without hepatic dysfunction selected blindly. chrorfic diseases, severity scores, eanse of admission, clinico-biologieal and hemodyunrrfic parameters, use of vesopressors, use of hepaiotoxic drugs, use of nutritional support and mortality were compared for hepatic failare and non hepatic failure groups.twenty nine patients had postmortem hepatic histologic examination, results: univaciate analysis: only parameters with p _< . are pre~nted. including these paramet~'rs in a multivariate analysis, anly c~hosis and vascular surgery remain independent risk factors for hepatic dysfunction. in particular, pao /fio , arterial lactate, do were not different between the two groups, some de~'ee of histological abnormalities was found in all liver samples, despite a normal bilirubin level in % of the cases conclusions: in our patients, conu'ary to previous studies, hypoxic and hemody~anfic parameters were not independent risk factors for hepatic dysfantion. this might be due to the inadequacy of the usual biologic definition of hepatic dysfunction as well as to the poor sensitivity of general hamodynamic parameters. critical states of various origin are complicated with the mldtiorgan farm (moi~ oceuzr~ce. due to their and functional features the lungs become the primmy damage target in various critical.states. ard that occurs in such states is associated with pulmonary edema development because of capillary permeability increase mediated by humeral and cenular responses to amag/~ factors exposure. r nmst be emphasized that mediators and effecto~rs of this respo~e affect not only puknonary capillaries, but other organs capiu~es as wellenhancing their permeability. orsans edema is a conmm~ finding at the autopsy of patients died from mof.clinical and radiolosial findings allow to have a diagnosis of pulmonmy edema before ~mi!ar lesions in other organs occm. additionally, there are some techniques that permit quantitative assessment of pulmonary edema flv.id (evlw) volume. in conclusion, we suggest that evlw changes in .dyn~rmcs in patients with mof are considered as a critical state severity measure which reflects indirectly the edema in other organs. objectives: we compared three different dialysis membranes to find out whether or not there were differences between their clearance characteristics on substances such as inuline, creatinine, urea, and phosphate to be eliminated in acute renal failure (arf). moreover, if a loss of clearance did occur we were interested in whether this was due to heparinization and a high production of the thrombine-anti-thrombine-complex (tat). methods: we carried out a randomized controlled study on consecutive critically ill patients presenting with arf, most of them in association with multi-organ failure, to be treated by continuous pump-driven arterio-venous renal replacement therapy on continuous low-dose heparinization. three different types of high-flux filter membranes (f tm [fresenius] , ct tm [baxter] , and filtra tm [hospal]) were assessed. each filter was changed intentionally after a hours" use. together the data of filters were evaluated, each at three different times (immediately after its onset [ hi, after h, and after h). the clearances of creatinine, urea, phosphate, and inuline were measured. results: there were some significant differences in clearance characteristics of inuline, creatinine, urea and phosphate between the filters (p< , ) showing the f tm membrane excelling filtra mand ct tm the more. the loss of inuline clearance ( mi/min/m ) after h, however, was insignificant for all filter types. a continuous low-dose heparinization scheme was applied without any relevant prolongation of the aptt. even lower losses were noted for the clearances of creatinine, urea, and phosphate. we found the tat-producfion increased after h (p< , ), but it did not rise any further. conclusions: as we could demonstrate in our study the clearance data of different types of filter membranes applied during continuous renal replacement therapy do show significant differences. on the other side, no relevant loss of clearance occurs during a hours" period indicating a high efficiency over time. to consider commercial aspects as well it shows that inexpensive conventional filter membranes can successfully be applied even for a longer renal replacement period, if needed. a retrospective study was performed on patients with acute renal failure (arf). we analysed survival in continuous (cd) and intermittent dialysis (hi)). mean age of the patients was years (y), patients ( % ) were < y, patients ( %) were >= y. the incidence of dialysed arf in our mixed intensive care departement is %/admission/y. statistics: fischer's exact test, mann-whitney-u test. efioloev: the contribution sepsis, cardiac failure and aminnglycosidcs was respectively %, % and %. treatment: cavh (cd) or cvvh (cd) was used in patients ( %), hemedialysis (hd) was used in patients ( %). data: mean apache scores were the same for cd and hd ( for both groups), patients treated with continuous dialysis techniques had significantly (p<o, ) more need for ventilation ( % vs %), elevated bilirubin ( % vs %) and a higher vasopresser need ( % vs %@ than patients treated with hemedialysis. there was a trend towards more sepsis ( % vs %; p= . ) mad coaguiation disorders ( % vs %; p= . ) in cd. taere were sign/ficantly more younger patients (< y) treated with cd ( % vs %, p< .os). mean apache score was significantly lower in patiants< y than as patienta>= y ( vs ; p< . ). patients< y had significantly (i}< . ) more coagulation disorders ( % vs %) and elevated bilirabin ( % vs %). there was no significant difference in vasopressur need and ventihatio~ between age groups. outcome:. hi) had a better sr compared to cd ( % vs ~ p< . ). patiants>= y had a comparable sr vs patients< y ( ") */e vs %; p----a.s.). tha global survival rate (sr) was % ( patients). conclusions : diaiysed arf has a well known lowsurvival rate ( %): hc~raedialysed patients had a better survival rate than patients treated with continuous dialysis. this can be explained by the fact that the latter were in a worse condition considering organ failure (more vantilatian, elevated bflirubin and need for vasepressurs), apache score couldn't illustrate that. patient~ y with arf have the same survival rate as patients< y: although patients >=- y have a higher apache score they have less organ faille. the avacbe score is not a good oredictor of survival in p with organ failure. departments of surgery and intensive care, guy's hospital, london, u.g-obiectives: a randomised controlled trial of a management protocol utilising the regular measurement of gastric intramucosal ph (phim) to control the administration of dopexamine. methods: patients admitted to a multidisciplinary teaching hospital intensive care unit (icu) undergoing insertion of a pulmonary artery catheter were managed according to a resuscitation protocol. randomisation was to either the protocol alone or to insertion of a nasogastric tonometer and subsequent management guided by phim. phim < . initiated volume and inotrope resuscitation and, if unsuccessful in elevating phim, dopexamine was commenced. approval was obtained from the hospital ethics committee. results: patients were considered for analysis and the two groups were well matched for age and sex. overall, there was a high hospital mortality of . %. there was no difference in icu or hospital mortality between the two groups (see table) . objectives: to compare cardiac output (co) measurements between continuous termodilution (cco) by thermal wire on pulmonary artery catheter (cco/svo vigilance. baxter critical care), and co measurement using a trans-esophageal doppler (dco) ultrasound system (odm ii, abbott laboratories), in the immediate postoperative period of cardiac surgery. methods: patients undergoing myocardial revascularization were monitored with cco by a swan-ganz catheter and an intra-esophageal dco probe, after induction of anesthesia. exclusion criteria were: aortic valve disfunction, previous valvular surgery esophageal disease, absense of sinus cardiac rhythm, and need of ventricular or intraaortic assistance. hemodynamic parameters, co by both cco and dco, svo . sao , diuresis, pha, and hemoglobin were repeatedly registered during the first hours after surgery, as the patients were kept under sedation and mechanical ventilation. results were compared using the method described by bland and altman. results: measurements of co were obtained, ranging . objectives: a decreased tissue oxygen delivery is responsible for a higher morbi-mortality rate among surgical patients; this diminished oxygen delivery/consumption rate (dojvo ) may origin the lactic acidosis observed in the gastrointestinal tract, reported in patients undergoing hypothermic cardiopulmonary extra corporeal surgery, and can be registered by tonometry as result of the gastric mucose ph. the purpose of this study is to evaluate the reliability of the intramucosal ph (phi) measurement by a nasogastric catheter as indicator of the do /vo > its co> relation to other parameters of do /vo disturbance, and with postoperative complications and clinical course. methods: patients ( male, female) undergoing cardiac surgical procedures were included ( myocardiai revascularizations, valvular substitutions, constrictive pericarditis). mean age was + years, mean weight _+ kg. a nasogastric probe (trie tonometrics) was placed after anesthesia induction; phi values were registered in the postoperative period ( ', ', ", ' and h after surgery end). the corresponding hemodynamic parameters, venous oxygen saturation (svo ), diuresis and arterial ph (pha) were also recorded. results: phi values ranged . to . (mean . ( . ); the mean values of clinical evolution were: extubation time, _+ hr.; discharge from postoperative care unit, - hr.; and hospital total postoperative time, _+ . days. complications registered were: perioperative acute myocardial infarctions, cases of respiratory insufficiency, occlusion of coronary bypass, an ease of hyperamilasemia. all patients with severe complications needing specific treatment showed either a low phi value, or a considerable descent in comparison with the initial register. statistic correlation between low phi and presence of complications was found; the low significance (p > . ) degree may be due to the low population size. conclusions: phi measurement in cardiac surgery patients is a non invasive, uncomplicated method for prediction of doz/vo disturbances, thus reflecting risk of increased major complications, and may precede changes in other usual indicators (svo , pha, cardiac output, ...). work-in-progress with a greater population size may offer more significant results. references: ( ) gutidrrez g: lancet ; : - . ( ) landow i: acta anaesthesiol scand ; : - . the haemoglobin-level (hb) is besides the arterial oxygen saturation and the cardiac index one of the relevant parameters of oxygen supply to the tissue. in contrast to otherwise healthy patients, there is no agreement on tile so-called transfusion-trigger in critically ill patients. in i?ont of this background the question arises, whether and to what extent blood transfusion in critically ill patients improves oxygen supply io tile tissue. this study was performed in critically ill/septic patients in the postoperative period alier an inlcclive/scptie revision operation of the hip or knee joint. on cardiac/seplic reasons monitoring consisted beside other measures of a pulmonary arlery catheter and of an indwelling arterial line li~r measurering/calculating standard haem~dynamic as well as systentic oxygen parameters. the indication for blood transfusion was given by hb together with the cliuical slatus of thc patienl (asa-scorc and multiple organ dysfunction (moi))). statistical analysis w~ks performed by mann-whitney-u-test. by fisher's exact-test and by wii.coxon-test: statistical significance was set with p< . . according tu the pretransfusion value of hb and of lactate (lac) palicnts ;,,'ere divided into groups as follows: a: hb< and b: >sg/dl: i: ac< . and ii: > .smm. in either group blood transfusion results in zt significant increase in hb (a: . _+ . to . + . g/dl; b: .(~ . tt, . + . g/dl; i: . -+ . to . -+ . jdl; i : . -+ . to . + . g/dl). wlailc, however, haemodynamic parameters do not difl)r significantly from each other before and alter blood transfusion, oxygen delivery (do, -ml/min x m-') increases significantly hi either group studied (a: -+ to -+ ; b: + to + ; : -+ to -+ ; i : -+ to -+ ), in contrast oxygen consumption (vo~ -ml/min x m e) does not change significantly in either group (a: i -+ to -+ ; b: -+ to -+ ; i: -+ tu -+ ; : -+ to +_ ); oxygen exlraction ratio decreases. this study in critically ill/septic patients demonstrates, that in this group of patients studied blood transfusion at a base-line-value of > . -+ . g/dl expectedly rises do~, however, it does not improve vo=; even not in septic patients with elevated lac-values. paclitaxel in a new anticancer agent, extract from the bark of the yew tree (taxus brevifolia), employed against breast and ovarian cancers resistant to chemotherapy. it promotes the polymerization of tubuline, and disrupts the normal microtubule dynamics. hematologic toxicity, hypersensitivity reactions (bronchospasm, urticaria and hypotension), and peripheral neuropathy are the main reported toxic effects. cardiac side effects are rare: atrioventricular blocks of higher degree are reported in . % of patients; congestive cardiotoxicity was discussed only in one trial in patients treated with paclitaxel and doxorubicin. we describe the history of a -years-old worn an with a breast cancer, diagnosed in , initial staging t nim , treated with mastectomy, axillary lymphadenectomy, andchemotherapy with a cumulative dose of anthracyclines of mg/m until august . the patient complained of dyspnea and severe hypotension immediately after an intravenous infusion of mg paclitaxel, given over hour for the treatment of bilateral, malignant pleural effusion. at echocardiography die left ventricular ejection fraction was reduced to %. she died days later because of a severe cardiac low output with hepatic and renal failure; an impressive hepatic cytolysis was observed. the post mortem examination confirmed the dilatation of the cardiac cavities, especially of the right ventricle, bilateral pleural fluid, and ascites. the histology was suggestive for a cardiomyopathy secondary to anthracyclines. the electron microscopy revealed a deposition of an unusual pathological pigment in the myocytes; subsarcolemmal deposition or membranous were absent. we hypothesize that paclitaxel was the cause of a major hypersensitivity reaction with shock and severe hepatic cytolysis, worsening the myocardial damage induced by anthracyclines. the possibility that a low doge of paclitaxel could directly increase anthracyclines cardiotoxicity -as decribed in the medical literature -will be discussed. objectives: activated endothelial cells release soluble intercellular adhesion molecule- (sicam- ), vascular cell adhesion molecule- (svcam- ), and e-selectin (selam- ). sicam- , svcam- , selam- , and inflammatory cytokines were determined. methods: sicam- , svcam- , and selam- were determined by elisa. tnf-a, il- , and il- were also measured by elisa. endotoxin was measured by an endotoxin-specific endospecy test after pretreatment of new pea method. results: the sicam- and s vcam-i levels were significantly higher in the septic multiple organ failure (mof) and sepsis groups than in the non-septic mof group. the selam- level was slightly higher in the septic mof group than in the sepsis withut mof group and non-septic mof group. the increases of soluble adhesion molecules were not in agreement with changes of plasma endotoxin level. levels of soluble adhesion molecules were correlated with the levels of plasma tnf-a and il- , but the level of il- . discussion and conclusion: the slcam- and svcam- levels in septic patients closely reflected the severity of the pathophysiological conditon. it was possible that the release of sluble adhesion molecules were not stimulated by plasma endotoxin, but endotoxin in the local infectious region. tnf-c~ and il- also were suggested to be involved in the release of these soluble adhesion molecules. obiectives: cardiopulmonary bypass (cpb) surgery is associated with a systemic inflammatory response attributable to the release of various inflammatory mediators and the activation of complement or coagulofibrinolytic system. in addition, adhesion molecules, such as icam- , elam- , and vcam- , appear to be of central importance in the inflammatory process following cpb surgery. we previously reported the effects of a synthetic protease inhibitor, fut- , reduced release of inflammatory cytokines (tnf, il-lg, il- ), activation of complement (c a, c a) or coagulofibrinolytic system (tat, pic, fpa) and protected platelet function (gpib, gpiib/llla) following cpb surgery. methods: in this study, we analyzed fut- on soluble adhesion molecules following cpb surgery. patients undergoing cpb surgery were divided into two groups, group a consisted of patients who received omg of fut- in priming solution, followed by a continuous infusion at mg/kg/hr during cpb in addition to initial heparin dose of mg/kg. group b, a control group, included patients who were injected with heparin only. the plasma slcam- , selam- , and svcam- concentration was measured by elisa. results: every soluble adhesion molecules decreased during cpb in both groups, and rose after cpb. selam- and slcam- reached their peaks on hours after cpb and on pod respectively in both groups, but they remained lower in group a (selam-i: . + . vs. . • ng/ml, p< . , slcam-i: • vs. • ng/ml, p< . ), svcam- , in both groups, remained lower than preoperative levels, but did much lower in group a. conclusions: fut- reduced adhesion molecules and suggested to be the effect on postoperative organ dysfunction. in the last few :,'ears the conditions of treatment in continuous hemofiltration/hemodiafiltration were discussed controversially. a significant removal of tnf-alpha and il-i could be demonstrated in cvvhd. the aim of our study was to investigate the elimination of tnf-alpha, l- , il- , il- , s-cd- and ifn-gamma in cvvh by measurement in plasma and hemofiltrate of critically ill patients with an acute renal failure. the patients of our study were treated with a continuous veno-venous-hemofiltration (polysulfone-filter, blood flow: - ml/h, filtration rate ml/h). the samples, hemofiltrate and plasma, were taken one hour after the start of treatment. the patients suffered from septic shock ( ), the so called hepatorenal s~aldrome ( ) and a severe pancreatitis ( ). the cytokine concentrations were measured with elisa-method. in contrast to elevated concentrations in plasma for tnf-alpha ( cases), scd ( cases), il- (l case) and il- ( cases), hemofiltrates contained no activities. only il- was removed in significant amounts with even higher levels in hemofiltrate than in plasma. this phenomenon was described so far for tnf-alpha and il- and may be due to the absence of metabolic properties (possibily enz~natic) in hemofiltrate. it can be shown, that tnfalpha, il- , il- could not be eliminated in cvvh with a filtration rate to ml/h. in contrast to findings of other investigators with a higher filtration rate (> ml/h), we found no significant concentrations of tnf-alpha and il in hemofiltrate. we conclude, that for a significant removal of important cytokines higher filtration rates (> ml/h) are necessary. objectives: multiple organ dysfunction syndrome including liver and renal impairment is a fatal complication in patients with the diagnosis of sever sepsis. this study focused to the effects of removing toxic substances from inflamnatory tissue by hemodiafiltration. ~ ethods: eleven patients were admitted to the icu in emergency center and met the criteria of systemic inflammatory response syndrome in association with infection. all patients developed liver and renal dysfunction and were treated by hemodiafiltration with high flux membranes (fb-u:nipro). the hemodiafiltration were performed times using nafamostat mesilate as an anticoagulant in hours with l of substitution fluid (hf-b:fuso). the serdm levels of endotoxin, cytokines, endothelin-i (et-]), human neutrophil elastase ~ -proteinase inhibitor complex (hne-pi), fibronectin (fn), lactate, and amino acids were measured before and after the hemodiafiltration. the hemodiafiltration would be effective to renal dysfunction by reducing endothelin and beneficial to tissue metabolism represented in fisher's ratio, but might be harmful to respiratory function by activating neutropila in patients of severe sepsss. background : intermittent hd may be poorly tolerated in the early phase of arf in hemodynamically unstable patients (pts). this technic may fail to achieve steady state urea low levels in hypercatabolic pts. method : nt = consecutive pts treated with hd; n = consecutive pts treated with cvvhf. hemodynamic unstability is defined by arterial hypotension and requirement of inotropie support despite adequate filling. rate of change in urea (u), ereatinin (cr), k + , ph were computed from a linear regression .analysis of data vs time in each treatment group during the first days of application of the two technics (anova). dally worst values were recorded. results : hd-group : apach% score = _+ ; mean number of organ system failure (osf) = . -+ ; mean blood pressure (mbp) = • mmhg (first day of application of hd). cvvhf-group : apachen score : + ; osf = -+ ; mbp = + mmhg (first day of application of cwhf discussion : during the first days of application of hd/cvvhf, u and cr decreased much more rapidly in the cwhf-group. k* and ph were maintained within normal range in the two groups. initial mbp which was much lower in the cwhf-group significantly improved during the application of cvvhf while mbp remained unchanged in the hd-group. conclusion : despite higher severity of disease in cvvhf group (apachen score, osf, lower initial mbp), we obtained a better performanco with cvvhf regarding the decrease of u and cr and the improvement of mbp. in relation to the different and continuous renal replacement techniques, the continuous venovenous one is the alternative method to continuous arteriovenous for critical patients with acute renal failure (arf). we present you our experience with cvvh in patients with mof. in our intensive care unit (icu) patients with mof were treated with cvvh in the period between january in to march in . the mean (• age of our patient population was , • years, being % male and % female the whole patient population was with mof iust at the moment the technique was accomplished; % was in mechanical ventilation, % needed vasopressor support and % required both of them (mechanical ventilation and vasopressor support) apache ii score mean of the patient population was , ~: , (range - ) and ati of them were with arf oligoanudc. technique: cvvh was accomplished using a single-d~al iumen catheter, ptaced in either a temoral or subclavian vein by the stand ard seld{nger technique. pol{sultone hemofitiers were also used, and the extracerporeal circuit used standard arterial-venous blcod tubing. blood flow and hence oltrafiltration pressure, within the circuit was generated by a roller blood pump. the modulus has a roller pump, a pressure transducer connected in an arterious and venous line, such as an air-transducer which is adapted to a drip-chamber in the return way. the replacement used was a peritoneal dialysis solution. medicine , st. george's hospital medical school, london. england. hepatic sinusoidal endothelium shows a major inflammatory response in porcine sepsis that can be attenuated by the administration of dopexamine hydrochloride. dopexamine is a beta and dopaminergic receptor agonist. the specific beta adrenoceptor antagonist ici has been shown to reduce the protective effects of dopexamine. we investigated the effect of this antagonist on hepatic ultrastructure in porcine sepsis. six pigs ( - kg) divided into groups were anaesthetised and intubated. cardiac output and portal blood flow were measured using standard techniques. the groups were; placebo, (peritonitis induced); blocker, (peritonitis induced and pg/kg ici bolus infused then given hourly). caecal content was aspirated and peritonitis induced. colloid was infused to maintain pawp at - mm hg for eight hours the animals culled, hepatic tissue removed and prepared for electron microscopy. in the placebo group hepatic endothelium was swollen and the sinusoids occluded by wbc. but in the ici blocker group, much of the sinusoidal endothelium was absent and there where large extra sinusoidal spaces among the hepatocytes. an assessment of the two groups showed worse hepatic architecture in the blocker group. the b antagonist blocked any protective effect of endogenous beta adrenoceptor agonist (adrenaline) on hepatic endothelium in porcine sepsis. george's hospital medical school, london. england. dopexamine hydr chloride, a beta and dopaminergic receptor agonist reduces hepatic damage in porcine sepsis. we tested dopexamine's effect on cerebral oedema. the beta adrenoceptor antagonist ici was infused to block any protective effect of dopexamine. nine anaesthetised pigs ( - kg) were randomised into groups; placebo, (peritonitis induced); dopexamine, (peritonitis induced and ~tg/kgdar of dopexamine infused); blocker, (as in dopexamine group but in addition pg/kg ici bolus given then infused at that rate hourly). caecal peritoneum was induced and colloid infused to maintain pawp at - mmhg for eight hours when the animals were culled, cerebral tissue removed, prepared for electron microscopy and digitisation. digitisation of the area of oedema surrounding the blood vessel and expressed as a percentage of the micrograph. . _+ . , dopexamine . + . ", blocker . + . . data expressed as mean + sd. significance p< . . * dopexamine compared to placebo and blocker. in the dopexamine group the area of tissue oedema was significantly lower than either the placebo or blocker groups. there were no significant differences between the placebo or blocker groups. the antagonist completely blocked the protective effect of the drug on cerebral oedema in porcine sepsis. beta adrenoceptor stimulation is protective of cerebral oedema in porcine sepsis. objectives: the hemodynamie~ of hepatic circulation during multiple organ failure (mof) have not been suffleienly studied. we investigated liver hemodynamics in two subgroups of patients with mof, those with either liver or lungs as the main organ of involvement. methods: three groups of patients were created: i) mof-hepatic involvement (mof-hi) ( patients) with bilirubin > . mg/dl and lung injury score < . , it) mof-ards ( patients) with respective values < . and > , iii) patients with head injury with respective values < and < , served as group control. all patients were in haemodynamieally stable state with an oxygen delivery index > ml/min/m prior to measurements. two swan-ganz catheters 'were inserted, one in the hepatic veins and one in pulmonary artery and the following measurements were determined: the hepatic vein free pressure (hvfp), the hepatic vein wedge pressure (hvwp), cvp, paop and co. the gradient of hvwp-hvfp represents liver perfusion pressures. by injecting contrast media at dose of iml/lokg with the balloon inflated to achieve sinusoidai image, the hepatic blood flow (hbf) was concluded by the time in seconds of media removal after balloon deflation. results: the co, cwp and cvp were comparable to all three groups. namely, for mof-hi, mof-ards and control groups the mean (+sd) value of co was . _+ . vs . _+ . (ns) and . _+ . respectively, of the paop was . +_ . vs +: (ns) and . + . respectively and of the cvp was .+. . vs . + . (ns) and . respectively. in contrast the two mof groups were different after the cut-offinclusion criteria ie the mean (+sd) value for bilirubin was . + . vs . + . ( < . ) and . _+ . respectively and lung injury score was . objectives: oxygen delivery (do ) and oxygen consumption (vo ) are increasingly monitored parameters in the icu. there still remain controversies about an oxygen supply dependency in critical illness particularly with respect to vo determination by either indirect calorimetry (vo m) or tick calculation (vo c). the purpose of this study was to investigate the changes in vo m and vo c following do increase. methods: the relatives of critically ill patients (mean age years, mean apache ii , mean mof-score ) gave their written informed consent to participate in this institutionally approved, prospective study. do was increased by fluid loading (hydroxyethylstarch %: mean volmne ml, mean duration of infusion min) and catecholamine support (dobutamine: mean dose , ~g/kg/min). changes in vo m and v c were recorded sinmltaneously before, during and following interventions. calorimetry was obtained with the metabolic monitor integrated in the ventilator (puritan bennett, carlsbad, ca adaptive endocrine response of organism to septic shock consisting in activation of the production of adrenal hormons, renin -angiotensin -aldosterone system (raas) and other hormonal systems has an influence over microvascular changes in these states and for development of multiple organ failure (mof). in patients with peritonitis of different origins ( nonsurvivors and survivors) were followed the changes in cortisol level and raas by radioimmunological methods and many variables for evaluation of respiratory, renal, hepatic function, coagulation etc. as a signs of mof. it was observed significant increase of the level of cortisol ( +_ , nmol/ i), aldosterone ( , • , nmol/i). by factorial statistical analysis we found significantly high correlations between hormonal changes and respiratory function (for example r=- , , p < , between cortisol and pao ; r = , , p < , between cortisol and d (a-v) ; olso renin -cao r=- , , p < , , renin d ~,vl o r = , , p < , ). such significant correlations was found and for raas with respiratory, renal function, byproducts of arachidonic acid thromboxan b and p fla, soluble fibrine degradation products etc. these correlations between the degree of endocrine changes and multiple organ failure in patients with septic shock produced by peritonitis suggest that their effects upon peripheral vascular resistance and constriction of the splanchnic, splenic, renal and other organ vasculatures are not always with physiologic expediency and there are perhaps the possibilities of therapeutic influence. intredu~on : dopexamlne has previously been shown to control hyperkalaemia ia patients with acdto renal failure (arf), however effects on the subsequent course of art are undomunente~ ob_iectlv~ : to evaluate clinical progress in patients with acute renal failure (arf) in an intensive care unit (icu) with regard to biochemical control, need for -and time to -dialysis, and outcome in patients receiving dopexamine. m~ods : consecutive patients meeting standard criteria for diagnosis of arf were included in the study. full cardiovas~dar, biechemical and intervention/outcome details were recorded. dopex.~min~ was infilsed at a dose of pg/kg/min in conjunction with a regimen of inotropir support and blood volume optimization. resn]~ : following the intzoduetion of dopc',~mine ilrinr vohlmes increased slightly over the next hrs fzom + ml/ hrs to + ml/ hrs (ns). data expres,uxl as mean + sem. three patients ( %) became polyuric with urine output > ml/hr within days and did not need dialysis. in the remaining patients the time to dialysis (to correct acid-base deficits or volume overload) was . + . days. serum potassium levels were well controlled. day or immediate pre-dialysis levels were . + . mmol/l compared with pre-lreatment . + . mmol/l overall mortality in this series was / ( %). duration of acute dialysis in survivors with renal recovery was . +_ . days. patients ( %) progressed into chronic renal failure and needed continuing renal replacement therapy. no adverse cardiovascular altects were seen at this low dopoxami~ dose although its competitive inhibition to adrenergic reuptake mechanisms meant that doses of pressor agents could often be reduced. : dopcx:~minr nsed in conjunction with inotropic support and blood volume oplimitntion, can safely postpone, or even avoid, the necessity for acute haemodialysis in icu patients. no evidence of tachyphylaxis to the effect on serum potassium levels was seen over the duration of the study. hen'era m., suarez g., dagn d., varela a., ramos j., garoia jm, aragdm c, jurado l, medina a. icu. hospital regional. malaga. spain. objective: to evaluate the haemodinamic tolerance to the veno-venous continuous hemefiltration (vvchf) system in patients with systemic inflammatory response sindrome (sirs), and the possible beneficial effect of this technique on the haemodinamics in these patients. material: patient admitted to the icu, with diagnosis of sirs and monitored with a pulmonary artery catheter at the beginning of wchf. we performed a complete haemodinamic study to all these patients (cardiac output, vascular resistanoss, ph and co in arterial and mixed venous blood samples, saturation of pulmonary mixed venous blood, do and vo calculations and temperature) and determined the respiratory mechanics (compliance and pao /fie relatinship) before starting the procedure, after minutes operating with the ultraflltrate branch closed (without filtered fluid production), afler and minutes of zero fluid balance bemofiltration and after minutes of filtration with negative balanos adjusted to the patients conditions. for the statistical analisis we have performed the anova test over the mentioned variables. results: we have not detected statisticaly significant differences of the analyzed variables before the beginning after operating the pun'@ for minutes without filtered fluid production and after minutes of zero fluid balance hf. only temperature shows a meaningful decrease in time. objectives: among many organs, playing the important role in pathogenesis of multiple organ failure, the particular place is taken by the intestine. ~ethods: the study was carried out in dogs !~n"~h pi was modelled by severe operative trauma (ot). the dcm was estimated by the indices values of work time (wt), contraction frequency (cf), mean amplitude of contractions (~ac) and motility index (mi) measured by method of tensography. "sl", created on the basis of sorbit and sodium lactate ( mosm/l), was injected in the dose of .o ml/ kg into v. cephalica antebrachii after hrs of ot. the results of the present study are the evidence of "sl" stimulative action on dcm and are experimental ground for "sl" using in complex therapy of pi in clinic. with splanchnic venous blood pc p.f. laterre p. goffette, j.p. fauville, a. poncelet, p. loneux, m.s. reynaert. intensive care unit, st. luc univ. hospital, brussels, belgium. determination of gastric intramucosal ph (phi) by gastric tonometry using the henderson-hasselback equation is expected to allow the detection of splanchnic ischemia in critically ill patients. because of bicarbonate concentration and acidbase balance influences on the calculation of phi, it has been proposed to use arterio-gastric pco,_ gradient [p(gast-a)co,] to assess splanchnic perfusion. htpothesis : pcoz in the gastric mucosa is in equilibrium with intraluminal co z and with co, in the blood leaving the stomach (mesenteric and portal blood). objective: mesure pco; and ph in portal vein blood and compare its value with pco and phi obtained simultaneously by gastric tonometry. material and method : in a patient ( y.), a fiberoptic catheter (baxter r) was positionned in the portal vein after transhepatic stent shunt repermeabilisation. hemodynamic parameters, do, (vigilance n baxter), gastric co and phi (tonometrics baxter) and portal blood gas were determined at regular intervals. results : sets of data were obtained and are expressed in mean + sd. gastric pco z was , + compared to , + . mmhg for portal pco . phi was . +._ , vs . +._o, for portal ph. no correlation was found for these parameters. p (gast-a) c was . + mm hg vs + . mm hg for p (portal-a) coz (no correlation). there was a good correlation between do e and p (portal-a) co z (r = , ) [figure] but no correlation with p (gast-a) c . obiectives: desaturation is a common finding during haemodialysis (hd). pulmonary oedema might be one cause for impaired gas exchange ( ). the aim of this study was to quantitate the amount of extravascular lung water (evlw) and gasexchange in chronic renal failure patients during and after a regular hemodialysis session. methods: chronic renal failure patients without symptoms or diagnosis of cardiac or respiratory disease were studied at the start (i), at the end (ii) and two hours after (iii) a regular bicarbonate hemodialysis session. the double-indicator dilution method, with indocyanine green and the stable isotope h as tracers, was used to measure evlw ( ). arterial bloodgases and endtidal co were registered. evlw data was compared to a group of renal healthy patients ( ). dcp n evlw, ml -pao , mmhg h~o +, nmol/l control group - -- l _+ "* -+ _+ crfgroup ii -+ ~ +- ns -+ "(" iii +- t _+ ns -+ t ** p < . dcp i from dcp , t p < . dcp li or i from dcp i, :~ p < . dcp ii from dcp i the evlw at the start of dialysis was larger in the crf group than in the control group. the evlw decreased significantly to a level not different from the control group in response to the reduction in weight after hd. pao~ was normal at the start of hd and showed a nun-signficant reduction after hd. paco ( . + . kpa) and etco ( . + . kpa) were unchanged while h o+ decreased and bicarbonate increased significantly. conclusions: the elevated level of evlw at the start of hd did not impair gasexchange. the decrease in evlw did not inhibit the decrease in pao . the reduction in h + followed by a fall in alveolar vantilation is the most plausible cause for the decrease in pao in bicarbonate dialysis. . prezant lung ; : - . . wallin j appl physio ; : - . a. dona~ d. battis& l col~ r danieli, d. achill~ l viglienz;~ c. giov-anaini, p. piaropao~ oblectives: to verify if intraoperative modifications of mtramucosal gastric ph (phi) below the normal lowest value . , can be predictive for important complications, as perforation, sepsis, mof or death. methocls: we have considered patients who andenvent major abdominal surgery. all patients received the same drugs in pre-anaesthasia, the same type of anaesthesia (balanced anaesthesia) and the same treatment with h -bloekers. after the induction of anaesthesia a gastric tonometer was positioned and a catheter was positioned in the radial artery. during the operation, every minutes, the following parameters were measured at the same time: phi, arterial ph (pha), blood lactate, mean arterial pressure. in follow up we considered death and complications happened during the hospital stay, in relation to intraoperative phi falls below . . results: among the patients, had a drop of phi below . during surgery. in three of them this fall was a single episode and happened within the first hour after the begiluting of the operation. after that phi rose to nomml values until the end of the operation these patients had a normal post-operative period, without complications, the other patients had a fall of phi during the demolitive manoeuvres. two paticots of them died. the first had a lowest phi= . and the second . . the first one ~zs operated on for hepatic istiecitoma, suffered a complete del'dseenco of the surgical wound on the th day after operation and died on the th day, the second one was operated on for a hepatic carcinoma had an intraoperative haemorrhage and died ~vo hours after the end of the operation. the other patients with a fall of phi had a lowest phi= . . . . . . . respectively.the first patient,operated onfor sigmoid carcinoma, underwent on a second operation for a transmural necrosis of the colic segment on the th day; the second one, operated for carcinoma of the right colon, had a cardiac ischelnia on the th pest-operative day and a dehiscence of the surgical wound on the th day: the third one, operated on for a sigmoid carcinoma, had melena in h post~ operative da b, and finally the fonrth patient, operated on for carcinoma of the tight colon, suffered a fistula of the surgical enteral anastomosis.all these patients were discharged alive from the hospital. the other patients, who had not reductions of phi ditring the operation, had a normal pest-operative period, without complications. conclusion: phi was able to predict the arising of some complications, probably due to intraoperative ischemic events. we can say that gastric tenometry, for its low invasivi.ty, can be included among the intraoperative monitoring in patients that tmdenvent on major abdominal surgery. (ttd),t"ea~rrerj.~ of hours duraticn. all l:atients nm.'-~ms_(~lly va~ ated in eantrol wcde ard_ la':'ad a a,~m--ganz catheter, with optic fibers for contirums mmsuremmt of svo mic studies were performed, c~e before the hegir~ of hd, c~e rain after the ~, ~ne at the middle, ~ne rain before lhe erd ard one rain after the erd of hd. paired t test ~as used far slatistical eval~ti~n. results: daring i~d there was a significant'reductton (p<o.~) of: c~tr~ venc~s eres~.re ( ve)emm to . ~ ~, ) ~arn'~w capil~ ~f~e pressure (i<~p) fr~n to ~mg hg, ) i~ from to . mmg hg. ~here was also a less pmnameed bat also-slatistics/ly significant r~ucticn (p<o. ) of: i) s~o~ from to patients undergoing surgical reconstruction of the aorta due to anenrysms of its abdominal part may develop transient and sometimes sustained episodes of dysoxia despite the conventional appeoreneas of being adequately resuscitated. indirect masurement of gastric mussel ph (phi) is being widely evaluated as a minimally invasive and sensitive means of assessing the adequaey of tissue oxygenation in these circumstances. the duration end degree of gastric intramucosal acidosis are related to the risk developing injured gastrointestinal tract end its putative consequences, i.e. translceation, cytokine release, organ dysfunction end failure, sepsis end death. measurement of phi is obtained indirectly by measuring pc in the lumen of the stomach with a silicone balloon tonometer (tonometrics, inc., worcester, ma, usa) and the bicarbonate concentration in arlz,,rial blood, end substituting these two values in the henderson-hasselbalch equation. objectives: to evaluate whether perioperative phi changes are predictive of complications end outcome end how they compare to standard haemodyuamic and oxygen trensport parameters. methods: patients ( males and female) urtderwent surgical replacement of abdominal aortic anonrysm with the aortic simple or bifurcated grail. patients were operated on eleetively, had an emergency surgery for raptured enentysm. haemodynamic, arterial ph and bicarbonate concentration measurements were taken before and after induction of anaesthesia, after cross-clamping and declamping of the aorta end at admission to itu. phi calculations ware done at the sime time except before induction, as tonometers were inserted after patient had been asleep. we used pulmonary utilizing a computer billing survey as well as hospital service transfer data from a month period ( / - / ), all obstetric patients that were transferred to the med/surg icu were identified, as well as their diagnoses and procedures. twenty-eight obstetric patients were transferred to the med/surg icu, compared to , deliveries occurring during that time period for a . % rate. the following procedural indications for icu transfer occured: insertions of endotracheal tubes, arterial catheterizations, temporary tracheostumy, and venous catheter. the following medical diagnoses were found: hypertensive/eclamptic episodes, hypotensive episodes, pulmonary embolism, mitral stenosis, and coagulation deficiency. there were four episodes of respiratory failure ( %). the historical rates of respiratory failure from the medical literature seen at a university hospital ore higher than those at our community hospital despite comparable icu transfer rates for critically ill ob patients. objectives: to evaluate the usefulness of several isss used in order to predict risk of death (rd). design: prospective data collection for months in a multidisciplinary -bed icu. methods: data from consecutive patients admitted to the icu from / / to / / were studied. all the necessary informations were stored in a computer using special software for every day computation of three isss (saps, saps ii, apache ii) and of the rd predicted by saps ii, mpm , mpm and odin. data from days of hospitalisation were used for comparative evaluation of isss and rd, while sensitivity and specificity in death prediction (cut off point of rd = %) were evaluated from data collected during the day of admission (saps ii, rpm , odin) or after hrs (mpm ). agreement between rd predictions was evaluated by bland and altman analysis. results:our results were the following: objectives: a) to create and validate software specially designed for the collection of demographic data and estimation of illness severity and predicted rd and b) to provide data necessary for the evaluation of the effectiveness and the efficiency of our icu. design: prospective data collection in a multidisciplinary -bed icu. methods: prospective data on specific physiological variables, selected demographic characteristics, comorbidity and the reason for icu admission were recorded every day for consecutive patients admitted to the icu from / / to / / . data were entered into a portable computer using specially designed software allowing the calculation of usual illness severity scoring systems (isss) and the corresponding predicted rd. results: we studied men and women, with a mean age of years. we conclude that, although observed mortality was higher than predicted rd, this difference was not statistically significant. the fact that the sd of predicted rd was too large, limits the usefulness of isss predictions for a comparative evaluation of different icus. objectives :the importance of objective evaluation of patients prognosis at their admission to an emergency department is clear. the aim of this study was to appreciate the prognosis of patients with the simplified acute physiology score ii (saps ii), and correlate this score to the prognosis and orientations of patients. methods : the score which is a reduced form of saps ii was calculated from clinical variables and laboratory items for patients. sensibili}y (se), specificity (sp) and accuracy (ac) were calculated to evaluate the performances of saps i . thresholds were calculated with the youden's test (se+sp- ). results : patients were admitted in the intensive care unit (icu)and patients in medical units; patients died. the saps ii was higher in patients which died than the survivors ( , + , vs. , + , , p< , ; respectively), the best threshold was , se was de %, sp was %, the ac was / . thus for patients admitted in icu than in medical unit ( , : : , vs. , + , , p< , ; respectively); the best threshold was , se was %, the sp was %, the ac was %. conclusions : these preliminary results suggest that saps ii can be used to determine the short term prognosis and the destination of patients seen in an emergency department. design: data was collected on all admissions over a six month period. all patients had a screening hiv elisa test. confirmatory ifa tests, western blot and flow cytometry were performed on hiv positive patients. the institutional ethics committee considered the major clinical impact of the study sufficient cause to waive the patient's right to informed consent, icu staff and patients were blinded to hiv results. following discharge patients were given the option of being advised of their hiv status. setting: the study was conducted in a bedded surgical icu at a tertiary care teaching hospital. patients, participants: all patients admitted during the period in question were included. interventions: "all patients were treated according to standard icu protocols. there were no interventions. results: most patients were admitted following trauma. there were no patients with aids, hiv positive and hiv negative patients. with respect to the latter two groups, the mean age and sex distribution was similar, there was a significant difference in organ failure ( % versus %, p < . ) and septic shock ( % versus %, p , : ) but no difference in icu/hosp[tal mortality or duration of icu/hospital stay. flow cytometry changes in hiv positive patients were consistent with the quiescent phase of hiv infection. conclusion: while morbidity is higher in hiv positive patients, there is no difference in mortality. hiv status should, therefore, not be an exclusion criterion for icu admission in this patient population. , n= ) and january-march (period , n= ) were studied. interventions: a resident micu team led by a trained intensivist took over the medical care from the primary physicians when the patients were admitted to the micu. the intensivist also vetted micu admissions and decided on micu discharge. in addition, there was a resident respiratory therapist to attend to vendlatory care during office hours. after office hours, the care of the micu was delegated to the on-call team on a rotational basis among the medical departments. results: the patients in the two groups were similar with respect to age, sex, race, source of admission and apache scores. the icu and hospital mortalities decreased from . % to . % (p=ns) and . % and . % (p=ns) respectively in period . the mean icu and hospital stay was also decreased from . __+ . to . + . days (p=ns) and . __+ to . • . days (p=ns) respectively. the improved micu length of stay for survivors in period was statistically significant ( . • . days vs . • . days = . ). there was no significant differences in the vse of peritoneal dialysis ( . % vs . %) and mechanical ventilation ( . % vs . %). however, utilisation of intra-arterial lines and pulmonary artery catheters increased from % in both to . % and . % respectively in period . conclusion: the duration of critical illness was reduced in period . hence, we believe that the closed |cu which was staffed by an intensivist and had the services of a respiratory therapist was beneficial for the care of the critically ill. the intensivist, was also more likely to utilise invasive monitoring. in the absence of liver transplantation, intensive care for patients with acute hepatic decompensation arising from alcoholic liver disease (ald) is indicated only for those who are likely to benefit from conventional means of organ support. we have attempted to elucidate potential risk factors and the efficacy of organ support in relation both to hospital outcome and icu costs so as to allow the earlier identification of relatively futile intensive care in this group of patients. methods: over a period of one year, ald patients ( males; females; median age years, median apache ii score ; hospital mortality %)with severe complications ( [ %] with bleeding oesophageal varices, [ %] with hepatorenel failure, [ %] with respiratory failure, [ %] with septic shock, and [ %] others) were studied in our liver icu. organ system failure, daily therapeutic interventions and icu costs were assessed using a patient management system (riyadh intensive care program). results: whilst survivors had a significantly lower admission apache ii score compared with non survivors (medians and , p< . ), or more organ systems in failure in the first hours of icu admission was associated with a % ( ) mortality. mortality was also related to child's grading and the numbers of organs supported. of the patients who received more than one form of organ support only one survived. organs supported mortality a (n= ) % none / ; % b (n= ) . % one / ; % c (n= ) . % two or more / ; % the icu costs for the patients were only s but s ( %, patient days) was utilised by the patients who died, giving an effective cost per survivor (ecps) of s . although the cost of treating the patients with or more organ systems in failure on the day of admission was s ( % of the budget) the ecps was s . conclusion patients with ald who have or more organ systems in failure early in their icu course have a poor prognosis and in the absence of liver transplantation, traditional strategies of organ support are ineffective. this begs the question whether such costly care is appropriate as although the ecps in this sub group is comparable with other patient groups with multiple organ failure, their outcome is considerably worse. objectives: to evaluate the economic impact to the healthcare sector of using dopexamine hydrochloride infusions to deliberately increase perioperative oxygen delivery in high-risk patients. methods: effectiveness data were obtained from a controlled clinical trial in which surgical patients were randomly assigned to either a control group (n = to receive best standard optimal fluid resuscitation perioperatively, or a protocol group (n = ) to receive, in addition, an infusion of dopexamine hydrochloride to increase the perioperative oxygen delivery index to greater than ml/min per square metre. resources consumed by patients during treatment were recorded prospectively and costs of resources at / prices were obtained retrospectively. cost-effectiveness ratios were estimated by dividing the total cost for each treatment group by the total number of patients in each group and by the clinical outcome of mortality days postoperatively. results: in the protocol group, there was a % reduction in mortality days postoperatively (p = . ), a significant reduction in the number of complications (p = . ) and a reduction in length of stay in the icu and surgical ward. the average cost per protocol patient to achieve a . % survival rate days postoperatively was s , , resulting in an average cost of s , per surviving protocol patient. the average cost of a control patient was s , to achieve a . % survival rate days postoperatively, resulting in an average cost of s , per surviving control patient. the use of dopexamine to deliberately increase oxygen delivery perioperatively generated a cost saving to the nhs of s , per patient together with a % reduction in mortality at days postoperatively. hence, the cost per surviving protocol patient was s . less than the cost of a surviving control patient. conclusion: the additional cost of dopexamine in protocol patients was offset by a lower incidence of complications and a shorter stay in the icu and on the surgical ward. hence, increasing oxygen delivery perioperatively in high-risk surgical patients saves both money and lives. objective: although king's college criteria are widely used, indication and timing for liver transplantation in acute liver failure is still far from certain. long-latency sensory evoked potentials -a proven measure of metabolic brain dysfunction (grimm et al. lancet ; : - ; madl et al. lancet ; : - ) -are markedly affected in patients with acute liver failure. serial sensory evoked potential recording should provide very useful information for the management of patients with acute liver failure. methods: recordings of standard bilateral sensory evoked potentials were performed in patients with acute liver failure. findings were focused on cortical n peak -a stable negative deflection occuring • ms after median nerve stimulation in healthy subjects, which can be recorded by skull electrodes over the sensory cortex. results: nine patients recovered spontaneously, patients were referred to emergency liver transplantation, and patients died. in all patients who recovered spontaneously, n peak was detectable between and ms. all patients who were referred to liver transplantation and of patients who died, developed a loss of a prior detectable n peak during the course of acute liver failure. objective: to determine high risk arid low risk patient groups in a medical intensive care unit regarding short term and long term outcome. methods: data on all admissions of the year were collected prospectively. according to their mode of admission patients were classified as admissions by the physician staffed ambulance service (as), admissions through the emergency department (ed)i referrals from non-intensive care wards (ni), and secondary referrals from other hospitals (oth). outcome was assessed in terms of in-unit mortality, in-hospital mortality, and long time mortality. patient groups were compared using the \ -test. life table analysis were per{ormed by kaplan-meier method comparing patient groups by log-rank test. the software spss for windows . . was used for statistical calculations. results: in-unit mortality: / patients ( . %) --oth . % > as . %> ni . % > ed . %; p = . . in-hospital mortality: / patients ( . %) --oth . % > ni . % > as . % > ed . %; p = , . mean survival time in days after discharge: as < ni < oth < ed ; p = . . conclusions: despite an excess in-unit mortality of secondary referrals from other hospitals the iongtime course of this special patient group is not different to others. solsuam, j, marrugat*, g, mirs, j, nolla, a, vazqu~z-sanchez, l alvamz, ~ioio s xndioina i~siw. ir~itate l(~icipal da l~sti~isn l~di~*, ~ospits dal objective: to study the influence of modifiable variables (complications derived from therapeutic activities) on the prognosis of ~atients admitted to the icu indapemently on thn severity of illnsss. patients am methods: between january asd ]lay data from , patients over years of aqe who retained in the icu for mare than hours ~ere pr~pectively regiatered. a cohort st~ly with follo~-~ nf patients durin~ ~eir stey in the hospital was deni~.el in all patients, reasons for a~issien, principal diagnosis sad severity of illn~s moasared by the saps scare vare recorded. fastens affecting patients' outcome that my be proventsd or modified included technical :omplisafioss, heapital-acqnired infections and in~pro~riate therapeutic decisions. a logistic regression model was used to assess the relative risk (l~} for in-heapital mortality adjusted for each variable. results: ic~ mortality ~s . % and in-hospitul mortality . %. patients who died showed a higher spas score then survivors ( , ~ i ,i). after adjusting hy severity of illness, co~;licetices that statistically increased the risk of in-hospital death were septic shock secomery to hoapitul-acqdired infection ( ~ . ; % el, . to . ), pmo~othor~x related to mocasnical ventilation (@ . ; % cl, . to . ) and delay in the insertion of a fln~-quidod catheter (ii~ . ; % ic, i.i to . ). col~lusien: registration of complicaticas derived from therapeutic activities is a valuable tool far quality central in the icu. g, ~i~ , j.l mle~ma, j, ~amqat*, j..~lla, a, vazquez-saltemz, f, alvamz , servioia de nndicina l~siu. i~stitutu ~icipal de ln~sti~acidn ~ i:a*, hospital dsl objective: to dstsr~ine the incidence of self-extebatien and its effect on ~ortality. patients and ]~etheds: betveen january and april , all i~tiente in whom selfextubatien w~s registered were inclnded in a prospective study. patients were divided into @nee who needed r~intabatinn within hoers and those who did not. in all patients, dsmoqraphie and ciinical data were recorded as well as icii mortality, in-hoapital mrtality and severity of illness according to saps score. eta were analyzed usi~ the cbj-square test for cathgorical verinbls, the analysis of varianc~ (anva) for aontinuc~ ~ria~les and a leqi tic regression anal~is to estimate the relative risk (iiii) for mortality as result of celt-nxtt~ation after adjusting for severity of illness. results: a total of intnmtsd patients amre stndied. self-extu~atien occurred in ( . %) patients and . % required reintuhot~pn. when a co,arise was made between patients who did not required reint@atinn and patien~.s who did, statistically significant differences in eqe ( . v_s . years, p = .~ ), ~verity of illness ( . ~ . spas score, p = . ), dia~isstia category ( s. % v_s . % of patients with res~iratury conditiono, p = , } and mean length of stay ( , ~ , days~ p = . ) were fo~m, a~ter ad~sti~ for severity, patients with self-ext@atinn who did not reqnired reintalatien showed a . iir for mortality ( % ci, .i to . ) as co~arod with patients in when self-ext@ation did mot occur. conclnsien: self-~extamtice that does not require reint@ation is associated with a isamr in-hospital natality probably dt~ to a prolonged period of weaming. patients' admissions to ices am often delayed doe to the shortage of beds available. @ile amaltieq icu admission, these patients are treated in observation nits of @e emergency services which bare ,either tile structure nor the trained ~reomenl that are available in leb~. objective: to daterdno the effect on the patient's proqusis of a delay in tile admission to the icu when criteria for icij admission are fulfilled. ~terials and methods: between jme am l?ece~ber all patients who fulfilled criteria to be almittod to the ic who for waste~r reason retained in tile observation unit for more than hours were included in a prospective stedy. in all patients, des~raphic end clinical dabs amre recorded as well as severity of illness aencrdi~j to saps score. a cesucontrol dasi~ was eend with a total ss~ln of , patients who suffered no delay is admission to icii over a period of years. data wen analyzed using the chl.-squ~re test (to aeons the association hetwenn in-patienty mortality end categorical vari~lns) and a maltipln logistic reqression model to sstimta odds ratio for) for in-hospital mortality as result of delay in icy admission as compared with early ad~issi| after adjusting for severity of illness end use of assisted mchenical ventilation. ~ &ults: a total of patients remained in the observation nit for more than hours with a del w in igd admission of . _+ . hoers. assisted mechanical ventilation was requited in % of patients and only monitericatien in %. itsse patients were cspared with ntients from the tet~l sample ratchod by age, sp~ score and rennoss of admission. in-hospital mortality for cases warn % as compared with . % for controls (p = s). after adjamtilg fen spas, age and mobamioal ventihtien, no statistically significant differences between both ~renpa were foam, altho~b there was a tendency towards a higher mortality amen@ patients with delay in icu admission (or = . ; % ci, , to , ). conclnnien: ~se findings suggest that prognosis of critically-ill patients is no worse as a result of admission to the loll being deln~d for borers. all data appropriate for the calculation of the apache ii score (aps) together wi'th other specific cardiac details relevant to these .patients were collected daily, verified and enter~ into a computer database. results: patients were studied. six patients died and five of thee underwent cardiac surgery. the mean aps was for survivors and t for non-survivors (p < . ). the mortality ratio was . and the major markers of mortality were apache ![ score, presence of chronic ill health, mean duration of ventiiation, mean length of icu stay and need for emergen~ surgery. sixteen percent ( ) of icu bed days were occupied by % of patients (non-sarvivors) which resulted in cancellation of cardiac sot#cat sessions in momhs. conclusions: this study concludes that apache t could be used as an audit tool in a cardiac surgical icu and demonstrates the severe compromis~don of cardiac surgical throughput by a few non-survivors, organ to determine the number of organ failure free days (offd) in a cohort of survivors and non-survivors with sepsis syndrome followed over a day period. ) to determine sample size requirements for clinical trials utilizing a increase in the number of organ failure free days as the primary outcome as opposed to mortality. methods: beginning december through to april , patients who met inclusion criteria of the "cardiopulmonary effects of ibuprofen in sepsis syndrome" and who did not have hiv/aids. brain death or moribund state were prospectively identified. presence or absence of failure of organ systems (pulmonary, cvs, renal, hepatic, gi, hematologic, & cns) was recorded daily until death or until days. a score of one was assigned to each organ system free of organ failure in patients still alive, ie, maximum daily off score= , maximum day off scorn= , sample size estimations were performed for variable detectable differences in off scores (delta). alpha was set at . (two-sided), with n/group = [(z a +z b ) o conclusions: a clinically relevant increase in off days may be detected with as small a sample size as to patients per group. this represents a significantly smaller sample size than needed to detect a change in mortality from % to % ( % relative risk reduction) where the n/group= . scoring patients in this manner prevents a lethal inte~entien from providing an improved organ failure score. in addition, an intervention that prolongs survival must also provide greater organ failure free days in order to be counted by this scoring method. survival as an outcome provides no information about the quality of that survival. off days provides a measurement of burden of illness. interventions which lessens this burden may be just as valuable as those that decrease mortality by providing a measure of the quality of survival and by decreasing costs of care. they may also prove to be an accurate surrogate marker of mortality. the advantage of this approach is that the event rote is much higher and sample size requirements are subsequently smaller. this would mean that clinical trials can be completed faster and at lower cost. outcomes such as mortality could then be assessed at a later date utilizing recta-analysis. we suggest that the use of off days is a valid outcome measure that may be utilized in clihieal trials of sepsis syndrome. the icu is perceived by many as being a stressful environment for both patients and staff. stress has been defined in three ways: a stimulus producing a particular response; the physiological and psychological response to a stimulus; an interaction butwom an individual and their environment. stress is currently thought to be a dynamic system of stimulus and. response which takes into account the individual's perception of the stimulus and their ability to respond effectively. stress may, therefore, be positive and allow personal development but an individual unable to respond effectively to a stimulus will experience negative effects or strain. critical illness is an intense stimulus to which the body needs to respond effectively. physiological responses are vital and most of intensive care involves supporting these. alternatively, blocking them, for instance with atom(date, increases mortality. psyehological responses are also vital but often poorly appreciated because of communication problems. many of the problems patients experience in an icu are evidence of psychological strain. this can be exhibited in various ways, for instance, anxiety, depression, passivity and confusion. dealing with critically ill patients is perceived as stressful. we recently studied occupational stress in our icu. most aspects of intensive care were not generally perceived as stressful indicating a self-selectien of icu staff. the most stressful aspects of icu work for nursing staff were the structure of the organization and career opportunities. medical and nursing staff had different stressors and different coping strategies. support for occupational stress, therefore, should focus on the individual and concentrate on information and communication. atmosphere, and especially at intensive care units, we face up to daily decision making. in most cases these are taken on the basis of personal opinion and the processing of a very limited amount of information. rising need to optimize the results of medical attendance becomes necessary to set structured system of d@cision making in which ethical basis have a sp@dial significance in view of next considerations: -we live into a pluralist society in which the importance of values is different. -most persons consider health as the first value only in the event of illness. -medical resources available are limited, whereas medical, attendance demand from population increases in a way many people consider it unlimited. in consequence, it becomes necessary to set up priorities in patients treatment. ehtical basis that rule decision making are essentially these ones: i. beneficence: to provide the patient that is being treated the highest profit. . non maleficence: it is our first duty to avoid hurting or damaging the patient."primum non nocere" . autonomy: in every particular medical attendance, the patient has ability to decide by himself. . justice: as equity: to provide the same treatment for those who have the same pathology, ignoring another factors such as age, sex or race. severe application of these principles can cause difficulty, which resolution requires a systematization of decision making. ( - ) . the lenght of stay between survivors and non survivors didn "t show statistical significance (p = . ). the mean aiii score when considering all admissions was , ( - ) . the initial score between survivors and non survivors showed ststistical difference ( . vs . ) respectively (p < . ). univariate logistic regresion analysis demostrated a % increment in death probability for every points augmentation in the aiii score with a sensitlbity of . % and specificity of . %, the roc curve showed that the best cut off point for death prediction was points with a sensitivity of . % and specificity of . %. if a patient is classified as high risk (> ) the bayesian analysis showed a . probability of death and for one class(fed as low risk (< ) a death probability < %. conclusions: the first day aiii score in this population showed to be a good discriminator between survivors and non survivors, and the risk of death augments as the aiii does. in this population an aiii score > points is asociated with a greater risk of death. using the aiii score in conjuntion with the clinical judgement will help clinicians reducing uncertainty in the every day decision making and better predict outcome, the results from this study should been taken with caution because the data were obtained from a small sample. objective: the quality of life has been considered a "uniquely personal perception" resulting from a mixture of health related factors and social circumstances [t. m. gill, jama , : ] . the aim of this study was to evaluate two measures of pqol in intensive care unit (icu) admitted patients. patients and methods: during icu stay and six-months after hospital discharge, co-operative icu admitted patients were directly interviewed about their pqol. we administered ftrstly the uniscale (pqolu) [sage et al crit. care med. , : - ] and then a step verbal scale (pqolv): best, good, fair, poor, worst. of the studied patients, at the first interview, were able to use both scales, but ( . %) understood only the verbal one. at the second interview, patients were not able to answer, used both scales and only pqolv. statistical analysis was performed using wilcoxon signed ranks, spearman rank correlation, student's t and chi square tests. results: of all cardiac surgery pts, pts ( . %) died in icu. they were males ( . %) and females ( . %). their mean age was (+ ) years and mean ef was . (+ . ). nineteen pts ( %) had low (< . ) preoperative ef. mortality was . % in the coronary artery bypass grafting (cabg) group (n= ) and . % in the valve replacement (vr) group (n= ). in the cabg +vr group, mortality was . % (n= ), and . % in the remaining pts (n= ). cardiogenic shock was the sole cause of death in pts ( %), septic shock in pts, whereas sepsis in combination with ards in pts, sepsis and stroke in two pts. in addition, pts died from cerebrovascular accidents, one from ards and one from pulmonary embolism. the pts who died in the icu had a significantly longer bypass and aortic cross clamp time and received more blood transfusions (p< . ) than a matched control group that survived to icu discharge. the duration of mechanical ventilation and length of icu stay were greater in the pts who died in the icu than in the control group. conclusions: . although cardiogenic shock is the main cause of death ( %)in cardiac surgery pts, sepsis and cerebrovascular accident are relatively frequent causes. . patients who died in the icu had longer bypass and aortic cross clamp time and received more transfusions, compared with the control group. . although renal or hepatic failure contributed to death in some pts, they were not the primary cause of death in any patient. objectives: evaluate the acute and follow-up outcome of patients (pts) treated with primary ptca (without prior thrombolysis) in acute myocardial infarction (ami) after and up to hours after onset of typical thoracic pain ("late" primary-ptca). methods and patients characteristics: from / to / consecutive pts with ami were treated by primary ptca in the wuppertal heart center pts ( , %) were admitted to our hospital > hours and < hours after symptom onset with ongoing chest pain and typical ecg-changes.mean age was years ( - ). pts were male, four female. % had an anterior wall myocardial infarction, % suffered an inferior/postero-lateral wall myocardial infarction.two pts were in cardiogenic shock at admission. singlevessel-disease was documented in . %, multi-vessel-disease in . %. average time of onset of pain to recanalisation was min ( - ). angiography revealed timi-flow in . % of the pts, timi-flow i in . %, timi-flow ii in . %. average follow-up (fu) period was months ( - months). timi iii lv-ef ~ -day major late re-late flow p.i.* aeute/fu mortality bleeds infarction mortality . % %/ % . % . % . % % early mortality occured in the two pts, who were in cardiogenic shock at admission no pt required emergency coronary artery bypass grafting.restenosis > % was seen in % of the pts. conclusions: "late" primary ptca achieves a favourable high recanalisation rate of about % (timi ill-flow) in our study group. additionally, there seems to be a trend for lv-ef improvement in follow-up. early high mortality is influenced by the patients admitted in cardiogenic shock. there might be a trend for increased major bleeding complications. objective: to assess the validity of saps ii (new simplified acute physiology score), comparing it with the previous version, (saps), in a sample of patients recruited by giviti, a network of icu's representative of the italian icu system methods: measures of calibration (goodness-of-fit statistics) and discrimination (receiver operating characteristics curve and area under the curve) were adopted in the whole sample and across subgroups differing in relevant prognostic characteristics. of the patients recruited during one month period, a total of patients were included in this study. for the purpose of the comparison of the two scores, patients with less than years, or having cardiac surgery or staying in the icu less than hours were excluded. vital status at icu discharge in the whole sample and at hospital discharge in half cases wher adopted as outcome measure. re$ ~: saps ii fits the data equally well compared to the older version (goodness-of-fit p= . and in the new and old versions, respectively) but its performance is somewhat better in terms of capability to distinguish patients who live from patients who die (areas under the curve . and . , respectively). furthermore, saps ii is better in terms of uniformity of fit across relevant subgroups, although substantial over prediction of mortality was observed in trauma patients and in patients admitted without organ failure to be intensively monitored. saps ii performed very wet] also in the subsample where hospital mortality was the dependent variable.satisfactory measures of calibration (goodness-of-fit p-- . ) and discrimination (receiver operating characteristics area= . ) were observed. c nr saps ii, a multipurpose scoring system developed in an international study, retains its validity in this independent sample of patients recruited in a large network of italian icus. although it has shown a good performance when adopted to predict icu and hospital mortality in the entire sample, further investigations are warranted. the observed over prediction of mortality in a few subgroups indeed call for a through assessment of the impact of confounders and biases on model performance when saps ii is adopted in samples that do not reflect the "average" icu patient. objectives: ) assess the effectiveness in a group of intensive care units by means of a quality performance index (qpi); ) assess the efficiency by means of a resource use index (rui); ) evaluate the performance of individual icus with respect to both indices (clinical and economical) while controlling for severity of illness. critical from ucis in catalonia patients alearic islands have been included in the study. inhospital mortality and weighted hospital lenght-of-stay (los) have been considered the outcome variables. severity of illness has been measured with the mpm ii at admission. in each icu, expected mortality has been obtained adding the probabilities of dying for its patients. expected los has been estimated adjusting a second order polynomial to the severity of illness. performance indices have been obtained by dividing the observed by the expected outcomes. re~ult~: the overall qpi was . and it ranged from . to . in the icus. the overall rui was and it ranged l~ont . to . . there was not a trade-offpattern between clinical performance and resource use. objectives: teaching hospitals often provide [cu care across a variety of specialized services. overall, this approach appears to result in the best risk adjusted survival rates, but at the highest cost (critical care medicine ; : - ): recently, there has been increasing focus on markers of overall hospital performance. however, in large teaching institutions, such markers may fail to detect intra-institntional variation at a large tertiary care medical center. methods: first intensive care unit (icu) day, acute physiology and chronic health evaluation iii (apache iii) and active therapeutic intervention scoring system (tiss) data were collected on random admissions to specialty icus with beds (range - ) between february i and december l, . post-operative solid organ transplant recipients were excluded. units included general medical, general surgical, and trauma, neurosurgery, cardio-thoracic surgery, and coronary care units. data were analyzed for risk adjusted outcomes: icu and hospital mortality and length ef stay (los); risk of requiring active cu treatment; and icu readmissinn using apache iii risk prediction models. results: the study icus cared for a diverse group of patients. mean apache iii scores ranged from . - . ; predicted risk of hospital death ranged from . - . %. standardized mortality ratios ranged from . to . with icus performing significantly better and performing worse than predicted (p< , ). los ratios and icu readmission rates ranged from . to . (ns) and . to . % respectively. patients predicted at low risk of requiring active icu treatment ranged from , to . % conclusions: there was wide variation in the mean level of patient severity between icus. after controlling for this severity, outcomes also varied widely. no clear pattern of overall institutional performance was evident. these data suggest that efforts to assess performance, improve quality, and maximize efficiency must be focused within individual units. programmatic evaluation of outcome allows for focused review of the processes of care contributing to good outcome (best practices) and where to focus ongoing quality improvement and cost reduction activities. background and method : we compared icu mortality in different age groups presenting with the same severity of disease. we assessed severity of illness by the physiological day -apache~ (physio-aa) score (thus excluding the age related points). for each of the following physio-a n score intervals ( - ; - ; - ; - ; > ) , we compared tcu mortality within age intervals (< ; - ; - ; - ; - ; > years - , - , - ) . in these groups mortality may be twice higher in the > years patients than in the _< years. mortality does not vary with age in low (physio a n = - ) and high (physio a n = > ) risk groups. in the low risk group, mortality is low in all the age intervals because of the begninity of illness. in the high risk group, extreme severity of disease probably blunts the impact of age and leads to high mortality rates in all age intervals. introduction: to access the actual social/clinical outcome of the patients who undenvent intensive care therapy oct) is rather difficult, quality of lilr is not easih.' defined and ohserver subjectivity is a prime factor in the evaluation. mortality ratio after discharge must be established and its causes understood. obieetives: the propose of this stud)-is to look into the mortality ratio that occurred on a series of patients that undorwent ict at our unit from of the ~iew point of severity of the original illness and the diagnostic groups. material and methods: during the period of one )-ear ( ), patients were treated at the unit, of them died, and ~ere not matched in our series because os incumpletc records. thirteen patients died in hospital after their reference to other departments, twelve patients were lost after discharge. thus. at the end. only patients were evaluated on the fu. the, were classified into the follov ng three groups: acute medical, elective surge d and acute and emergency postoperative. the patients were seen at , and months after discharge. the, were evaluated in accordance to their abili~, to being self supported in their daily life and capecity to fully return and hold to their pre~ ous jobs. apache scores were evaluated for each of the three groups and correlated to the icu dead, hospital dead, and mortality after hospital discharge, spss package was used for statistical analysis. remlts/conclasions: data shows that / patients died after discharge from the hospital, of ~itch nine died in the first three months. seventy-eight per cent of the patients were fully self supported in their daily life and % showed some kind of handicap. fosty-nine per cent of the patients wore on retirement either due to age or some form of chronic disease, when admilled to our unit. thirty-two peg cent had not been able to return to work, because the" were incapacitated on discharge. only % had return to their fully jobs but the period of the stu~, is not enough for all of them to be fully physically recovered. preliminmy statistical analysis shows us significant differences among groups. the aim of the present study is to compare the prognostic performance of five general severity indices ou coronary patienta and to find out if a proper ntatistical hundling of these indices could provide better results in these patients. methods: saps ii, mpm ii (mpm ii i mpmp ii ), apach ii end gaprik were evaluated o~ patients with acute myocardial infurction admitted to intensive care units from catulunye. calibration and discrimination were calculated for each index. calibration was calculated by th bosmer-lemeshow test. discrimination was evaluated by the area under the relative operating characteristic (roc)curve. if a model did not show a good performance it was customized using multiple logistic regression. finally, tworeduced models were developed, one fro~ the mpm series (mpm ii cor) and one from the group apache-saps (sapsiicor).their performances were again evaluated. results: discrimination was high enough for all models. neverthelees, oelibration of apache ii, saps ii and mpm was not satisfactory. thus,mpm ii , saps ii and gaprik were customized for coronary patients using the logits of both models, and obtaining good calibrations. mpm ii , and apache-saps were adapted and reduced to (mpm ii cor) end to variables (sapsiicor), respectively . both models showed better oalibrutions end discriminations than the original models. conolusion| models developed for multidisciplinary patients show a good discrimination when applied on aoronar i patients, but some needed customization in order to improve calibration. the number of variables of the principal model can be reduced (even to or variables) without loosing prognostic accuracy. objective: to compare the ability of two methods to predict outcome for intensive care patients. methods: we included consecutive intensive therapy unit (itu) admissions with an itu stay> hrs in a month prospective study (exclusion criteria: burn injury and age < yrs). data were couectsd applying the criteria described by the developers [ , ] . the definition of coma (mpm ii) was modified and the best assessment within in's, rather than the admission score, was used. statistical analysis included classification tables and receiver operaung characteristics (roc) curves to assess discriminative power, and lemeshaw-hosmer statistics and calibration curves to test accuracy of prediction. results~ average abe was yrs (ranse: - ) with a male:female ratio of . : . the actual hospital mortality was . %, mean predicted death rates were . % (mpmz ii) and . % (ap hi). non-survivors had siguitlcanfly higher predicted risks than survivors applying both methods (p< . l, t-test). the total correct classification rates (tccr) for apache iii were bett~r for all decision criteria applied (tccr, decision criterion %: apache ]/i . %, mpm ii . %). the area under the roc curve was . (ap iii) and . (mpm ii) confirming the better discrimination of apache ill. accuracy of risk prediction was similar for both models (ap nl ~ - , mpm b ;( - , lemeslmw-hosmer). showing some fluctuation, calibration curves lay close to the ideal line for predicted risks -< % with increasing deviation for higher risk groups (s. figure) . apache iii underestimated the risks of hospital death for almost all risk groups (curve above diagonal), whereas considerable overestimation for predicted risks > % ceenred with mpm~ii. objective: to assess the goodness-of-fit of the apache iii model for british itu patients. methods: we prospectively studied a cohort of adult patients consecutively admitted to a medical-surgical itu over a period of months. patients with burn injury, age < yrs and itu stay < hrs were excluded. using a eomputerlsed database, we routinely recorded hrs apache ill scores. predicted risks of hospital death were computed by critical audit ltd, london. accuracy of risk prediefion was assessed by hosmer-lemeshaw chi square (;( ) statistics and calibration curves [ ]. discrimination was tested employing classification tables and receiver operating characteristics curves (roc). restths: the mean age of the male and female patients was yrs (range: - yrs). of these patients, % were medical admissions, % were admired after emergency and % after elective surgery. the observed hospital mortality was . %, the overall mean predicted death rate was . %. mean predicted risks were siguifieanfiy greater for nonsurvivors ( . %o) than for survivors ( . %, p< . l, t-test). apache iii showed good calibration (z -~ , lemeshaw-hosmer). however, the calibration curve lay above the diagonal for almost all risk groups reflecting the tendency to underestimate actual mortality (s. figure) . the best total correct classification rate (tccr) was . % (decision criterion: %). the area under the roc curve was . % confirming the good discriminative ability of the model. objectives: the aim of this study is to point out the discrepancies between needs and actual treatment of less severely ili patients admitted in italian intensive cam units (icus) requiring only intensive monitoring, and verify the substantial likelihood of data comparing those collected from a national short term study with a regional long ternl use. ~: less severely ill patients ("observed patients") were only monitored; they did not require intubation, even if for a short period (less than houm) or major cardioeiranlatory supports, and were neurologically normal. epidemiologieal national data were obtained from giviti group (gruppo italiano valutazione interventi in terapia intensiva); this cohort study, collected patients, in two months in summer in all over italy. regional data were echieved in a three years entlection ( -i ) in lombardia' icus from archidia group (arehivio diagnostieo), including patients. mortality, severity score, diagnostic category and some typical intensive procedures were analysed and compared in both studies. patients' disgunstie categories were defined as surgical, medical and trauma, according to the main diagnosis and the presence/absence of surgical procedures. rr observed patients account for . % and % of all icu's patients respectively in national and regional data. very tow mortality rate was found in national data ( . %) and extremely low mortality in regional data ( . %). in both studies mortality, s.a.p.s. and length of stay were much lowor in "observed patients" than in general icu's population (mortality: . % and . %; .a.p.s. score: . and ; iength of stay: % and ). homologous distribution of patients in the two studies was noted for what concern their diagnostic category, aside from a slight prevalence of tranmatised patients in the giviti study. in the two groups the surgical patients were respectively % vs. %, medical patients were % vs. % and traumatised were % vs. %. % of "observed patients" in national study and % in the regional did not received any intensive procedure. only a minority of these patients availed haemodynamie eonu'ol with swan-ganz or renal haemofiltration. conclusions: these results underline that about one fourth patients admitted in italian icus benefit an oversized slructure i, relation to the real needs of their pathology. in hot more than % did non received any advanced treatment and mortality and s.a.p.s. score were substantially lower respect to general population. the results obtained from these two studies are similar, suggesting an uniform distribution of the case mix in italy, even if a different recruitment period and a different gengraphieal distribution were used. some discrepancies in the two studies were found in the diagnostic categories moreover regarding the tranmatised patients ( % vs. %); this can be explained from the seasonal (summer) characteristic of the national study. mutuality, yet very low, is different in the two groups, but these data do not allow any definite explanation. finally these epidemiologieal survey suggest need of further studies settling more strict criteria of admission in icu. this study aims to evaluate patients outcome, quality of care and effectivity of therapy in our intensive care unit. the main goal was to indentify factors that the most influence that outcome. during . the authors collected data of patients outcome and predictor variables. overall mortality rate was , %. the most common causes of death were infection. the diagnosis of sistemic inflammatory response syndrome (sirs) and multiple organ dysfunction syndrome (muds) significantly correlate with death ( %). average length of stay was . days ~. % patients died in the first ten hosiptal days and only % after days. age was directly correlated with death % of dead were older then sixty years. an analysis of physiological variables showed that serum levels of gl~cose ( %) and natrium ( %) were in optimal physiological values. serum proteins ( %) and haemoglobin ( %) levels were inversely related to death. multivariate showed that alveolo-arterio difference in content was the most informative of all mortality predictors (mean value , mmhg in % patients io>mrnhg). factor that most influence the patients outcome was infection (sepsis) and muds. use of predictive indicators of outcome in critically ill patients may help to assess treatment regimens and to compare patient groups. acute physiology and chronic health evaluation (apache if) score (crit. care had. ; : - ) and the sepsis score of elebute and stoner (br. h surg. ; : - ) have been used, objectives: to compare sepsis score and apache ii score in predicting outcome of critically ill patients. methods: overall survival during the past years for patients in our icu was calculated = % (prior probability). the outcome of patients who were admitted to our icu for > hours was observed. apache ii score on admission, patient predicted risk of death (apache ii risk) and the sepsis score on the first day of antibiotic course were prospectively recorded. discriminant function analysis of the scores in relation to outcome was performed. results: apache ii and sepsis scores in the survivors were significantly lower than in those who died ( . i . v~s . • . and . • v's . • . respectively p < . ). correct prediction of outcome by each score is shown in discussion and conclusions: although both scores have been previously evaluated in predicting outcome of icu patients, studies of the sepsis score were conducted in small numbers of patients or involved additional measurements not routinely available. this study demonstrates that the sepsis score alone or in combination with apache ii score is more effective than apache ii score in predicting outcome. objective to test the hypothesis that resuscitation titrated against gastric intramucosal ph (phi) improves survival in critically ill patients as suggested by gutierrez et al~. method emergency admissions to the intensive care unit were randomized into control and intervention groups. in the control group phi was measured at , and h while in the intervention group phi measurements were made hourly for h. both groups were managed according to the same guidelines to achieve the following targets: mean arterial pressure > mmhg, systolic arterial pressure > mmhg, urine output > . /ml/kg, haemoglobin > g/dl, blood glucose < mmol/ , arterial oxygen saturation > % and correction of uncompensated respiratory acidosis. if the phi was < . after achieving these targets, or after maximal therapy to achieve the targets, patients in the intervention group were given fluid to ensure an adequate cardiac preload and then dobutamine at then mcg/kg/h, titrated against phi. this additional therapy was continued until h after entry into the study. in each year patients were subdivided in two series with random selection, so that the st series contained abeat / and the nd / of the patients. the st series of all the years constituted the devdoping data set and the nd series the validation data set. with data of the st series ( patients), we created the predictive model, using stepwise logistic regression (bmdp, usa). each patient has been evaluated in die st, th, th and th day, calculating for each lime the apache ii score (for a total of records), independent variables were, besides time and apache ii of the time ( michaloudia g,, melissaki a., alexias g., gogafi c., kolotoura a., krimpeni g., pamouktaoglou f, filias n. objectives: to determine the medical staff's attitude towards various ethical issues methods : between january and february , anonymous questionnaires were sent to intensive care units, all over greece. results : questionnaires ( , %) were replied and returned back. of them , % were answered by male and , % by female. the doctors replied in the following rate : , % aged up to , % aged between and , % aged over . questions were answered and were divided into main topics, as following: . admission criteria: limited bed availability was the main cause for refusing admission in , % of icu's. , % evaluated each case's viability and only , % used some prognostic score system. , % of icu's accepted all cases and a significant percentage ( %) gave in to pressure coming from their colleagues ( , % female and , % male). . informing the patient/relatives: only , % was willing to tell the whole truth, while , % had given selective information.. in the case of iatrogenic incident, , % withheld it, because either they feared legal implications ( , %), or lost of trust ( , %). doctors are asking consent from the patient and/or his family, in order to include him/her in research protocols, in a rate of , %, while only , % found informed consent necessary for the proposed treatment procedure. . withdrawal of therapy/dnr orders/organ donation: , % were willing to withdraw complex treatment in patients with short life expectancy, except of administi'ating intravenous fluids, feeding and analgesics. in , % such a decis~n was unanimous, while the percentage of those carrying it out was , % ( , % female, , % male). in case of brain stem death , % ( , % female, , % male) withdrew any life support. , % would like therapy withdrawal to be legally established, while only , % would perform euthanasia, if there was substantial legal cover. for these cases, relatives' consent was considered to be necessary from a percentage of only , %. , % considered organ donation to be a necessary proposal, while , % refused to ask the patients' relatives for an organ donation, either because they didn't have the psychological strength for it ( , %), or because they doubted the procedures' objectivity ( , %). note: in greece, icu beds are less than % from the total number of hospital beds available. only a percentage of - % of these admissions comes from the same hospital, with a potentially direct evaluation. usually an icu doctor has to be informed through the telephone. finally, employment conditions in greece are such that any changes of the medical and nursing staffare limited. conclusions: the mathematical model we found has been validated also in the second series and the discrimination capability increases with time. using this model we can evaluate the probability of survive at every, time. its application at different times permits a better evaluation of haemodinamically instable patient trend. introduction: the feasibility to assess pulmonary capillary pressure (pcap) offers the opportunity to determine the longitudinal distribution of pulmonary vascular resistance (pvr). the purpose of this study was to measure pcap and to calculate pvr to determine whether relevant shifts in the distribution of pvr could be expected after routine cardiac surgery. methods: the study population consisted of consecutively admitted patients after cardiac surgery. surgical procedures included coronary artery bypass graft (cabg) (n= ) and mitral valve replacement (mvr) (n=t ). pcap was estimated by analysis of the pressure decay tracing after pulmonary artery occlusion. after estimation of pcap precapillary (ra) and postcapillary resistance (rv) was calculated. a complete set of hemodynamic variables was obtained at hour and at hours after operation. results: there were no significant hemodynamic changes during the first hours after surgery. the mvr group maintained pulmonary hypertension and higher levels of pcap. ra/rv, reflecting the longitudinal distribution of resistances, remained unchanged. however, rv predominated ra during the postoperative period in both groups. objectives: evaluation of the influence of long-term continuous i.v. administration of the ace-inhibitor enalaprilat on regulators of circulatory homeostasis. methods: t trauma and sepsis patients randomly received either . mg/h (group i, n= ) or . mg/h (group , n= ) of enalaprilat i.v. or saline solution (control, n= ) as placebo for days. plasma levels of endothelin- (et), atrial natriuretic peptide (anp), renin, vasopressin, angiotensin-ii, and catecholamines were measured before injection of enalaprilat (='baseline' values) and during the next days. results: except for et, plasma levels of all vasoactive substances exceeded normal range at baseline. angiotensin-ii significantly decreased during enalaprilat infusion ( . mg/h: from . • to . • pg/ml; . mg/h: . • to . • whereas it remained significantly elevated in the untreated control patients. vasopressin increased only in the control group (p< . ) and decreased after . mg/h of enalaprilat. et remained almostunchanged in group , whereas et increased significantly in the control patients (from . • to .t• on the th day). catecholamine plasma levels (epinephrine, norepinephrine) markedly increased in the control group (p< . ), but they did not change significantly throughout the study period in both enalaprilat groups. conclusions: continuous i.v. administration of the angiotensin-converting enzyme inhibitor enalaprilat beneficially influenced systemic and local vasoactive regulators of the circulation, which are normally increased in the critically ill. thus patients at risk of (micro-) circulatory abnormalities may profit from enalaprilat infusion. objectives: to determine the time taken for hemodynamic and gas exchange variables to a reach stady-state after a change from supine to trendelenburg position (trp). methods: we prospectively studied adult patients with severe sepsis or septic shock requiring hemodynamic monitoring. usual cardiorespiratory parameters were measured at baseline, min after the patient was placed in a trp and again min after the return to a supine position. a fiberoptic pulmonary artery catheter (svo~ oximetrix, abbott) allowing continuous svo monitoring wa~used. during the protocol we also continuously measured sao~ by pulse oximetry and vco~ and vo by monitoring partial concentration of o and co ir~ inspiratory and expiratory gases (deltatrac metabolic monitor, datex). therefore, we were able to monitor cardiac output variations by dividing vo~ with arteriovenous difference according to the fick equation (co-fick). results: no significant difference in hemodynamic status was observed min after the patients were placed in trp. despite the fact that no significant change was observed in co and vo~ estimated by thermodilution, co-fick had a tendency to dedrease continuously in trp and then to return to its initial value when patients regained supine position. respiratory gas analysis showed a small but persistent continuous increase in vco without a similar trend in vo values. conclusions: we conclude that no significant hemodynamic effect was detected in our patients after min in trp. evaluation of vo from respiratory gases analysis after a change in body's position should be interpreted with caution, since the patient may not yet have reached a stady-state after rain. since vo did not change, vco~ increase was probably due to position related changes in-pulmonary gas exchange and not to a change in patient's metabolic status. objectives: to determine whether changes in svo and/or other hemodynamic parameters during weaning trials could be used to predict successful weaning. methods: we prospectively studied adult patients with a history or clinical evidence of cardiovascular dysfunction, who were unable to tolerate spontaneous breathing (sb) for hours. for all these patients right heart catheterisation was considered necessary in order to detect hemodynamic alterations during weaning. a fiberoptic pulmonary artery catheter (svo ximetrix, abbott) allowing continuous svo monitoring was sod. hemodynamic status was evaluated ~t baseline and after one hour of spontaneous breathing through a t-piece. patients were assigned to one of two groups depending on whether they tolerated sb for hours. data were analysed by analysis of variance and unpaired student's t-test we also used multiple linear regression analysis to determine which hemodynamic variables were correlated with the magnitude of svo~ change and multiple discriminant analysis to determine if asy of the above variables were associated with toleration of sb for hours and/or successful weaning (s-w). (j physiol ; ." - ) . we tested the hypothesis that the ventilatory stimulation by dead space (vd) loading and % co inhalation is accompanied by a proportionate cardiovascular change. methods: six healthy subjects, mean age, year, performed three incremental exercise tests in a randomized order: ) inspiring air without vd (air control, ac); ) inspiring air with vd of ml (avd); ) inspiring % co ; % oxygen, balance nitrogen. the ventilatory responses were examined at matched heart rate (hr) equivalent to % peak hr. results: ventilation (vi) was significantly greater (p< . ) during the avd and co tests than during the ac test at the same work rates. end-tidal co (petco ) and estimated arterial co (paco ) were significantly greater (p< . ) at w and w. oxygen saturation was significantly lower (p< . ) during the avd test than during the ac and % co exerdse. at matched hrequivalent to % peak hr, vi was significantly greater (p< . ) during the avd and % co tests than during the ac exerdse ( l, l, and /). conclusion: we conclude that the increase in xri and petco due to vd loading and % co inhalation is not associated with an acceleration in hr. sup.ported by mrc (canada). objeetlve: the production of large amounts of oxygen radicals from the onset of ~en may be responsible, st least in part, for peroxidative damage to myocardial tissue. the aim of this study was to evaluate the time dependence of plasma tbars in patients with am] receiving thrombolytie therapy (tt). patients and m~hods: filiy eight patients admitted in icu ( men and women; mean age . - . years) rec~ving systemic tt for possible am] were ~died. all patients received recorabinant haman tissue-type plasminogen activator (r-tpa). the mean time fi'om the onset of symptoms and the be~nning of tt was . - . hours. peripheral veao~s blood samples were obtained fi'om each patient before and serially after tt ( , , and hours). tbars levels woe determined by using a spectrophotometrie technique. rq~r fusion was identified by the timing of ereatine phosphate kkmse (cpk) peak (< hours). table i list the variation of plasma eoneenlrations of tbars (mean -sd) in groups (a,b, and c) as a function of time from the beginning of tr. co,arisen oftbe time cuncentzatiens reveal a difference p<o. between group ami (a and b) versus group c. the between-groups (a-c) comparison showed significant differences (p<~). ) at , and hours fi-om de begin~g of'it. comparison ofthe , and hours eonocnlratiens did not reveafl any difference becween n~ (group b) and angor (group c). condmien: we have observed a maintenance of high level of tbars in reperfused patients enly. these results suggest that the increase of tbars levels may be due to phospholipid derangament of reperfused myocytes. objective: to detemline if tbars levels may be an early index reflecting peroxidative damage in ami patients. patients and methods: by using a spectrophotometric teclmiqtm, based on a modificatien oftbe buege and aust method, tbars levels ware determined in healthy subjects (group i) , patients with several risk factors of ischaemic cardiopathy (group if) and icu patients with possible aim ( were confirmed ami (group ma) and were angor (group iiib)). peripheral blood was obtained at the time of the icu admission in the group i . electrecardiographic data and eehocardiographic wall motion were used to identify ami location (anterior, inferoposterinr and indeterminate). statistical analysis was made with a standard statistical package (rsigma, horus hardware). , h , s, , h statistically significative increase of ' ix~e/ea.ee x~* tbars levels (p< . ) only in pstients with con/tuned ami (group ilia) . no differences were found between the rest of the groups. the figure shows the relationship between the tbars values and the time from the onset of the aim's symptoms. conclusion: we have found that in am/patients the tbars levels can be used as an early index reflecting peroxidative damage occurring to ischemie tissues. perioperative risk in patients with ischemic heart disease. the protective role of diltiazem. lg. hatziantoniou, p. tassiopoulos, c. fakiolas, ! g. foussas~ m. lycourinas perioperatlve morbidity and mortality are mainly of cardiac origin, particularly among elderly patients (pts) ~ith ischemic heart disease. we performed a randomized trial, in order to investigate the possible protective activity of diltiazem (d). methods: pts (aged zo• with a history of coronary insufficiency were subjected to major urological operations. they were randomized to two groups: group i ( pts) -p:ithout d and group ii ( pts) -d ~as administered . mg/kg iv bolus hours prior to anesthesia and . mg/kg/h upon the end of operation and for at least hours post-operatively. iv was substituted by oval administration of x mg starting the next day and for the rest of hospital stay. all pts underwent daily clinical, electrocardiographic and laboratory (ck-mb) control. results: are shown in the following interactions between the heart-lung unit and a cardiac assist device are the crucial issues for successful outcome of patients under mechanical circulatory support. several factors have been indicated and we can diffrentiate between anatomical and hemodynamic interactions. especially right heart failure and pulmonary hypertension have been proven to limit left ventricutar assist device (lvad) performance. in order to study the effects of right heart failure (rhf) and pulmonary hypertension (pht) on mechanical circulatory support, we designed an experimental study in dogs and combined in six groups left heart failure (lhf) and rhf and pht, respectively. in the control group we induced lhf with subsequent occlusions of lad and cx followed by reperfusion: in the next group we supported with an lvad during minutes starting minutes after cx occlusion (group if). in the next group we added right ventricular ischemia by occluding the rca simultaneously to the cx (group iii). these both groups we repeated only with pre-existing acute pht induced by the injection of o fm glass beads (groups vi and v). a last group was similar to group v (lhf and rhf and pht), but instead of a lvad, we used bvad support. in i and v no animal survived the second perfusion period ( hours). in ii and iii (no pht), all dogs survived in stable conditions. in iv (lvf, pht), % survived, the remainder died of incomplete recovery. in vi with bvad, only % survived. in iii and v, during occlusion of the rca, the right ventricle stopped contracting and right atrial pressure and mean pulmonary pressure equalized. this lead to a significant drop in inflow on the left heart side, which still provided sufficient lvad performance in case of absent pht (iii) and lead to death due to "low lvad output" syndrome in v. this "low lvad output" snydrome could be partly overcome by bvad support. we conclude that heart-lung interactions play a major role in lvad performance. by optimizing the perioperative management, they all can be overcome, except the occurrence of alveolar leakage syndrome. obiectives : we propose a new approach of transmural pulmtmary artery occlusion pressure (paoptm), in presence ef peep, from the folluwing hypothesis : if respiratory swings of paop are compared to those of alveolar pressure (apalv = plateau pressure -total peep), an index of transmission of airway pressures to pulmonary vessels is obtained (i t = apaop/apalv). the value of the product of it by peep can be substracted from end-expiratery paop (paopee) to obtain a calculated paoptm (paoptrnc). thus paoftmc = paopee -(apaop/apalv x peep). methods : we tested this hypothesis in patients by comparing paopee, paoptmc, and paopnadir obtained within the first seconds after disconnection from the ventilator, value of reference of paoptm in absence of intrinsic peep (peepi). at zeep, peepi was absent in patients (group a) but present in others (group b). patients were studied under the peep level chosen by the attending physician-(gr a : +_ ; gr b : _+ cmh ). results : l. gr a : paopee ( _+ mmhg) differed (p < - ) from paopnadir ( • mmhg) and paoptmc ( _+ mrahg). gr b : paopee, paopnadir, paoptmc differed between themselves ( " - , + , + mmhg respectively). . good correlatkm (r - (i. ) and agreement between paopnadir and paoptm were found in gr a, while there was no agreement in gr b (bland and altman analysis). . lung compliance (v t/bpah,) correlated well with i t in both groups (r - . ), suggesting that our hypt)thesis was strtmg even in patients with peepi. conclusions : paoptm under peep ventilation can be obtained, even in patients with peepi, from parameters easily measured at the bedside. introduction: cardiopulmonary bypass (cpb) may cause ischemia in several organ systems like the heart, brain and kidneys. reactive oxygen species (ros) are formed by subsequent reperfusion and stimulation of neutrophils by cpb. ros are harmful to biological systems e.g. cellular membranes, enzymes~ dna. many natural antioxidant systems protect against the effect of ros. in patients undergoing cabg we evaluated the production of ros by total plasma antioxidant status (tas) and lipid peroxidation measured by lipofuscin flip). methods: tas (randox, uk) and concentrations ofllp (tsuchida et al.) were measured in consecutive patients undergoing cabg. all the patients had a normal serum creatinine clearance (> ml/min). serum samples were obtained a) before operation, b) after removal of the aortic crossclamp, c) at admission to the icu, d) hours after operation, e) hours after operation. results: tas was significantly decreased after removal of the aortic crosselamp ( b, c and d lower than a), followed by a subsequent significant increase of lip ( c and d higher than b). the levels of tas and lip returned to baseline hours after operation. methods: patients with preoperative lvef< % undergoing coronary artery bypass grafting were studied. after surgery, a f femoral artery catheter was inserted and connoted to a fiberoptic monitoring system (cold z- t; pulsion medizintechnik, germany); this allows, with a double-indicator dilution technique, the calculation of cardiac index (ci,l/min/m ), intrathoracic bood volume (itbv,ml/m ), pulmonary blood volume (pbv,ml/m ) and extravascular lung water (evlw,ml/kg). with a f pulmonary artery catheter, wedge (w,nunhg) and central venous pressure (cvp,mmhg) were measured, while extraction ratio (o exr,%) and oxygen delivery (do ,ml/min/m ) was calculed. peak inspiratory pressure (pawp,cmh ) and mean airway pressure (mawp,cmh ) were measured with a varflex flow transducer (bicore,sensormedics,us). the patients were studied after minutes (to) of volume controlled standard ratio ventilation (vc), and after minutes (ti) of stabilisation period of pcirv ( % inspiratory time, % pause). vt,ve and total peep were held constant in every mode of ventilation. +_ . " *'p < , versus to conclusions: these data show that pcirv : is a safe ventilatory support also in cardiac patients with impaired ventricular function, and monitoring of itbv is more reliable to measure and optimise circulatory volume status, than w and cvp. c.ledeki-,g.rldisis,s.karotzai,c.micheilidis,m.agioutantb, g.beltapaulos. objeolivee:to evaluate the influence of lvswl on the well known correlation of sr and svo . paw eight patients ( melee end females) were included in this study regerdlen of the icu ~h"niseion couse. all paints were ,'~theta~ with e fiboroptir pulmonary artery catheter connected with an oxymetfir (r)~ so /co abbot computer.for any pulmonary artery catheter insertion, two pain= of sr and svo were obtained, one dudng inserlion and one during taking the catheter out. for any pair obtained, we eleo collected the deta concemig with the pedient's hemodynamir and oxygenation end we calculated the lvswi. were significantly (p<o. ) higher in group i. conclusion: breathing room air, the less hypoxemic patients had a rather satidactory do and tissue oxygenation (pro ) because they had properly adapted their ci. the absence of this mechanism (right heart impairement ?) in the hypoxemic patients, in spite of higher blood ne and e, was responsible for the poor do and pro . objectives: acute massive pulmonary embolism (pe) increases pulmonary artery pressure (pap) and dght ventdcular aftedoad, which may lead to dght ventdcular failure. inhaled nitdc oxide (no) selectively dilates pulmonary vessels. thus, inhaled no may be of potential therapeutic value in the management of the acute phase of pe. methods: following institutional approval, ten piglets (body weight + kg) were anaesthetised (midazolam/piritramide) and ventilated using a modified servo ventilator (siemens elema, sweden; fio . ). the following variables were obtained: systolic pap (spap), mean pap (mpap), diastolic pap (dpap), mean artedal pressure (map), central venous pressure (cvp), pulmonary capillary wedge pressure (pcwp), cardiac output (co), and artedal (pao ) and mixed venous (pvo ) oxygen saturation. pulmonary vascular resistance (pvr) was calculated. to induce pe, pm microspheres (sephadex g coarse, pharmacia biotech, freiburg, germany) were injected in an amount sufficient to increase mpap to mmhg initially. rain after embolisation when haemodynamics were in a steady state (control ), inhaled no was administered. further measurements were taken min after ppm no (no ), min after ppm no (no ), and min after discontinuation of no administration (control ). the administration of inhaled no resulted in a significant decrease in spap, mpap, and pvr. the cessation of no inhalation led to a complete return to pretreatment control values (table) . co, map, cvp, pcwp, pao?, and pv~ remained unchan no administration. objectivss:evsluate the yield of recombinant tissue plasminogen activater(rt-pa, actiiyse ~ oehringer ingelheim) as a thrombolytic agent ie pulmonary embolism ie .kr~auma patients. methods: in five patients with diagnosed pulmonary embolism(blood gases, chest x-ray, echocardiography, perlesion lung scans) were given rag of actij.yse as ~ hrs infusion, followed by heparin~single j. ihjaction snd iefusion ie doses - j./kg/day).the effectiveness of rt-pg thrembo].ytic sctier~ was svaluated im . . hrs(blood gases) amd on the third dayafter t~eat-,ment(~chocardiography and lung scan). results: three hrs after actilyse was given we observed significant clinical imprevemeet,confirmed by elevation of pao and sao in dlood.echocardiegr~m on the third day showed regression of pulmonary hypertension and lung scans showed % improvement in total lung perfusien. except slight bleeding from the site of injection nc~ more thromboiytic complicatioos were observed. conclusiens: actilyse used in five hrauma patients with pulmonary embol.ism was a very effsctive and safe thrombolytic agerlt, inducing rapid and evident clinical improvement. intermittent positive pressure ventilation ( ppv) by increasing pleural pressure, decreases the pressure gradients for systemic venous return and left ventricular (lv) ejection. we have previously shown that when inspiration is synchronized with systole using synchfjv in patients with severe cardiomyopathy, that cardiac output (co) was increased relative to both ippv and non-synchronized hfjv ( ). however, it is not known if similar effects could be realized in ventilatordependent patients with lesser degrees of lv dysfunction and fluid overload. we thus compared synci-ifjv to ippv in stable patients following coronary artery bypas grafting. methods: normothermic who were stable (< % variance/h.) were studied following admission to the sicu. heart rate (hr), mean arterial (pa), pulmonary artery occlusion pressure(ppao), co by continuous thermodilutiun (vigilance, baxter, usa), mixed venous saturation (svoz) and arterial blood gases (abg) were measured after rain. of each step. ippv was simv adjusted for a rate, tidal volume and fit to insure both normal abg and a peak airway pressure < cm h,o. syncfifjv was delivered by a hfjv ventilator (acutronic) during systole with ventilator parameters adjusted to optimize abg. protocol: patients received three ippv runs (ippvi. , ) with synchfjv runs interposed (hfjvz ). statistics: anova for repeat measures and paired t-test were used to compareruns and demonstrate variability. results:no significant difference in any of the measured hemodynamic variables were seen among the ippv and synchfjv runs (table) . post hoc separation of patients by preoperative ejection fraction (ef; ef > % ; n= and < %; n= ) did not alter these results. back~ound: in man, vascular endothelium-bound ace is expressed in concentrations greater than x that in serum and is believed to be the site of synthesis of circulating angioteusin il it is unclear whether ace inlubitors interact similarly with ace in different vascular beds. coronary vessels possess all the components of the renin-angiotensin system, including ace which may be involved in normalcardiac homeostasis, as well as in the pathogenesis of various cardiomyopathies. obiecfive: to develop a method for assaying the interaction of ace inkibitors with coronary endothelium-bunnd ace in man, methods: ace a~aty was meas~ed in five patients undergoing cabg surgery, from the transeuronary hydrolysis of the synthetic ace substrate h-bpap. trace mnou~ of ~fi-bpap ( gci) were injec~d as a bolus in the root of the aorta and simultaneously blood was withdrawn from a coronary sinus catheter into a syringe containing protease inhibitors which prevented the convession of umeaet~ ai-i-bpap by blood ace. the sample was later centrifuged to separate cells from plasma and the radioactivities due to formed product (~rl-bphe) and total sh were astimated in a [b-counter. two additional such determinations of ace activity were perform~ the second in the presence of . pg/kg e (coinjected with ~-i-bpap) and the third ten minutes after e. results: all subjects were hemodynamically stable throughout the course of the there were no noticeable hemodynamic effects of e. control transcorunary metabolism of~-bpap averaged g -a: %, in agreement with previously reported data. in the presence of e, % metabolism of ~-bpap was reduced to • reflecting a • inhibition of normal ace activity. ten minutes after e, ~ri-bfap metabolism had partially recovered to :l: %, representing a -a: % inhibition of control ace activity. from this data, the dissociation constant of e for coronary ace in vivo was estimated as . x " sec "l. conclusions: we have demonstrated the feasibility of repeated, reproducible measures of coronary endothelium-bound ace activity and of its inhibition by e. this procedure is safe and can be used to study the role of ace in normal cardiac function and in card pathologies. objectives. primary pulmonary hypertension (pph) is a progressive fatal disease of unlmown origin, with median life expectancy of less than three years after diagnosis. the responsiveness of pulmonary hypertension to a variety of vasodilator agents led to the speculation that, concomitant with vascular renmdelling processes, persistent vasoconstriction is an important feature of the disease. long term use of ca-channel blockers and intravenous pgiz may improve mortality in certain populations of pph patients, but both of these treatments lack selectivity for tire lung vasculature. the aim of this study was to test the efficacy of aerosolised prostacyclin and its stable analogue, [loprost for selective pulmonary vasodilatation in pph. methods: in three patients with pph, we compared aerosolisation of prostaglandin iz (pgi ) and iloprost to a battery of vasodilatory agents (diltiazem, nifedipin, inhaled nitric oxide, intravenous pgiz). results: nebulisation of pgi and iloprost tumed out to be most favourable for achieving effective and selective pulmonary vasodilatation. pulmonary vascular resistance decreased from + to -+ dyn*s*cm (p< . ) and pulmonary artery pressure from . + . to + . mmhg (p < . ), cardiac output increased from . + . to . _+ . i/rain (p < . ), mixed venous oxygen saturation from . _+ . to . + . % (p < . ) and arterial oxygen saturation from . + . to . _+ . % (mean _+ sem of trials in patients). -month iloprost nebulisation in one patient ( gg/day in six aerosol doses) demonstrated sustained efficacy of the vasodilator r~men. conclusion: aerosolation of pgi or its stable analogue may offer as new strategy for selective pulmonary vasodilatation in pph. endothelial adhesion molecules may play an important role in the pathogenesis of myocardial cell damage, and may contribute to the progression of heart failure. we measured the plasma soluble intercellular adhesion molecule- (sicam- ), vascular cell adhesion molecule- (svcam- ), and e-selecfin (selam- ) levels in patients with acute myocardial infarction admitted within hours after onset. peripheral venous plasma-samples were collected at the time of admission, , , , , and hours after onset. plasma soluble adhesion molecule concentrations were determined by elisa. patients were divided into groups as follows: group ; killip's class (k) and without thrombolytie therapy, group ; k and with thrombolytic therapy and group ; k and . both plasma sicam- and svcam- concentrations in group and were elevated rapidly and significantly and maintained at a high level during the first days. plasma selam- level did not change in any of the groups. these results suggest that the adhesion molecules icam- and vcam- may play a role in the pathogenesis of myocardial reperfusion injury and may indicate its severity in myocardial infarction. objectives: nitric oxide (no) is known to exert cytotoxic and negative inotropic effects on cardiomyocytes. no synthase activity has been reported to be increased in infarcted area in animal model of myocardial infarction. these findings suggest that no may be an important regulator for myocardial damage and cardiac function after myocardial infarction. we measured plasma no no -(nox) levels and estimated serial changes in acute phase of myocardial infarction. methods: subjects were patients admitted within hours after onset. venous blood samples were collected at -hour intervals on the first day, -bour intervals on the nd day and -hour intervals on the rd day and th days after onset. plasma nox concentrations were determined by griess method. results: the time course of the plasma nox levels (mea~+sem) displayed a tendency to gradually increase and to make a biphasic pattern with two peaks about hours and - days after onset (basal level; . _+ . , first peak; . !-_ . , second peak; . + . ram/l). plasma nox concentration was not influenced by the thrombolytic therapy, and nox values at the time of hours after onset were significantly correlated with maximal plasma creatine kinase level (r= . , p< . ). the levels of plasma nox in the early stage of myocardial infarction (from admission to the th day after onset) did not correlate significantly with the hemodynamic parameters (left ventricular ejection fraction, pulmonary capillary wedge pressure). conclusion: the early and late increase in no production after myocardial infarction may be implicated in the deterioration of myocardial contractility and induction of myocardial damage in the early phase of myocardial infarction. range - ) fullfilling the high risk criteria of shoemaker (colectomy , gastrectomy , pancreaticoduodenectomy , others ). patients were admitted to the icu preoperatively. arterial and pulmonary artery catheters were inserted and hemodynamics and oxygen transport were measured at admission and after stabilization to predetermined physiological end points. patients were considered stable when ci > . l/min/m , pcwp > mmhg, hb > g/l, sat >. . objectives: evaluate the acute effects of , mg ipratropium bromide and , mg fenoterol (ibf) inhaled dose on pulmonary function in nonsmocers (nb:m) and smocers (s) with sever (new york heart association class ii-iii), stabile congestive heart failure(chf) and healthy subjects. methods: pulmonary function tests were performed < h postprandial. the tests consisted el arterial blood gas aspiration followed by routine spirometry and pletismography, and single-breath gas analysis. after performance of these maneuvers, the patients was administred puffs-ipratropium bromide ( , rag) and fenoterol ( , rag). for , h, spirometry was repeated. results: in resting, pulmonary abnormalities observer in the s group were more severe then abnormalities observere in the nsm group. after treatment with ibf the improvement in pulmonary function was even more marked in patients who had smoked. the mean changes by forced expiratory volume in second(eevt) was , % (p< , t) improvement and , % (p< ,ob), forced expiratory flow betwen % and % of the forced vital capacity (fef . ) was , % (p< , ) and , % (p< , ) and maxamal voluntary ventilation (mw) was , % (p< , ) and , % (p<o, ) improvement. the mean changes in normal subjects were mild (fev increased by , %,fef - by , % and mvv by , %). we observed no adverse heamodinamie conseevence and no arrthytmogenicity. conclusion." in patients with chf the airway response to ibf is highly significant. further study is needed to determine whether therapy with ibf will load to improvement quality of life in patients with chf with dearly documented venti/atory defects. compared with unilateral ima grafts, usage of bilateral ima grafts in cabg patients causes greater thoracic trauma and further reduces blood supply to the sternum and intercostal muscles. these effects may be associated with increased postoperative respiratory complications obiectives: to study the effects of bilateral ima grafts on postoperative respiratory morbidity in cabg patients. methods: two groups of patients undergoing cabg were prospectively studied: group (n= ) received both imas as grafts and group (n= ) received only the left ima. the two groups were matched for age, smoking habits, total number of grafts and preoperative ejection fraction. pao /fio ratio was measured in all patients in the icu, at , , minutes on mechanical ventilation (mv), at , , minutes after extubation (ex) and on postoperative day (pod) . in addition, radiografic scores of atelectasis and pleural effusion were assessed postoperatively. results: group had significantly longer bypass ( _+ vs + . p< , ) and aortic cross clamp time ( +- vs + , p= , ). pao /fi ratio was significantly higher in group only at minutes after extubation (table) . there was no difference in the incidence or severity of atelectasis or pleural effusion between the two groups. the duration of mv was longer in group ( +_ vs _+ , p= , ), but the length of icu stay and hospitalization were similar. there was one case of pneumothorax in group and one case of pulmonary infection in each group. after extubation m[n min min min* rain min pod ' _+ _+ + -+ - -+ _+ _+ _+ _+ -+ _+ _+ -+ mean_+sd, *p=o, o conclusions: compared with unilateral ima grafts, the usage of bilateral ima grafts in cabg patients is not associated with increased postoperative respiratory morbidity. the diagnosis of rv ischaemia is difficult in the critical care setting. the purpose of this study was to determine the ability of rv endomyocardial and interstitial ph electrodes to detect rv ischaemia in an animal model of rv infarction produced by fight coronary artery ligation. we hypothesized that changes in transmural and interstitial ph would accompany the induced tissue hypoperfusion. rv interstitial (phint) and transmural endomyocardial ph (phendo) were measured in adult anaesthetized pigs before and serially after coronary ligation. phendo and phint fell progressively and significantly following coronary occlusion. this was accompanied by ischaemic ekg changes. the absolute and relative rates of change were greater for phendo compared to phint, particularly after minutes of ischaemia, ph was unchanged in control experiments when the electrode was placed within the fight atrial or ventricular chambers, as well as when coronary ligation was not performed. these data suggest that rv myocardial ph measurements reflect coronary ligation induced rv ischaemia, ph recorded from an electrode placed against the rv endomyocardial wall via a central vein was as useful as an interstitial measurement that required a thoracotomy. this newly described technique may be helpful in clinical settings where differentiation between ischaemic and nonisehaemic causes of rv dysfunction is important. centre. depts of cardiac surgery and anesthesiology, university of bari, italy. lung injury secondary to cardiopulmonary bypass (cpb) is well recognized. nevertheless, mechanical respiratory changes after cpb have been poorly investigated. aim of the study was to evaluate the effects of cpb on respiratory mechanics. elastance of the lung (est l), chest wall (est cw), and total respiratory system (est rs) were measured in patients without previous pulmonary disease undergoing elective cardiac surgery. there were males and females; no pleurotomy was done. cpb was carried out with membrane oxygenator. mean cpb and cross clamping times were _. and • min. flow (pneumothacograph), airway opening and esophageal pressures (pressure transducers and esophageal balloon) were measured on patients sedated and paralysed. measurements were obtained before sternotomy, at the end of cpb after chest closure, four and seven hours later. est rs, est l and est cw, were measured by occluding the airways and the end of a tidal breath. before end cpb- h after h after stemotomy closed chest cpb cpb estrs (cmh /l) . + . . - . " . _+ . * . - . * est cw " . _+ . . • . + . . -+ . est l " . -- . . - . " . -_ . " . _+ . " x + sd. * p < . : anova vs "before sternotomy". our data show that cpb induces increase in est l, whereas est cw remains unchanged despite sternotomy. impairment in est l after cpb evolves within the first hours and stabilizes hours later. during the clinical course of human imunodeficiency virus infection (hiv), patients (lots) can develop congestive and dilated cardiomyopathy syndrome (dcm). the aim of our study was to analyze the prevalence of dcm among an hiv population, its clinical and echocardiographic changes under a long term follow-up and therapy with an ace-inhibitor agent (captopril). we prospectively studied pts, % ( / ) developed dcm during the follow-up study. among this group % ( / pts) were in class i and ii and % ( / pts) in class iii and iv of the nyha classification. we divided this population in a acei+ ( pts) and acei-( pts), according to the administration of captopril. we analyzed the following parameters: age, gender, race, ivda, type of hiv, cdc classification, number of t and t type cell population and its t /t ratio, therapeutic with acei, type and number of opportunist infections, mycobacteriosis and neoplasm's. we analyzed several echocardiographic parameters, which included the initial (i), final (f) and variation (a) of the lv internal diastolic (lvdd/mm) and systolic (lvsdimm) diameters, lv % of fractional shortening (lv%fs/%), ivs and posterior wall thickness and lv mass (lvm/g). the two groups differed significantly in the following parameters: left ventricular functional indices of patients (pts) with iscjhemic dilated cardiomyopathy (idcm) have a critical value in terms of clinical prognosis of this disease. three-dimensional echocardiography ( -de) is a recently developed method that gives a better evaluation of lv morphology and function. the purpose of our prospective study was to assess the clinical applicability of this new -de method in a population with ischemic dcm diagnosis and calculate its lv indices of global and regional function. we studied a group of idcm pts, mean age _+ yrs, % male gender, using a combined -de tee approach. all pts were in class ill/iv of the nyha classification. first, ekg gated images were acquired through a mechanical pullback of the tee probe, and each set of data was transferred to a conventional ibm-pc system using a dedicated -de software. computer processing and analysis of the tee images led to a -de reconstruction matrix and off-line calculation of several lv global and regional indices. in all idcm lots the lv cavity was clearly reconstructed and end-systole and diastole in several orthogonat projections, out in multiple planes and its function quantitated using -de derived indices. mean values of -de lvesv= -+ ml, lvedv= _+ ml, lvstv= _+ ml and lvef= -+ % were obtained. for all these -de parameters, good correlations were observed with -de lv measurements obtained through biplane tee analysis of the lv cavity (r>. ; p<. ) as well as regional analysis of sequential -de cut planes. conclusion: in our group of patients with the diagnosis of ischemic dilated cardiomyopathy, this new -de method could be applied. our results show that this method allows a better assessment of the lv morphology and spatial geometry, with the calculation of global and regional indices with critical clinical and prognostic value in this particular cardiovascular pathology. simultaneous left atrial (la) and left ventricle (lv) inflow analysis assessed by pulsed doppler tee illustrate the loading conditions and reflect the hemodynamics of the left heart. we performed a prospective tee pulsed doppler study with recordings of the transmitral lv filling and pulmonary venous (pv) flow drainage in a group of patients with dilated cardiomyopathy (dcm). a group of dcm patients, mean age _+ yrs, % male were studied. this population was divided according to tee severe lv dysfunction (group slvd+ % pts; group slvd- % pts) in each pt we measured the peak velocities (vel/m/sec) and time velocity integrals (vti/m) of the transmitral early (e) and late (a) filing waves, the vel and vti of the pv systolic (s), diastolic (d) and atrial contraction (c) reversal flows. -de tee evaluation of the lved, lves, lvst volumes and lvef were obtained. we calculated other parameters, such as e/a, s/d and a/c ratios and the sum of c+a vel, that refelect la systolic function and lv compliance. + -_ . simultaneous and quantitative analytical approach of the pulmonary venous and transmitral flows and ventricular volumes improve the non invasive assessment and understanding of left ventricular diastolic function and cardiac performance in dilated cardiomyopathy patients. objectives : to assess the hemodynamic effects of fluid loading (fl) in acute circulatory failure (acf) due to acute massive pulmonary embolism. methods : hemodynamic measurements (fast-response thermistor pulmonary artery catheter) were performed at baseline (baseline) and after a rapid fluid loading with (fl ) and (fl ) ml of dextl'an (rhemacrodex| in patients free of previous cardiopulmonary disease ( • yrs) with acf (ci < . l/rain/m ) due to angiographicalty proven mpe (miller score > ) . results : are expressed as mean _+ sem and compared by anova. a significant negative correlation (r = . ) was observed between baseline rvedv[ and the effects of fl on ci. such correlation was not observed between baseline rap and the fl induced increased in ci. conclusion : fusibmificantly increases ci in acf due to mpe. however, the simultaneous decrease of arterial content due to hemodilution, limits the benefits expected from improved ci on peripheral oxygenation. obiective: to examine the hemodynamic effects of external positive endexpiratory pressure (peep) on right ventricular (rv) function in acute respiratory failure (arf) patients. methods: incremental levels of peep ( - - - cmh ) were applied and rv hemodynamics were studied by a swan-ganz catheter with a fast response thermistor for right ventrieular ejection fraction (rvef) measurement in mechanically ventilated arf patients (lis = . ~- . sd). according to the response to peep , two groups of patients were defined: group a ( pts.) with unchanged or increased rv end diastolic volume index (rvedvi) and group b (h pts) with decreased rvedvi. results: in the whole sample cardiac index (ci) and stroke index (sj) decreased at all levels of peep, while rvedvi , rv end systolic volume index (rvesvi) and rvef remained anchange d. at zeep the hemodynamic parameters of the two groups did not differ. in group a, ci decreased at peep , rvef decreased at peep (~ . %)~ rvesvi increased only at peep (+ . %) and rvedv[ reded unchanged. in group b, ci and rvedvi started to decrease at peep , 'rvesvi decreased only at peep (- . %), anf rvef was unchanged. individual behaviors of the hemodynamic parameters at the levels& peep were studied. rvedvi and ci were significantly correlated in out of:l patients in group b, and in no patient of group a. on the contrary, mpap and rvesvi were significantly correlated in out of patients in group a, and in no patient of group b. the slope of the relationship between rvedvi and rv stroke work index (rvswi) expresses rv myocardial performance. this relationship was significant (no change in rv contractitity)in patients of group b and in patients of group a. in some patients of group a, increments of peep shifted the rvswi/rvedvi ratio rightward inthe plot (rv function decrease). conclusions: in arf patients peep causes more often a preload decrease with unclmnged rv conctraetility. on the contrary, the finding of increased rv volumes during the application of peep is related to a decrease in rv myocardial performance. thus, these data suggest that application of peep might be considered as a stress test to assess rv function. right introduction: after heart transplant (ht), the right ventricle can be subject to an acute pressure overload, especially in cases where there is a preexisting severe pulmonary hypertension. this provokes right ventricular failure and, occasionally, circulatory collapse in intensive care unit. desire the advances that have been made in systems for preserving the donor heart and in post-surgical management, we have failed in our attempts to totally avoid this problem. the right ventricular function, although it usually remains within tolerable limits in these patients during the post surgery period, represents a factor which limits the results achievable in clinical transplant programmes. objectives: to determine the maximum tolerance of the right ventricle (mxtrv) when faced with acute pressure overload. to study the function of both ventricles of the healthy heart (donor) when faced with different degrees of pulmonary hypertension. to detect possible interactions between the ventricles in the absence of the pericardium to approximate the experimental model to the clinical model of ht. materials and methods: the pulmonary artery is progressively constrained in an experimental model until biventricniar failure is detected. this experiment is performed in two diffferent situations: with and without pericardial integrity. results: when pericardial integrity is maintained the mxtrv faced with a pressure overload is . + . nun hg. when this pressure is exceeded there is a circulatory collapse with a sharp fall in the cardiac output and in the aortic pressure. however, when pericardectomy is performed (model similar to ht), only • . nun hg is tolerated (p < . ). conclusions: with the pericardium open, as in heart transplant, the maximum pressure that the right ventricle can support is significantly less than with the pericardium closed. the pericardium has a positive effect in protecting the systolic ventricular interaction. it is, therefore, advisable to close the pericardium after heart transplant. jb prrez-bernal, a ordrfiez, a. heroandez, jm borrego, map camacho, c cruz, mac s~nchez, j monterrubio, c garcia, e. gonz~lez. hospital uulversitario " virgen del rocio ". sevilla. espaiqa. introduction: nowadays cardiomyoplasty isused incases of cardiac insufficiency as an alternative to cardiac transplant. after surgery the patients show a noteable improvement with the aid of this "biological circulatory assistance". some researchers suspect that the improvement could also be due to the formation of new blood vessels from the muscle that wraps the heart, nourishing the ischemic myocardium. objectives: our cardiovascular research group has proposed as an objective, the detection of any possible myocardial neovascularization through the muscle used for cardiomyoplasty. in the case that there are new blood vessels to the diseased myocardium through the wide dorsal muscle in which it is wrapped and which aids it mechanically, it would be possible to confirm the worldng hypothesis that cardiomyoplasty not only improves the cardiocirculatory funcfinn mechanically but also by facilitating a better blood flow to the ischemic myocardium. materials and methods: the cardiomyoplasty technique is described using an experimental model of myocardial ischemia. the vascular cast is achieved by injecting methacrylate simulataneously into both the coronary tree and the wide dorsal muscle, in five experiments the connections between the coronary vascular system and the vascular structure of the wide dorsal muscle are demonstrated, conclusions: we have demonstrated that cardiomyoplasty, as well as improving ventricular function, favours the revascularization of the myocardium. cardiomyoplasty could be indicated for cases of ischemic cardiopathy in patients in whom it is not possible to perform direct revacularization using conventional methods. a the therapeutic cardiological manouevres necessary in cases of ischeima reperfusion have increased considerably: fibrinolysis, transluminal angioplasty, coronary revascnlarization surgery and cardiac transplant. the appearance of a specific pathology ht acute reperfusion has been related to free oxygen radicals (for) generated by oxidative damage. objectives: to evaluate the appearance of for during a conti-olled process of ischemia-reperfusion in an experimental biological model and compare it with that in clinical cases. materials and methods: transitory cardiac ischemia was performed in five rabbits by reversible surgical ligation of the descending anterior coronary artery. after minutes coronary reperfusion was performed. blood samples were taken in the basal situation, at the end of ischemia and at , and minutes after the start of reperfusion. malondialdehyde (mda) was measured to evaluate the degree of lipid peroxidation (oxidative damage to the membrane). in ten patients undergoing conventional cardiac surgery the production of for was measured after aortic clamping. results: we observed that after minutes of reperfusion there was a highly significant increase (p < . ) in the mda values (mean = . /zmols/l). these returned to basal levels after and minutes of reperfusion. conclusions: an "explosion" of oxygen free radicals was detected very quicldy, just a few minutes after post-ischemia reperfusion. thus, if antioxidant agents are to be used to reduce the toxic effects of the for, these will ordy have a therapeutic effect if they are administered in the early phases of reperfusion. introduction: aortic connterpulsation is a ventricular assistance widely used in intensive care units in patients with cardiogenic shock as a provisional ventricular assistance. paraaortic or external aortic counterpnlsation is been investigated as a definitive veutricular assistance in those cases of terminal congestive heart failure and when heart transplantation is counterindicated. aims: to assess the haemodynamic effects of an aortomyoplasty in a biological model of congestive heart failure. material and method: as specimens, we used "large white" pigs. mean weight was kg. after the administration of conventional anaesthesia, dissection of the ladssimns dorsi muscle was performed on the samples at the laboratory of experimental surgery of our hospital. then we performed a thoracotomy at the level of the fourth intercostal space to reach the thoracic aorta. the aorta is dissecated centimetres from the exit of the subclavia and it is wrapped by the dissecated muscle. a cardiomyostimulator is provided in order to allow the synchronization between the diastole and the muscle contraction. the model of heart failure was provoked using verapamil plus propanolol i.v.. results: a significant increase of the aortic diastolic pressures and a significant decrease of the left ventricle telediastolic pressures were observed. this improvement in the parameters (dpti/tti) implies an increase of the coronary perfusion in a model of heart failure. conclusions: using the external aortic counterpulsation, the aortomyoplasty improves the coronary perfnsion and the heart efficiency in patients with heart failure in whom no conventional therapeutic action is possible. the permanent character of the paraaortic counterpulsation is it main advantage. the appearance of specific pathologies as a resuk of myocardial reperfasion has been related to the oxidative damage secondary to the release of oxygen derived free radicals (ofr). during the myocardial ischemia induced during heart surgery with extraeorporeal circulation, severalsubproducts of the oxygen are produced that shall cause toxic effects after the reperfusion which could be counteracted by the physiological antioxidant systems and/or provided by the medication. aims: to asses the ofr during heart surgery. to check whether an antioxidant treatment administered in the preoperative period make decrease the levels of ofr before and after the myocardial reperfusion and to verify whether its administration have any beneficial effect on the intra and extraoperative management. material and method: the study comprehends patients studied as two groups of individuals each (a and b). all patients underwent conventional heart surgery of valvniar substitmion or myocardial revaseularization. group a patients were administered rag/ hours of vitamin e (tocopherol acetate) hours prior to the intervention as antioxidant treatment. group b patient were not administered vitamin e. we assessed the quantity of malondialdehido (mda) to assess the degree of lipidic peroxidation or oxidative damage of the membrane during the myocardial ischemia and nm after the reperfusion. conclusion: patients who underwent heart surgery and were treated with tecopherol acetate in the preoperative period presented levels of rlo significantly lower than those who were not administered the drug, both during the intraoperative period and after myocardial reperfusion. we detected in these patients a need for antiarrhythmicals and pharmacoiogical support with catecholaminas, although not significant, both in the introaperative period and the immediate postoperative period. recommendations for the treatment of pulmonary embolism (pe) in the presence of right atrial thrombus (at) are conflicting. because of a significantly higher mortality rate due to fulminam or recurrent pe, there is a necessity to treat patients (pts) with mobile type a thrombi compared to pts with adherent type b thrombi. therapeutic strategies include anticoagulation, thrombolysis (t) or surgical thrombembolectomy. combination thrombolysis (cot), predominantly used for the treatment of acute myocardial infarction proved to prevent reocclusion of the infarct related artery at a comparable rate of hemorrhagia. benefit has been related to the alteration of hemostatic proteins by non-fibrinspecific thrombolytic s. administration of cot in pe has been performed sporadically. in the present case, a -year old male with no history of prior cardiovascular disease developed acute dyspnea which was related to pe in the presence of deep vein thrombosis of the left femoral vein. therapeutic anticoagulation was installed for a couple of days until there were several bouts of deterioration. biplane transesophageal echocardiography (tee) was performed and revealed a large, wormlike, hypermobile thrombus within the right atrium. computer tomography (ct) of the chest detected a saddle embolus in the bifurcation of the pulmonary tmnk almost occluding the entire left pulmonary artery (pa) and parts of the right pat consisted of mg frontloaded rt-pa and the subsequent continuous administration of urokinase in a dosis of . u/hr for hrs followed by therapeutic anticoagulation. symptoms, blood gases and ecg improved steadily during infusion, no adverse effects, i.e. minor or major hemorragia were registered. follow-up ct promptly after termination of t showed almost complete resolution of the saddle embelus, whereas tee showed complete dissolution of the at. ' finally, the patient was switched to oral anticoagulants and had an uneventful clinical course until he was discharged. conclusion: in the present case, cot was effective for the treatment of a complicated pe without any adverse effect. introduction: nowadays we can assist hearts with problems of insufficiency by techniques other than transplant. many researchers believe that the best way of assisting insufficient heart muscle is with another muscle from the patient. this technique of ventficular assistance is known as cardiomyoplasty. we describe the surgical technique of cardiomyoplasty using a biological model. the transformed skeletal muscle is transferred to the thoracic cavity where it wraps the heart and assists it. the choice and preparation of this muscle is currently under investigation. our group has focussed on the development of protocols for electrical stimulation to transform a skeletal muscle into a muscle which resists fatigue and which is functionally similar to the myocardium. we detect the optimum time at which this muscle has been transformed, by studying the transmembrane action potentials using intracellular electrodes. when the action potential of the trained muscle behaves like cardiac muscle we consider it ready for cardiomyoplasty. conclusions: cardiomyoplasty is an alternative surgical technique to cardiac transplant, which has a great future in the treatment of patients with advanced cardiac insufficiency. we describe methodology which, by intracellular techniques, allows selection of the optimum moment of transformation of a skeletal muscle trained to perform,like cardiac muscle, without suffering fatigue. purulent pericarditis is a rare disease. its treatment associate systemic antibiotics and drainage of the pericardium. we report a ease of purulent constrictive pericarditis in which intraperieardial fibrinolysis was use. a years old patient admitted in our icu for a constrictive pericarditis as a complication of a purulent pericarditis diagnosed seventeen days before. he had also an aehalasia and the o'esogastric endoscopy had found an oesophageal neoplasm. a fistula was not seen, indeed pericardial of flora was the same that oropharyngeal. hemodynamie and echographic study had confirmed a constrictive pericarditis. because of the poor state of the patient an intraperieardial fibrinolysis was prescribed ( . ui of streptokinase on days , , , ). fluid drainage was improved and cardiac output was also improved (day : . .min "i, day : . l.min'l). no change ofhemostasis was noted. a pericardeetomy and an oesophagectomy were performed after days of evolution. eighteen months latter the patient was still alive. intraperieardial fibrinolysis seems an interesting therapeutic way if rapidly prescribed in the purulent pericarditis course. the decrease in the systolic pressure following a mechanical breath, termed ddown (delta down), has been shown to be a sensitive indicator of preload ( , ) . however, the clinical use of this method necessitates the introduction of a short apnea. we have therefore developed a respiratory systolic variation test (rsvt) which obviates the need for apnea. the test is based on the delivery of successive breaths of increasing magnitude ( , , , and ml/kg). a line of best fit is drawn between the minimal systolic values (one after each breath) and the downslope calculated as the decrease in blond pressure for each increase in airway pressure ( mmhg / cmh ). in mechanically ventilated patients the rsvt was performed during controlled mechanical ventilation under sedation. the test was repeated after the administration of ml/kg of plasma expander. the initial mean downslope of the rsvt was -. + . mmhg/cmh . following volume loading the downslope decreased to -. + . (ns). at the same time, cardiac output (co) increased by . + . l/min (p<. ), end-diastolic area (determined by tee) increased from . + . to . + . cm (ns), and paop increased from + to + mmhg ( p < . ). the preinfusion downslope value of the rsvt correlated significantly with the increase in the co (r = . ) and the eda (r = . ). methods: an expert system has been constructed running on a multimedia computer with the two objectives in mind, viz training of inexperienced staff, and protocol guidance with treatment regimes for all staff. the system is based on experience gained from two previous systems, the one for dealing with acid-base and electrolyte problems in icu patients; the second for stabilisation of patients with heart rate and blood pressure abnormalities. the training section takes the form of a stage-by-stage account of the insertion of the pac and displays of correct waveforms, coupled with indications of possible incorrect placements, and guidance when failing to achieve the perfect positioning. the treatment protocol section extends an existing protocol for correcting abnormalities in heart-rate and blood-pressure, and now takes account of all the indices as measured by the pac. the system will suggest treatment to correct such things as abnormal wedge pressures concomitant with parameter values throughout the rest of the cardiovascular system. the type of patient eg post-operative cardiothoracic or i. c. u. trauma, will be taken into account when recognising abnormal parameter values and when prescribing treatment. results: a working system which will be improved by the finetuning being carried out. the results and lessons learnt will be presented at the conference. method: septic shock was defined as severe sepsis with either persistent hypotension (mean arterial pressure; map < mmhg) or the requirement for a noradrenaline (na) infusion ~ . g/kg/ rain with a map --< mmhg. cardiovascular support was limited to na + dobutamine (db). c was given for up to h at a fixed dose-rate of either , . , , or mg/kg/h iv. during c infusion, na was to be reduced and if possible withdrawn, whilst maintaining map above mmhg and the cardiac index (ci) as clinically appropriate. assessments were made at baseline (t = ); at i h from the start of treatment (t - ); and at the end of treatment (t - ) with c . conclusions: c does not appear to increase mpap or worsen pulmonary gas exchange in patients with septic shock, when given by infusion for up to h. c is a novel vasoactive agent for the treatment of septic shock which will now he evaluated in a randomised, placebo-controlled safety and efficacy study. objectives : to compare cardiac output (q) data obtained for thermal indicators in pulmonary artery (qtpa) and aorta (qtao) and for the stable isotope hzo in aorta (q v~ o) with indocyanine green (icg) in aorta (qicg) as reference. methods : an indicator solution of ice cold h ( . ml), h ( . ml) and icg ( mg) was injected as bolus via the injection port of a swan-ganz catheter. qlco and qzmo was measured using a dual optical system (penn lab instruments, philadephia, pa, usa). qtpa and qtao was measured using a in contrast to the recoveries of thermal indicator in pa and h in aorta the :~covery of thermal indicator in aorta was significantly increased in group ii (n= boluses) over group i (n= boluses) ( . <- . vs. . +- . , p= . ). conclusions: the "overrecovery" of thermal indicator in aorta is in agreement with " biscks deconvolution study (i) and results in erroneous values for q. the most pausible explanation is the distortion of the thermal curve caused by the slow response time of the thermal detection instrument as shown by ganz ( ) objectives: to compare data obtained with the double indicator dilution method using indocyanine green (icg) and the stable isotope h for the estimation of extravascular lung water (evlw hzo) to gravimetriu lungwater data (evlwg~). methods: an indicator solution oflcg ( rag) and h ( . ml) was injected as bolus via the injection port of a swan-ganz catheter. dilution curves for icg and zh was registered in aorta with a dual optical system (penn lab instruments, philadephia, pa, usa). cardiac output and mean tranist time was measured for both tracers (qico, tlco, q n o, t o) ( ). data analysis: evlwg~av was reference for evlwzhzo calculated as q hzo times the difference in mean transit time between t nzo and rico (atm n). as reference for atzn o evlwg~,v was divided by q~cg to obtain atg~,. a reference distribution volume for h was calculated as the sum of central blood volume and evlwg=v. boluses were administrated in a group (i) of anaesthetized pulmonary healthy sheep while q was altered. another boluses were administrated in a group (ii) of anaesthetized sheep with stable oleic acid induced pulmonary oedema. evlwg~v measurement was performed postmortem. results: for boluses h parameters were not significantly different from their respective reference parameter: at vao . +_ . s vs. atg~, . + . s, evlwzh o -+ ml vs. evlwg~,~ + ml. in group i the ratio between hzo parameters and respective reference parameters (n= ) were independent of qlco from . to . l/min. obiectives: to assess the thermo dye method using indocyanine green (icg) and thermal indicator for the estimation of lung water (evlwt). methods: ice cold indicator solution of icg ( mg) in water ( ml the aim of the study was to assess left and right ventricular function in the early postoperative period after orthotopic heart transplantation to elaborate therapeutic approaches of heart function abnormalities correction. mathefial and methods. haemodynamic monitoring data of twenty one patients ( men, women ) age from to were studied. cardiac output, pulmonary artery, right atrium and pulmonary wedged pressure were measured with swan-gans catheter. central haemodynamic indices were calculated with the help of computer-based monitoring system. relations of ventricular stroke work index to it's end-diastolic pressure were used for ventficular function assessment. results. in most cases right ventricular disfunction was the main problem. isolated fight ventficular failure with high pulmonary vascular resistance (pvr) was observed in % ( pts), without high pvr-in % opts) and with left ventricular failure-in % ( pts). one of the most important reasons for fight ventricular failure was the time of heart ischemia more than min, which is of great importance in the ease of distance harvesting. the most effective treatment for cardiac failure was combination of dobutamine with i oprotherenol, atrial pacing and vasodilatators in case of right ventfieular disfunction. all cases with isolated right ventricular failure were treated sucsessfully. biventricular heart failure was a sighn of bad prognosis and the reason of death in cases. conclusion. right ventfieular disfunetion is the main problem during transplanted heart adaptation in the early postoperative period. optimal therapeutic management of cardiac disfunction includes infusion of dobutamine in combination with isoprotherenol, atrial pacing and vasodilatators. cardiology-department of clinical centre-kragujevac institution for occupational health "zastava"-kragujevac, sr yugoslavia the aim of the investigate is analisis five years survives patients with a.i.m.in dependence of locality and risk-factors. we ana~sed- ~-pat~e~ts ( males and woman), average , years. for statistic evaluation we used life-table slstem in oder to estimate prognostic determinants. patients with respkatory muscle paralysis may benefit from respiratory assistance by abdomino-diaphragmatie pneumatic belt. we used a non invasive technique, m-mode sonography, to assess the effect of this device on diaphragmatic excursion. we measured the amplitude of right diaphragm motion in seven patients with duehenne muscular dysl~ophy in supine position with various thoracic posture ( ~ ~ ~ without and during pneumatic belt respiratory assistance. without respiratory assistance, the thoracic posture had no significant consequence on the amplitude of diapttragm motion, either in quiet or deep breathing. the pneumatic belt increased the diaphragm motion amplitude from . +__ . mm to . +_ . ram (p = . ) at ~ tilt angle, and from . + . mm to . + . mm (p = . ) at " tilt angle. the tidal volume increased from + to + rut a * tilt angle, and from + to + ml at * tilt angle (p = . ). two patients could not bear the horizontal position ( ' tilt). in the five other patients, the pneumatic belt increased but not significantly the amplitude of diaphragm motion ( . + . mm to . + . ram). after an overnight respiratory assistance, pao increased from . +_. . to + . mmhg ( = . ), sao increased from . + . % to . +_. % (p = . ), and paco decreased from + . to . +_. mmhg (p = . ) according to the ventilatory pattern result, m-mode sonography allows to measure non invasively the improvement of diaphragm kinetics obtained by pneumatic belt respiratory assistance, and may be helpful for its adjustment. objective: to study the effect of flow triggering (flow sensitivity and l/min) vs pressure triggering (-lcmh ) on inspiratory effort during pressure support ventilation (psv) and assited/controlled mode (a/c) in stable copd patients non-invasively ventilated with a full face mask. methods: the patients were studied during randomized min. runs using a bird st ventilator at zero peep (zeep). trigger values for pressure (-lcmh ) and flow ( l/rain) were the lowest allowed by this ventilator. the transdiaphragmatic pressure time product per breath (ptpdi), dynamic intrinsic peep (peepi,dyn), maximal airway pressure drop during inspiration (apaw) andl ventilatory variables (ti,te,ttot,rr,vt and minute ventilation) were measured. results: no major problems due to airleaks or to auto-triggeriffg phenomena were observed in the patients, so that all of them were able to perform all the protocol runs. minute ventilation and respiratory pattern were not different using the two triggering systems. the ptpdi was significantly higher during both psv ( . + . cmh: x sec) and a/c ( . + . ) with pressure triggering, as respect to psv ( . + . , p< . ) and a/c ( . + . , p< . ) with flow triggering ( l!m). no differences were observed between and l/min flow triggers. apaw was also significantly larger during pressure triggering; peepi,dyn was reduced during flow triggering being . + . cmh (psv flow trigger) vs . + . (psv pressure trigger) and . +_ . (a/c flow trigger) vs'f~ +l (atc pressure trigger). conclusions: in stable copd patients non-invasively ventilated, flow triggering reduces the respiratory effort during both psv and aic mode as compared to pressure triggering. this may be partly due to a decrease in peepi,dyn using a flow-by system. objective. cardiac output is higher during alternating ventilation (av) (i.e. differential ventilation of the lungs with a phase shift of half a ventilatory cycle) than during synchronous ventilation (sv) of both lungs . we verified the hypothesis that the higher cardiac output depended on a lower central venous pressure and intrathoracic pressure, due to a lower mean lung volume, which we attributed to part of the expansion of the inflated lung at the expense of the expiring, opposite lung . we studied this interaction between the lungs during one-sided inflation, which we called cross-talk. method. in anaesthetized and paralyzed piglets we applied short periods ( s) of one-sided ventilation ( breaths per rain, bpm), while the other lung was open to the ambient air. the air flow into the non-ventilated lung during expiration of the ventilated lung was integrated to volume. we studied -to-r and r-to-i cross-talk at ventilatory rates of , and bpm. the amount of cross-talk was the volume displacement in the non-ventilated lung. results. during bpm the r-to-i crosstalk was _+ . % (mean +__ sd) of the tidal volume to the right lung and the -to-r crosstalk _ . % of the left tidal volume. both values increased at bpm to _ . % (p < . ) and _ . % (p < . ) respectively. the values at bpm were in between., conclusion. we concluded that the lower mean lung volume and lower thoracic expansion during av compared to sv depends on partial expansion of the inflated lung into the non-inflated lung, resulting in a lower mean intrathoracic pressure as the main reason for the higher cardiac output during av. obiective: natural surfactant given for rds in premature infants leads to a rapid improvement in oxygenation, but lung compliance did not improve in most studies. however, acute effects on lung mechanics during and immediately after surfactant administration have not been studied before. methods: a total of administrations of bovine surfactant in recommended doses was given via a small catheter into the distal endotracheal tube either as a bolus (n = ) or as a slow infusion (n = ) in infants with established rds. static compliance (c), resistance (r) and time constant (tc = cxr) of the lung were measured every minutes with a lung function cart (sensormedics ) without interrupting ventilation. infants receiving synthetic surfactant were studied as controls. results: after surfactant as a bolus or during infusion c first decreased but then increased, whereas r increased immediately with great fluctuations but did not return to baseline. this pattern was more pronounced in infusion than in bolus administration. change of c and r varied greatly in the individual case, maximum c was > %, maximum r > % of baseline value. retreatment was followed by an increase in r in all patients, but c increased only in the one who was responder. patients receiving synthetic surfactant had no change of c or r and were non-responders. ob~i ctives= acute lung injury (ali} sometimes induces severe hypoxernla which may be refractory to conventional modes of mechanical ventilation (mv). the elm of this study was to observe some cardio-pulmonary effects of an alternative method of ventilatory management of severe ali. five patients with severe ali (murray scores > ) requiring mv were studied. protocol inclusion was considered when a control-mode of mv (with a pzo~=l. and a peep level < cme=o} was not able to get either a p.ojf=o= ratio > or a s.o= > %. patients were sedated, paralyzed, and a ventilator (serve c) was used for pressuz'e-control ventilation (pcv). fio= was maintained at . and peep removed. continuous gas flow ( • ml/kg] was humidified and jet delivered through a tube ( ram id, ml capacity, . ml/cm h=o compllancel ended in a nozzle ( . mm is) attached to the endotracheal tube connector. a thermodilution flcw-dlrected catheter was inserted in pulmonary artery. following variables were recorded minutes before and after protocol started: tidal volume (vt), minute ventilation (vz), intratracheal pressures (p~w), wedge pulmonary artery pressure (wp), central venous pressure (cvp), mean arterial pressure (map), cardiac index (ci), arterial and mixed venous oxyhemoglobin saturation (sao=, svoa) , oxygen delivery (do~) , oxygen consumption (vo ) , intrapulmonary shunting (q./qt) , and oxygen extraction ratio (ero). this observation suggests that hfpv could allow to ventilate at lower fin and improve blood oxygenation during the acute phase after inhalation injury reducing toxicity risk related to high fin . further studies are necessary to confima these results and evaluate the possible implications on mortality alter smoke inhalation and for other icu pts. objectives: to design a system for volume controlled high frequency ventilation (hfv) and to estimate the dependence of the tidal volume (vt) on frequency (f) in normocapnic ventilation in rats at frequencies - hz. methods: a new system for volume controlled hfv was devised consisting of the generator of the constant flow during inspirium and the constant pressure during expirium. the ventilator allows ventilation at frequencies - hz with the relative inspiratory time (ti) . - . . the airway pressure was measured at the proximal port of tracheostomic cannula , at the same site inspiratory and expiratory flow was measured using modified lilly-type of pressure-differential flow sensor. non-linearity of flow sensor was compensated on line by derived equation based on calibration at static and dynamic conditions. flow and pressure data were evaluated on line using original software. value of the positive end expiratory pressure (peep) was serve-regulated by analogous feed-back. in animal experiments white wistar rats ( - g) narcotized with ketamine/xylazine with cannulated carotid and femoral arteries were kept at the rectal temperature ~ the arterial pressure was monitored. after traeheotomy the metal cannula ( mm [.d.) was inserted, animals were curarized and ventilated at the following condition: peep = . kpa, ti = . . the dead space of ventilator including canula was . ml. the initial frequency was hz and rain after each change of the ventitatory regimen the blood gases analysis was performed. the frequency was changed according to the following schedule : hz--> hz--> hz--> hz--> hz--> hz--~ hz--> hz. vt for each frequency was regulated to maintain normocapnie ventilation with arterial pco = + mm hg. the arterial po was always above mm hg. results: for normocapnie ventilation in rats the following tidal volumes vt [ ml/kg] were found : vt = . --+ . ml/kg for ft = hz, vt = . + . mukg for fz = hz, vt = . +_ . ml/kg forf = hz, vm = . + . ml/kg forf = hz andvmt= . + . mukg for fs = hz (presented as mean values _+ s.d., n = ). the regression analysis using the mean values resulted in the equation for normocapnic vt in rats in our experiments : vtn = . * f-e. . conclusions: the described system allowing ventilation in a wide frequency range - hz with accurate measurements of airway pressures and vt might be useful for optimisation of artificial ventilation in new-barns with different lung pathologies. supported by grants iga mz cr nr - and gacr nr . s intensive care unit. university. hospital of south manchester, uk. methods: measurements were conducted on ventilated patients (puritan bennett ac with metabolic monitor pb set to measure end tidal co ). all measurements were repeated with the patient stabilised at cm. cm and cm peep. inclusion criteria were: ) haemedynamic stab(l( .ty for hr; ) pulmonad" anon" flotation catheter in situ: ) volume control ventilation with plateau of . s: ) fio ~ > . to maintain pao~. > kpa with em peep: ) qs/ot > %; ) pao /fio ratio < . measured variab!es included: r minute volume: plateau ainvay pressure: applied and intrinsic peep: fractional end tidal co ; arterial and mixed venous blood gases and hacmod).ttamic variables. results: statistical analysis was performed using repeated measures anova. significant decreases in cardiac index (ch p< . ), compliance (p<t). ) and o,'gcn deliver, index (do , p< . ) occurred with increasing peep (table ) . no other variable showed any significant change. methods : all consecutive patients orally intubated in the micu between january to april were studied. etts were positioned according to the reference marks mentioned above (measurements from upper incisors). the position of the ett tip in relation to the carina was measured on the chest radiograph done with the patient's head in neutral position. results : a total of men and women were studied (n= ). the mean distance of ett tip to carina was . cm. using the reference markings, cases ( . %) had the ett tip < cm from the carina and cases ( . %) > cm. one case resulted in an endobronchial intubation. the mean height of all patients were cm ( - ) for males and cm ( - ) for females. of the patients with ett tip < cm from carina, the mean height was cm and cm respectively. ~ onclusion : adopting the above quoted reference marks did not result in ideal positioning of the ett in a significant proportion of cases ( . %). we postulate that [s because our asian population is generally shorter than those in previous studies. objectives: to measure the changes of pulmonary mechanics before and after tracheostomy in patients with prolonged mechanical ventilation and to determine factors that predict the outcome of liberation from mechanical ventilation. design: prospective. setting: respiratory intensive care unit (ricu) in a tertiary hospital. patients: twenty patients with chronic lung disease requiring long-term mechanical ventilation. tracheostomy is indicated for further care. intervention: tracheostomy. measurements and results: pulmonary mechanics including respiratory rate (rr), tidal volume (vt), peak inspiratory pressure (pip), intrinsic positive end ex~ piratory pressure (peepi), lung compliance (cld), mean airway resistance (rawm), work of breathing (wob), pressure time product (ptp) by bicore cp- pulmonary monitor were recorded hours before and after tracheotomy. ventilator setting parameters remained the same during surgical intervention and were also recorded for comparison. generally, the mechanics including pir wob, raw~x and ptp showed improvment after tracheostomy. but only pip was significantly reduced (pre . _+ . to post . _+ . , p < . ). changes of wobp showed significant correlation with pre-operation rr, minute volume (mv), wobp, and peep(. changes of raw m were also significantly correlated with pre-operation peep, vt, and raw m. the patients were divided into two groups according to their outcome after two week follow-up. group included eight patients who were completely weaned from ventilator; group included twelve patients who still remained ventilator-dependent or were mortality. there was no difference in age, duration of mechanical ventilation, pro, post or changes of several lung mechanics between the groups of patients. pre-tracheostomy peep i and cld showed significant difference between these two groups ( . _+ . vs . + . in peepi; . _+ . vs . _+ . in cld, p < . ). pre-tracheostomy ventilator setting in mode of assist/control also showed significant higher percentage in group ( % % in group vs . % in group ). conclusion: in prolonged mechanical ventilation patients with chronic lung disease, tracheostomy will significantly improve pip and slightly reduce wobp, raw m and ptr patients who used pressure support mode before tracheostomy had better underlying lung conditions (lower lung compliance and auto-peep) will have better chance to wean from mechanical ventilation. forty-eight infants with congenital diaphragmatic hernia presenting within the first hours of life, who underwent surgical rapair,were analysed prospectively in order to produce a reliable inde x of severity of disease that would reliably predict eventual outcome. there were survivors and deaths in this series (mortality %).using arterialpco values measured hours after surgical repairand correlating them with an index of mechanical ventilation,we have been able to clearly define two groups of diaphragmatic hernia based on their response to hyperventilation. the first group, with co retention and severe preductal shunting,was unresponsive to hyperventilation with high rates and pressures the mortality was %. the second group responded well to hyperventilation and demonstrated reversable ductal shunting only. survival in this group was %. arterial co accurately reflects the degree of lung development in this disease and separates those patients with severe pulmonary hypoplasia where the outcome is invariably fatal, from those with a well developed contralateral lung where there is excellent potential for survival. respiratory failure unit, dpt medicine, univ. thessaloniki, thessaloniki, greece the variability of arterial blood gases (po , pc ) and the ph (abg) was examined in stable icu patients, few hours before a successful weaning from the ventilator. all patients were lightly sedated and the ventgatory conti~ons were pressure support (ps) for and ps plus intermitted mantatory ventilation in ii. [n each patient, speciments of abg were measured at min intervals during a - study period. at the same time with abg the arterial blood pressure (bp), the heart rate (cf), the tidal volume (tv) and the respiratory rate (n r were measured. for all the patients, the mean coefficient of variation (c) was . percent for po , . percent for pco and . percent for hco . the average sd for ph was . , the corresponding c for systolic bp, diastolic bp, cf, tv, rf were . , . , . , . , . percent. we conclude that the spontaneous variability of arterial blood gases in icu patients is not substantial ~hen they have stable the heamodynamic and the ventilatory parameters. deptx?fa'aaesthesioiogy and reanimation, rhe sechenov medical academy, moscow, russia objective: ~he prevention and treatment of hypoxia in the critical patiems. methods: i~fusions of perphtoran -a blood substitute with gas-transporting fimclion based on perphtorhydrocarbon -in patients with acute hypovolemia, microcirculatory distnrbance~ tissue gas exchange and metabolism; pulmonary iavage in ; iongterm extrapulmonary oxigenation with tleoroearboa oxygenator in combination whb ~trafiltra!ion, hemosorption and hemodialysis -in patients. results: pe~htoran increases blood volume, co,sv, decreases svr, improves capillary blood flow, increases the blood oxygen capacity, tissue oxygen tension, del, vo by improving the rheologic properties of blood and plasma, normalizes ext., prevents and eliminates fat embolisation and ards. decreases the need for blood transfusions and infusions of plasma expanders by . - . limes. alveolar venti!ation-perfusion ratio remains unchanged with its increased effective utilization. there was no surfactant destruction during lavage. extrapulmonary oxygenation of small volumes of venous blood eliminates venous destruction and then arterial hypoxia and increases pulmonary oxygenation. the use of lluorocarbon cxygenators during hemosorption and hcmodialysis provides the atraumatic and iongterm oxygenation of arterial blood and increases elimination of co which prevents the development of hypoxic complications. conclusions: perphtoran and fluorocarb~n oxygenators are effective in the correction of hypoxia in the criticat patients. objeqtives: to determine if there are differences in oxygen consumption (vo ) during weaning from mechanical ventilation (during total ventilatory support and spontaneous ventilation with cpap), and to compare different predictive parameters of weaning in predicting success of weaning. methods; prospective study in critically ill patients treated with mechanical ventilation for at least h, who fulfilled at least of standard weaning criteria (vt> ml/kg; respiratory frecuency (f) < ; pimax > cm h ; pao /fio > ). baseline measurements: t, vt, p . , pimax, f/vt, p . *(f/vt), p . /pimax. study protocol: measurement of vo , vco (medgraphics), vt, f, ve, and arterial blood gases during total ventilatory support (cmv), and after and minutes of spontaneous ventilation with cpap cm h . the weaning trial was stopped, failure to wean diagnosed, and mv resumed it a patient presented significant tachypnea, tachycardia, bradycardia, cardiac rythm disturbances, hypertension, hypotension, hypoxemia or hypercapnia. results: four patients did not complete the weaning trial, were extubatad, and of them had to be reintubated before h, being considered also weaning failures. during cmv, vo /kg was . + . ml/kg/min, and . _+ . mlo- /kg/min after ' on cpap cm h (p < , ). of patients ( %) with standard criteria were extubated, while only of ( %) with criteria (p< , ). next objectives: compare the extent and distribution of lung injury in dogs preinjured with oleic acid (oa) and ventilated with high tpp and adequate peep in the prone and supine position. methods: lung injury was induced with oa ( . - . ml/kg) in anesthetized, paralyzed, and intubated dogs (n= ) during volume controlled ventilation: rate= /min, peep= cmh , ti/ttot= . , fio = . , vt= ml/kg. animals were rotated during the oa infusion and the following minute stabilization period to assure uniform injury. in the supine position, peep was set - cmh above the lower inflection point (as determined by the pressure-volume curve), and vt was set to obtain a tpp of cmh : animals were ventilated in either the prone (n= ) or supine (n= ) position for four hours. pulmonary artery occlusion pressure was maintained constant ( - mmhg) with saline infusion. at the end of the protocol the lungs were removed and divided by template into dependent (d) and nondependent (nd) sections for wet weight/dry weight (v~n/dw) and grading of nstologic lung injury (hli; scale - ). oseillatron | is a pneumatic device that generates high frequency, oscillation by means of a reciprocating system in the form of a membrane. it generates sinusoidai wave form at ( to ( cycles/rain. the system does not deliver gas but must be adapted to the proximal respiratory, circuit of a conventional ventilator, resulting in ci-ifo. it was developed to enhance intrapnlmona~ diffusion during mechanical ventilation and to mobilise endebronchial secretions. methods. we measured arterial blood gases and haemedynamics during a first period of conventional ventilation (cppv) followed by. two rain periods of chfo (sequences : ( and ) c/rain : group l, n = l: and c/rain : group , n = ). measurements were made at the end of each period. cardiac output was measured using thermedilution method: flu and peep were kept unchanged throughout the study. intrinsic peep was also evaluated by, means of an occlusive valve. results. pa is not significantly modified during chfo at or c/rain. paco is slightly decreased at c/rain (p = .( ). however, intrinsic peep remains unchanged. there is no sequential effect (gr. l vs gr. ). there is no more effect of chfo for patieets who are at a flu higher than . (n = ). no changes in haemodynurmcs are observed except a slight increase in central venous pressure (cvp) during ci-ifo (p < .ol). obiectives: to examine the effects of inspiratory muscles unloading on neuromuscular output at controlled levels of chemical stimuli. methods: the ventilatory response to co was examined in ten normal subjects using rebreathing method. ventilation ~) and respiratory muscle pressure output (pmus) at the same end-tidal partial pressure of co (petco~) were compared with and without combined flow and volumeproportional pressure assist in two protocols (a and b). protocol a (n = ): two levels of assist were studied; flow assist (fa) of cmh /i/sec and volume assist (va) of cmh /i (assist ), and fa of cmh /i/sec and va of cmh /i (assist ). all conditions were applied randomly. v~, tidal volume (vt) and breathing frequency (f) were measured breath by breath and plotted as a function of petco~. protocol b: in subjects, in addition to above measurements, esophageal (pes) and gastric (pg) pressures were measured and the time courses of transdiaphragmatic pressure (pdi) and pmus were calculated. one level of assist (assist ) was studied in this protocol. results: in both protocols inspiratory muscle unloading did not change the f response to c%. compared to control, with assist v t response was displaced upwards; at petco of mmhg v t was increased significantly by . + . i and . + . i in protocol a with assist end , respectively, and by . _+ . i in protocol b with assist (p< . ). ~/~ responses showed similar changes as vtresponses. in both protocols the slope of v~ response (s did not change significantly with unloading. at low petco~ ( mmhg), pdi and pmus waveforms did not differ with and without assist. with unloading, at high petco ( mmhg), pdi and pmus at the end of neural inspiration decreased by . -+ . % and . + . %, respectively, from control values. neither change was significant (p> . ). by theoretical analysis we estimated the expected changes in vt and ~/~ when the levels of assist used in both protocols were applied in the absence of : any change in neural output response to co z. the predicted response was similar to that observed, indicating that the small difference in pdi and pmus between control and unloading runs was due to intrinsic properties of respiratory muscles end respiratory system. conclusions: these results suggest that when chemical stimulus is controlled, respiratory motor output is not downregulated with unloading. the determinants of the response of the respiratory output to inspiratory flow rates (v~) were examined in awake normal subjects. subjects were connected to a volume-cycle ventilator in the assist/control mode and v~ was increased in steps from to i/min and then back to i/min. v~ pattern was square, and all breaths were subject-triggered. in six subjects the effects of breathing route (nasal or mouth) and temperature and volume of inspired gas (protocol a) and in subjects the effects of airway anesthesia (upper and lower airways, protocol b) on the response of respiratory output to varying v~ were studied. in protocol b, in order to calculate muscle pressure during inspiration (pmus), respiratory system mechanics were measured using the interrupter method at end-inspiration. independent of conditions studied breathing frequency increased . significantly and end-tidal concentration of c% decreased as v~ increased. the response was graded and reversible and not affected by breathing route, temperature and volume of inspired gas and airway anesthesia. with and without airway anesthesia (protocol ) neural inspiratory and expiratory time and neural duty cycle, estimated from pmus waveform, decreased significantly as v~ increased. at all conditions studied the rate of change in airway pressure prior to triggering the ventilator tended to increase as v~ increased. the changes in timing and drive were nearly complete within the first two breaths after transition with no evidence of adaptation during a given ~/~ period. we conclude that v~ exerts an excitatory effect on respiratory output which is independent of breathing route, temperature and volume of inspirate and airway anesthesia. the response most likely is neu~'al in origin, mediated through receptors not accessible to anesthesia such as those located in chest wall or below the airway mucosa. it has been shown, in mechanically ventilated awake normal humans, that increasing inspiratory flow rate (~/~) exerts an excitatory effect on respiratory output. it is not known if this effect persists during sleep. to test this seven normal adults were studied during wakefulness and nrem sleep. subjects were connected through a nose-mask to a volume-cycled ventilator in the assist/control mode and ~/t was increased in steps ( - breaths each) from to i/min and then back to i/min. v~ pattern was square, and all breaths were subject-triggered. forty-one trials during nrem sleep and during wakefulness were analyzed. both during sleep and wakefulness minute ventilation increased and total breath duration (ttot) decreased significantly in a graded and reversible manner as ~' increased. these changes were complete in the first breath after v{ transition. the response was significantly less during sleep than during wakefulness (p< . ); at i/min ttot, expressed as % of that at i/rain, was . +_ . % during sleep and . +_ . % during wakefulness. during wakefulness, at i/min, the rate of change in airway pressure prior to triggering the ventilator, an index of respiratory drive, was % of that at i/min (p< . ). the corresponding value during sleep, was % (p> . ). in four sleeping subjects the increase in v~ was sustained for . - min. there was no evidence for adaptation of the response; tro t, averaged over the last three breaths, did not differ from that obtained when vj was sustained for only - breaths. we conclude that ) vt exerts an excitatory effect on respiratory output, mediated by a reflex neural mechanism and ) the gain of this reflex is attenuated by sleep. chest radiographs is a common complementary technique for patients in critical care units, with a low cost and easily available. however, it has certain well-known limits in diagnosis, the most important derived from the low quality of some pictures. in this paper we make a general review of some new technical approaches developed for improving the quality of the images, and so incrensing the diagnostic value of conventional radiology. we begin deaeng with the correct positioning of the patient, trough the filtering techniques, the synchronization of radiology and ventilation, and we make reference to the new computerized systems for digital image processing. conclusions: the portable radiographic system is a device that probably with maintain for many years in critical care units as a basic non-invasive diagnostic tool. but we need an increase in the efficiency of it, applying means as simple as a correct positioning of the patient, or the use of fitlers or synchronizers. thus we should improve the general standards of portable radiography. "are circular circuits safe? quantifying undelivered tidal volume in pediatrics patients". objectives: to evaluate the overall influence of internal compliance of circular circuits on delivered tidad volume (vt). methods: we studied prospectively asa i pediatrics patients ( to yr. old) scheduled for elective general surgery. mechanical ventilation was supplied by an ohmeda excel (circular circuit). the internal compliance of the circuit (cc)-anesthesia machine plus external circuit-was determined by the supersyringe method: corrugated dar tubes of mm. id and . m. long (children < kg), and a corrugated dar set of mm. id and . m. long (children > kg) were respectively used for ccl an cc values of . and . ml/cm h . a vtof mlg/kg and respiratory frequency was adjusted for an end-tidal co (etpco ) between mmhg. tidal volumes (measured by spirometry) and airway pressure (paw) data were recorded every ten minutes. volumes and thorax-lung compliances were calculated as follows: (vt delivered = vtadjusted-vol compressible, being vol. compressible = co x ppeak (aw). apparent compliance (ca) = vt adjusted/pplateau(aw), and true compliance (ct) = =vt delivered/pplatean(aw)). comparative statistics were separately designed between calculated compliance data and tidal volumes on a paired sample ~test basis. results: calculated values for volumes and thorax-lung compliances were: conclusions: due to the elevated internal compliance of the circular circuit there is a remarkable dilference between adjusted and delivered vt: mean undelivered vt was . % and reached as high as . %. teere is also a significative error in calculating true thorax-lung compliance: its overestimation can be as high as . %. circular circuits are considered safe and cost-saving for anesthetical practice. nevertheless we conclude that anesthetists should bearin mind vt losses when using circular circuits, due to compressible volume. tracheal stenosis is one of the most serious complications of patients submitted to prolonged endotracheal intubation, in which the decrease in inner diameter of upper airway makes it very difficult to achieve a correct ventilation. objectives: compare the results of applying high frequency jet ventilation (hfjv) to some of these patients with conventional controlled ventilation (cmv). methods: we used a prototype of high frequency jet ventilator (santiago- ) developed in our university, and we developed a tracheal tube in wich we modified the distal tip (conic tip). we applied this system to two patients which were initially ventilated in the operating room with usuai controlled mecanical ventilation (cmv) following the standards of our department, and then intubated with the special endotracheal tube and ventilated with hfjv. results: we could verify a proper ventilation of both patients with cmv and hfjv. during hfjv, the airway pressures were lower than those recorded during cmv. a lower airway pressure prevents lesions due to high pressures. conclusions: hfjv is a good method of ventilation for patients with significative stenosis of the trachea, not only during surgical procedures, but also during ventilation for long periods in critically patients. the ventilatory setting is pressure support mode. the pressure level and fit were kept constant during h/d. arterial blood gas, wbc count, and mean bp was checked according to the schedule: '(immediately before h/d), ', ', ', ', ', '. respiratory drive (represented by poa), tidal volume(ti) and minute ventilation(ve) were continuously recorded by pulmonary mechanics monitor (bicore cp- ). the mean value of the breaths minutes before blood sampling were used to represent the ventilatory status of that period. anova test is used for comparison between groups. for poa, hierarchical cluster method is applied to divide the cases into two groups of similar change. conclusions: our data suggest that pl is very useful, non invasive and low-expensive emergenc e support for arf, expecially in the elderly with severe chronic pulmonary disease and relative controindications to eti. pl seems to be an effective alternative when it is not immediatly possible to perform etl. the multiple inert gas elimination technique (miget) can be used to assess the effects of any given mode of mechanical ventilation on the pulmonary and systemic factors determining arterial po and pco> however, a potential problem in mechanically ventilated patients is that the l mixing box (mb- l) placed in series in the expiratory side of the circuit of the ventilator to sample mixed expired gas may provoke substantial discrepancies between the tidal votume set in the ventilator and the effective tidal volume delivered to the patient, due to the increase in the compression volume (vc) of the circuit. the effects of the mb- l on the v c were compared with those produced by a new l mixing box (mb- l) specifically designed to produce adequate gas mixing and to prevent loss of the two most soluble gases (ether and acetone) used in the miget. at any given peak cycling pressure (p~ak, cm h~o), the v c (ml) provoked by the mb- l was substantially higher (vc= . *ppeak) than that provoked by the new mb- l (vc= . *ppeak). at a ppeak = cm h ~ the v c were ml (mb- l) and m{ (mb- l), respectively (p< . ). in a group of subjects ( m/ f, _+ years), for each of six the gases used in the miget, the regression line between the mixed expired partial pressures simultaneously obtained from mb- l and mb- l fell on the identity line. it is concluded that the new mb- l allows adequate assessment of the effect of different modalities of mechanical ventilatory support on pulmonary gas exchange, with less potential for gas compression and thus hypoventilation. objectives evaluate the influence of different pressure support ventilation (psv) levels on cardiovascular and respiratory funcion in icu polytrauma patients. metbed&we studied polytrauma icu patients , who were in weaning process , after long term mechanical ventilation for acute respiratory failure . mean age ( - ) yrs . they all were connected to servo ventilators siemens c , and all were in stable condition , without sedation , inotropes or diuretics. the hemodynamic studies were done with continuous svo , swan ganz catheter (oximetrix, abbott). they all were in spontanuous mode (spent) with cm h cpap for at least one hour. we turned them to psv with inspiratory assistance (psv cm h ) and after rain we applied psv cm h , and after min psv cm h . hemodynamlo and respiratory measurements were done before and after the application of insiratory assistance. the results were statistically analyzed with anova. resets . respiratory variables . no significant changes in minute volume (ve). tidal volume (vt) and mean airway pressure (mpaw) increased statistically significant (p< . ) . respiratory rate (rr) decreased significantly (p< . ) . blood gase showed no difference . cardiovascular variables. cardiac output (co) decreased ns , heart rate (hr) had no change , central venous pressure (cvp) , mean pulmonary artery pressure (mpap) , pulmonary capillary wedge pressure (pcwp) , increased ns , oxygen delivery (do ) decreased ns, oxygen consumption (vo ) decreased ns. conclusions. psv is a very useful respiratory mode helping patients to be weaned from long term mechanical ventilation . it has beneficial effects on respiratory function and oxygen consumption without affecting seriously the hemodynamic parameters, possibly due to a decrease of the work of breathing. a. michalopoulos, a. anthi, k. rellos, j. kriaras, s. geroulanos intensive care unit, onassis cardiac center, athens. objectives of this study was to examine the effect of different levels of peep on postoperative svo and pvo values in a group of patients, following open heart surgery. methods: upon transfer to icu, patients ( males and females) of mean age _-+ years, were randomly assigned to receive (n= ), (n= ), or cm of peep (n= ). there were no statistically significant differences in demographic data or preoperative respiratory status among the three groups. all patients were ventilated on the assist control mode with a tidal volume of ml/kg. the fraction of inspired oxygen (fio ) was adjusted to keep a pao around mmhg. mixed venous po and svo were measured at min, and hours after application of mechanical ventilation in the icu, just before extubation (be), half hour after extubation (ae), and at hours post-extubation. differences at each study time were analysed by anova. results: mean svo and pvo values among the three groups, for all study intervals, are presented in the table. conclusion: we found no differences (p=ns) in tissue oxygenation (expressed by svo and pvo ) among the three groups, at any study interval, in the early postoperative course of patients following open heart surgery. intrinsic peep (peepi), and high elastance and resistance increase inspiratory work load in copd. cpap reduces work of breathing by counterbalancing peepi. pav provides flow (fa) and volume (va) assistance proportionally to patient resistance and elastance and inspiratory effort. we studied the effects of partitioned support (cpap-fa-va) on breathing pattern and inspiratory effort in five copd patients on pav compared to spontaneous ventilation (sv) and full support (fs: cpap+fa+va). flow, volume, minute ventilation (ve) respiratory rate (rr), inspiratory swing in esophageal pressure (apes), and its integral per breath (pti/b) and per minute (pti/m) were measured. objectives: to evaluate airway pressure fluctuation (apf) during spontaneous breathing in a high compliance cpap system. methods: the cpap system consisted of two l weighted balloons in a wedge shaped holder. ventilating gas flowed from one balloon through a low resistance one way valve into a tracheal tube (ett) provided with a pycor co sensor to monitor rebreathing. the ett was connected to a piston drive mechanical lung. expired gas flowed through a low resistance valve into a second weighted balloon, from where it was exhausted through a peep valve connected in parallel with the second weighted balloon. we evaluated system performance at v r from to ml, at rr from to bpm, while closely monitoring cpap airway pressure swings. at v v of and ml the rr was limited to bpm. for comparison we explored aps of a one l balloon cpap system, the cpap mode of the puritan bennett , and siemens ventilators, when connected to a healthy adult volunteer breathing through an ett. results: the compliance (cpl.) of one l balloon system was linear over a range from . to . l, with a cpl. of . l/em h .the cpl. of the l balloon ( . l/em h ) was linear between a volume of and . l. apf of the weighted balloon system was under em h at all v r (except at a v r of ml aps was . em h ), while the apf in the l balloon was up to em h . apf witli human volunteers with the two commercially available ventilators in the cpap mode was about cm h ; while under identical conditions apf in the l balloon system was . emhzo; and in the two l balloon system was below lcm h . conelusions: cpap using the two balloon system exhibits lower airway pressure fluctuations than a single balloon system; and is substantially lower than found in the two commercially available ventilators when used in the cpap mode. objective: to perform independent lung ventilation (ilv) with individual tidal volume (vt) set at a value generating a plateau airway pressure (pplat) < crnh~o and to evaluate the usefulness of the continuous monitoring of endtidal co (etco ) as a guide to titrate individual lung vt during ilv and for the weaning from ilv. methods: in seven patients, ilv was performed with ttvo ventilators set with the same fio: and respiratory rate. each lung was ventilated with a vt that developed a pplat < cmh~o. this setting led to a lower vt on pathological lung (pl). vt was increased in pl following etco~ and paco -etco variations. ilv was discontinuated when etco~., vt and statical compliance (cst) were similar in both lungs. results: one hour after starting ilv (ti), pl mean vt was significantly lower than in normal lungs (nl) ( + ml vs + ml, p< ) two individual behaviours were observed on tl in pl: four patients presented low etco: (range - mmhg)and normal pacoz (range - mmhg), while three patients had normal etco (range - mmhg) with high pac (range - mmhg). one hour before stopping ilv (t ), vt, etc and paco were the same in each lung. the pao /fio: ratio improved in all patients from the beginning ofllv cst of pl was + % of the normal lungs' cst on ti and improved to . + % ofnl's cst on t (p< . vs conclusions: setting vt of pl to a value not overcoming a pplat threshold does not impair oxygenation and is helpful in avoiding barotraumatism. measurements of differential etco and of the differential paco -etco gradient can be used to titrate vt allocation during ilv and as a guide for the weaning from ilv. total respiratory resistance in mechanically ventilated patients exceeds values obtained in normal subjects, due to the added and highly flow dependent resistance of the endotracheal tube (rett). this can adversely effect the efficacy of pressure regulated modes of assisted ventilation, such as pressure support (psv) and proportional assist ventilation (pav). recent work demonstrates that the influence of rett during psv can be overcome by using tracheal (ptr) rather than airway opening (pao) pressure to regulate the pressure applied (intensive care med :$ , ) . the purpose of this study was to see if this approach would also be effective during pav. flow, volume, pao, ptr, and transdiaphragmatic pressure (pdi) were measured in intubated patients in which either pao or ptt were used to regulate the pressure applied during pav where volume assistance was varied from to % of respiratory elastance. representative results (mean + se) are shown below. compared to spontaneous breathing (pav %), pav increased tidal volume (vt) while reducing respiratory rate (rr) so that minute ventilation ('~e) also rose. this was associated with a reduction in inspiratory effort, as reflected by a decrease in the pressure-time integral ( [ p) of pes and pdi both per minute and per liter ~re. the effects on breathing pattern were similar for pao and ptr regulated pav. in contrast, the reduction in inspiratory effort was always greater for ptr regulated pav. in conclusion, the volume assistance provided by pav is more effective when ptr rather than pao is used to regulate the pressure applied. pav methods: retrospective data analysis of adult patients with normal pulmonary function before operation and uneventful course following coronary artery bypass graft surgery over an month period. we compared assist/controlled mandatory ventilation (s-cmv, patients), synchronized intermittent mandatory ventilation with inspiratory pressure support (s-imv/psv, patients) and biphasic positive airway pressure ventilation (bipap, patients). results: patients ventilated with bipap had a significantly shorter mean duration of intubation ( . h, p< . ) than patients treated with s-imv/-psv ( . h) and s-cmv ( . hi. with s-cmv . % of the patients required single or multiple doses of midazolam but only . % in the s-imv-/psv group and . % in the btpap group. the mean total amount of midazolam of these patients was significantly higher in the s-cmv group ( . mg) than in the s-imv/psv group ( . mg, p< . ) and in the bipap group ( . mg, p< . ). the consumption of pethidine and piritramide did not differ between s-cmv and s-imv/psv but was significantly lower during bipap (p< . ). after extubation the paco patients was highest in the s-cmv group. conclusion: ventilatory support with bipap reduces the consumption of analgesics and sedatives and duration of intubation. unrestricted spontaneous breathing as well as fully ventilatory support allow adequate adaptation to the patients requirements. bipap seems to be an alternative to s-cmv and sqmv/psv ventilation not only in patients with severe ards but also in short term ventilated patients. _objectitives: after end-inspiratory airway occlusion we examined the ensuing gradual decrease in tracheal pressure (ptr) with the following equations proposed by bates et al. and hildebrandt: pv = p'v e'~cccl~ +pst, rs (bates) [ ] where p'tr is tracheal pressure immediately after occlusion, to= is occlusion time, "r is viscoelastic time constant of respiratory system, and p t is static elastic recoil pressure of respiratory system. p~(t) = h -h log t (hildebrandt) [ ] where h~ and h are parameters depending on lung volume, and initial time is s for analytical reasons. materials & methods: we studied healthy patients intubated, anestethized with propofol, paralyzed with vecuronium, and mechanically ventilated with constant flow ( . i/s) at zeep for minor surgery. pressure was measured in the trachea. flow was measured with a pneumotachograph and volume was obtained by numerical integration. the rapid occlusions were produced by an external valve. the signals were sampled at a frequency of hz and processed on a pc. the influence of the cardiac artifacts during the occlusion time ( s) was reduced by a software low-pass filter kaiser finite duration impulse response of elevated order. results: the mean (+ sd) coefficient of correlation using eq. was , -+ . , and using eq. was . + . . the values ofz~ (eq. ), however, decreased with increasing the tidal volume (vt) according to the following equation: "~ = . - . v t, similary, the values of h~ and h increased with increasing v t according to the following functions: h~ = . + v i and h = . + . v t. conclusions: the behaviour of "% of eq. suggests that the linear viscoelastic model is not sufficient to further describe the mechanical properties of the respiratory system over the vt range ( - ml/kg) in ventilated patients. infect this model predicts that "c is constant and independent of tidal volume. on the other hand the plastoelastic model is not sufficient to further describe the mechanical properties of the respiratory system. in fact "r obtained by fitting an exponential for data of eq. , is determined by the time of endinspiratory airway occlusion. obiectives: according to the viscoelastic model, the viscoelastic pressure of the respiratory system pv=rs during lung inflation with constant flow e~ is t/ r wh t lsms ira tlmeand r given by:pv~c.~ = d~( -'e-~ )[ ] ere " ' p" tory " and "r are resistance and time constant of viscoelastic unit. in the past, the viscoaletic constants were determinated by performing a series of occlusions at different lung volumes, or a sedes of occlusions at a fixed lung volume achieved with various inflation flows. in the present study we have developed a new method for determining "c and r which requires a single constant flow inflation. our method is based on determination of pv~r, during a single breath constant flow inflation, and of z during the ensuing end-inspiratory airway occiusion. dudng the occlusion the tracheal pressure p~, declines according the following function: ptr = p'lr e " too= " z + e~t.r= [ ] where p'~r is tracheal pressure immediately after occlusion, toc c is occlusion time, p,i.rs is static elastic recoil pressure of respiratory system, and ~ is viscoelastic time constant. we first determinated "~ by analyzing the time-course of ptr according to eq and next determining r according to eq. , using the expedmental values of p,i=~, ~ and ti, as well as "~ obtained with eq. . materials & methods: we studied healthy patients intubated, anestethized with propofol, paralyzed with vecurenium, and mechanically ventilated with constant flow ( . i/s) at zeep for minor surgery. pres-sure was measured in the trachea. flow was measured with a pneumniachograph and volume was obtained by numerical integration. the rapid occlusions were produced by an external valve. the signals were sampled at a fi'equency of hz and processed on a pc. the influence of the cardiac artifacts dudng the occlusion time ( s) was reduced by a software low-pass filter kaiser finite duration impulse response of elevated order. results: the mean coefficient of correlation with eq. was . . with v t of ml/kg, the mean values (+ sd) of ': and r of the subjects amounted to . • . s and . • . cmh i "~ s. with the traditional multi breath method the corresponding values were . + . s and . _+ . cmh i " s, respectively. with the t-test the difference between new and traditional "~ was statistically significant, between new and traditional r was not significant. conclusions: with the single breath method it is possible to compute ': and r . the mean values of r with v t of nd/kg, however, was slighuy different than those obtained with the traditional multi breath method. the application of modem principles of respiratory care and mechanical ventilation in icus has resulted in increased survival of critically ill individuals with neuromuscular, skeletal and irrevers~le pulmonary diseases. in these chronically ill individunts mechanical ventilation, long term therapy (ltot) and continuous home care is considered a chronic life supporltng technique that can not be withdrawn after their discharge from an icu. the aim of this study was to present the results of a rehabilitation programme and home care that runs in our ward. twenw three patients were referred to our clinic f~om icus during - . a specific rehabilitation programme designed according to individual's needs was performed. patients that benefitted from this programme were grouped into the following disorders. ) post tb respiratow failure ( %) ) neuromuscular diseases, ( %) } undiagnosed sas { %) ) cope) ( %) ( patients had a overlap syndrom). the programme consists of : ) assessment and mechanical support ff needed of the respiratonj system with non invasive methods (nasal or via tracheostomy). ) group and individual respiratory therapy ) mobilization ) nutritional support ) educational classes for the members of the family. three from the patients passed away (during the year), are under nippv during night with or without supply, pts recieve ltot. conclusion: the development of a programme for chronically ill individuals in especially designed wards in hospitals and the overall care at home is considered necessary at least in hospitals with icus. a rehabilitation programme and home care permits the fast but safe discharge of these patients from units of acute medicine that the cost of treatment is high and besides permits beds that are invaluable. we considered that the rehabilitation prod'amine and home care in our ward is the first performed in greek chronically ill pts and even though there is no special administxative support we think that the results are quite saltsfactory. objective: we postulated that the product of the respiratory frequency (f) and the ratio of inspiratory pressure (ip) to maximal inspiratory pressure (mip) would predict the weaning outcome in deeompensated copd patients better than either variable alone or other indices previously proposed. methods: in decompensated copd patients with difficult weaning, we measured, daily, respiratory mechanics data both during mechanical ventilation and after ten minutes of spontaneous breathing. then we calculated weaning indices reported in literature and some new integrated indices. according to the results of the discriminant analysis, we considered the integrative index crop (acronym of compliance, rate, oxygenation and pressure), the rapid shallow breathing index f/vt, the load/capacity ratio ip/mip, and the following new index: f x ip/mip. we used receiver-operatingcharacteristic (roc) analysis by calculating the area under the curve considered as the overall probability of correct classification. results: main results are reported in the following objective: to evaluate the reliability of some indices of endurance in predicting the weaning outcome of decompensated copd patients. methods: in decompensated copd patients with difficult weaning from mechanical ventilation (mv) we measured, daily, blood gas analysis, ventilatory and airway pressure pattern during mv, breathing pattern (frequency (f) and tidal, volume (v~)), inspiratory pressure (ip), and maximal ip (mip) during spontaneous breathing (sb). thereafter we calculated the following weaning indices: crop (compliance * mip * (pao /pao ) / f), flvt, ip/mip. data obtained the day at which the patient was considered ready for a trial of sb on clinical grounds but weaning failed (wf) and those obtained the day of the successful weaning (ws) were compared statistically through the wilcoxon rank-sum pair analysis. in order to quantify the predictive accuracy for each index with respect to successful weaning we calculated sensitivity, specificity, and diagnostic accuracy according with the standard formulas. methods : five patients ( + yrs) suffering from ards (lung injury score > . ) for hours or less entered into the study. irv (volume controlled, decelerating flow, % inspiratory pause, lie = / ) was compared to conventional ventilation (cv) (volume controlled, constant flow, no inspiratory pause, iie= / ). these two modes were applied for hours in a randomized order, with the same levels of total peep (peept = peep + peepi), tidal volume ( . • . ml/kg), respiratory rate ( • "bpm) mad fit ( • %). measurements (respiratory mechanics, hemodynamics, arterial and mixed venous blood gases) were performed after , , and hours of application of each mode. rvsuils : are expressed as mean + sem and compared by anova. backeround and methods: periodic breathing (pb) is characterized by repetitive cyclic variation in minute ventilation. pb is considewxl to be provoked by an instability in the respiratory control. inintubated, spontaneously breathing patients conventional modes of pressure support ventilation, i.e., triggered inspiratory pressure support ps), do not allow patients to breathe with theirinherent breathing pattern. therefore, pb, if existing, will appear mainiy after extubation. since our new mode of pressure support ventilation" automatic tube compensation" (atc) continuonsly corrects for the flow-dependent tube resistance during insnmdon and expiration ("electronic" extubatim), it pemaits patients to maintain their own inherent breathing pattern. then, ff necessary, tracheal pressure can be additionally supported by volume-proportioead and/or by flow-proportional pressure support (proportional assist ventilation, pav). (~as~: we report the case of a -year-old male patient who was intubated due to acute respiratory insufficiency after acute myocardial infarction with left ventricular dysfunction. during ips of mbar the patient showed a regular breathing pattem which became periodic during atc. in addition, proportional assist ventilation of mbar/l increased periodic breathing in such a way that the typical cheyne-stokes breathing pattem occurred (see figure) . baqkground: the hering-breuer reflex (hbr) is characterized by an inhibition of inspiration during lung inflation. this response has been recognized as an important vagally mediated mechanism for regulating the rate and depth of respiration in newborn mammals. in adult man the hbr is considered to be active only at lung volumes well above functional residual capacity, i.e., at tidal volumes above ml. assessment of the hbr requires specialized methods such as single breath or multiple occlusion technique. methods; in the presence of desynchronization between ventilator and patient, which frequently occurs during triggered inspiratory pressure support ventilation (ips)(see figure) , prolongation of the interval between inspiratory efforts (indicated by negative deflection of the esophageal pressure) due to lung inflation exposes an active hbr. we examined the occurrence of hbr in intubated critically ill patients. strength of hbr was assessed by the formula: prolongation [%] = ((inspiratory interval of interest -preceding inspiratory interval)/preceding inspiratory interval) * ( . rr of patients examined showed moderate to severe desynchronization. in of these patients a (re)activation of the hbr was found. the strength of hbr amounted to + %. there was a significant correlation between tidal volume and strength of hbr. in contrast to previous reports, an active hbr was shown during lung inflation well below ml. b pck~round: triggered inspiratory pressure support ventilation (ips) is commonly used to support inspiration in intubated spontaneously breathing patients. despite its usefulness ips shows some disadvantages which can be deleterious in crificauy ill patients: -additional work of breathing to be performed by the patient due to the flow-dependent tube resistance -desynchronization between patient and ventilator due to inherent triggering failures of the ips mode suppression of the patient's inherent breathing pattern -inability to predict successful extubation in difficult-to-wean patients methods: based on the known flow-dependent tube resistance our new mode "automatic tube compensation" (atc) compensates for the pressure drop across the endotracheal tube ("electronic" extubation). then, if necessary, tracheal pressure can be supported by volume-proportional pressure support (vpps) and/or by flow-proportional pressure support (fpps). results: hitherto, we have examined patients after open-heart surgery and patients with acute respiratory insufficiency (ari) or ards using atc with/without vpps/fpps. preliminary results suggest that the new mode avoids additional work of breathing due to accurate compensation of the pressure drop across the endotracheal tube during in-/expiration prevents desynchronization between patient and ventilator allows patients to breathe with their inherent breathing pattern accurately predicts the outcome of extubation even in difficult-to-wean patients due to "electronic" extubation conclusions: the new mode atc with/without vpps/fpps allows to support ventilation in a more physiologic manner and overcomes the disadvantages of conventional modes of pressure support in intubated patients. backgound: cheyne-stokes respiration (cs) is characterized by regula]; recurring periods of hyperpnea and apnea. in normal subjects, cs may occur after hyperventilation, after arrival in high altitude, or during sleep. it has also been observed in patients with prolonged circulation time due to congestive heart failure, as well as in some neurological patients. there is no report about the influence of sedative drugs on periodic breathing (pb) and cs. methods: in intubated patients conventional modes of pressure support do not allow patients to breathe with their inherent breathing pattem. therefore, periodic breathing and cs are rarely seen. since our new mode of pressure support ventilation "automatic tube compensation" (atc) continuously corrects for the flow-dependent tube resistance during inspiration and expiration ("electronic" extubation) it permits patients to maintain their own inherent breathing pattem even if pathological, e.g., periodic. results: using this new mode of pressure support ventilation, periodic breathing was unmasked in of intubated patients, of which showed cs. in of these patients the occurrence of cs was linked to impaired left ventricular function with increased circulation time. normal left ventricular and neurologic function was found in the remaining patients. in of these patients cs disappeared after intravenous administration of the benzo-diazepine antagonist flumazenil (figure). consequently, in this patient cs was induced by benzodiazepine sedation. objecti',~s: in contrast to conventional rhodes for pressure supported spontaneous breathing, our newly developed ventilatow mode ,,automatic tube compensation" (atc) completely compensates for the flow-depandant pressure drop tlpm-r across endotracheal ttlbe (ett). in the atc mode, the ventilator supplies a flow v' in order to maintain a constant tracheal pressure p~,,~. to this end, pk,,= has to be oontinuousiy determined. since continued measurement of p,,~ by introducing a catheter via the ett is not reliable, we opted for its continuous calculation socordng to the following equation: p~ = p,,, -aperr, pw being the continuously measured airway pressure. this also requires the continual measurement .of flow v' to calculata apm-r using the non-fineer approximation: aport = kvv' + k .w. the constant tube coefficients k~ and k are mathematically determined by mesns of a least-squares-fit procadum based on laboratory investigations. tracheal secretions, however, reduca the omss-saction of the ett. consequently, ~ values of ki end k are changed rendering the p~,ch calculations inaccurate. therefore, k and ~ have to be pedodcally updated to ensure an a~urete monitoring of pn,~ and a complete tube compensation under atc at any time. background: one of the first steps in weaning patients from controlled mechanical ventilation is to stop muscle relaxation and to reduce sedation. it can take several hours, however, until the patient is able to trigger the ventilator and to breathe spontaneously. during this period, many patients display a sudden increase in peak airway pressure of up to %. patients and methods: to investigate the reason for this potentially dangerous effect, we continuously measured lung and chest wall mechanics in post-operatively ventilated patients. lung mechanics (airway resistance and lung compliance) was measured using the esophageal balloon technique as described in [ ] . chest wall mechanics (tissue resistance and chest wall compliance) was calculated from lung mechanics and total respiratory system mechanics as described in [ ] . results: we found a decrease of chest wall compliance (cw) to be the main reason for episodes of sudden airway pressure increase while lung compliance (cl) remained unchanged. the decrease of c w can be inter- gil cano a, san pedro jm ~, sandar d, herntndez . , carrizosa f, , herrero a. emergency and intensive care department, hospital of jerez, spain objective: ) to determine the incidence of hypoteasion (h) associated with emergency intabatian of mechanical ventilation, and ) to establish its relauonship with respiratory mechanics (rm) and arterial blood gases. mechanical ventilation performed in the emergency room, in a prospective eans~eative manner, were evaluated. data collected included patient demographics, diagnoses, blood pressure and arterial blood gas levels before and at~er intabatian, and p_m, including calculated pulmonary end-inspiratory volume above functional residual capacity (veic) and calculated dynamic hypetinflatien (dhc). all patients received midazolen and awaanrinm to facilitate tracheal intubatien and rm measurement. hypotension was defined as a decrease in systolic pressure higher than mmhg or an absolute decrease in systolic blood pressure below to mhg within hour of intabatian. patients were excluded because met at least one of the following exclusion criteria: preexisting shock or h ( ), cardiac arrest ( ) . there weren't any association between peepi or other airway pressures (paw) and h, but calculated pulmonary volitmes had tendency to be larger in patients with h (p < . ). high paco before lrasheal intubatian ( . - mmhg) with a quickly decrease alter starting mechanical ventilation was a usual finding (p < . ) in patients who developed h. paw. ) thexe was a good relatienship between h and high arterial paco before traqueal intahatian and its fast "washing" with mechanical ventilation. ) because cao patients had the highest incidence of h, controned mechanicel hypoventilatien driven by paco changes and pulmonary volumes monitoring instead paw, should be attempted in these patients to avoid this cemplication after tracheal intubatiert. introduction: the endotracheal tube (ett) and demand valve devices cause an added work of breathing (wobadd), which is the work necessary to overcome the resistive load of the ett and the breathing circuit ( ). application of ips has been shown to partly compensate this added work ( ). since tbe amount of wobadd is flow dependent, a fixed ips is not adequate to completly compensate the wobadd ( ). therefore, atc has been developed as a new form of assisted spontaneous breathing ( ), which provides a flow-dependent pressure support. thereby, it theoretically should compensate all the wobadd due to the tube. the purpose of this study was to evaluate the reduction of wobadd with ips and atc for different ett. methods: a mechanical lung model (ls , dr*alger, liibeck, frg) was used to generate a constant spontaneous breathing pattern. the ls was connected to an artificial trachea (at, cm long, mm id). the at was intubated with three different tubes of . , . , . mm id and connected to an evita ventilator modified to provide atc as an option (dfager, liibeck, frg). flow and airway pressure were measured between the y-piece and the ett for four different modes of ventilation: cpap, ips of and cm i and atc all with a peep of cm h . the tracheal pressure (ptrach) was measured in the at. total wobadd was calculated as the area subtended by the ptrach-volume curve below peep. results: the results for total wobadd in nd/ are shown in the figure for the three different ett: breath/mln, s=success, f=failur% *~p<. , **-p< , ns = non significant, f versus s neveltheless, in / patients, invasive ventilation was necessary in mean . _+ hours after beginning of fmpsv. there was no significant difference between the two groups (success, failure) in following parameters : sex, age, previous histoly, medical treatment, saps & , clinical signs (rr, spo , heart rate, blood pressure, glasgow score...), radiological and echocardiographic findings and standard biological parameters. only two parameters were related with failure : .a low value of pac on admission until the patients were intubated. . an increased level of cpk in relation with an acute myocardial infarction ( / cases in the failure group, vs / cases in the success group, x~(with continuity correction) : p<. ). conclusion : fmpsv is a noninvasive, safe, rapidly effective method of treatment in acpe, which may avoid tracheal intubation. further studies are necessary to precise if association of arf and low paco (< mmhg) and/er acute myocardial infarction represents an indication of immediate invasive ventilation. introduction: since the added work of breathing (wobadd) imposed by the endotracheal tube (ets and the breathing circuit is regarded as an important contribution to the total work of breathing, considerable effort has been tmdettaken to compensate for this added work. ips has been fotmd to decrease the wobadd imposed by different ventilators ( , ). because of the flow dependent pressure drop across the etf the tracheal pressure (ptr) should be measured to estimate the total imposed wobadd (wobtut) ( , ). the aim of this study was to assess the circuit imposed work (wobcirc) and wobtot (including ett) for different demand valve ventilators during cpap and/ps. methods: a mechanical lung model (ls , driiger, lfibeck, frg) generated a constant spontaneuus breathing pattern. the ls was connected to an artificial trachea (at), intubated with an . nun et]', end connected to one of four ventilators (servo c and servo , siemens,-elema, sweden; evita , driiges, liibeck, frg; pb ae, puritan bennett, carlsbad, usa). three different modes of ventilator settings were tested (cpap, ips and mbar; trigger set at maximal sensitivity, peep always mbar). flow and airway pressure (paw) were measured between the y-piece and the etr; tracheal pressure (ptr) was measured in the at. wobtot was calculated as the area under the ptr-volume curve below peep, wobcirc was calculated as the area under the paw-volume curve below peep. results: in the foti g., patroniti n., cereda m., sparacino me., giacemini m., pesenti a. inst.of anesth.and intensive care-univ.of milan -sgh monza i aim of the study was to assess cpl,rs measurement obtained by the airway occlusion method during psv. we therefore studied paralyzed cppv ventilated ali patients (lung injury score = . • that were weaned to psv. we performed end inspiratory and end expiratory airway occlusions using the hold function of the ventilator (siemens serve c), first during cppv and then within the th psv hour. airway pressure and flow signals were recorded (cpi bicore) for subsequent analysis. an airway pressure plateau was defined as a flow tracing in which airway pressure was stable for at least . sec. end inspiratory (pel,rsi) and end expiratory (pel,rse) recoil pressures were then measured as the mean airway pressure during plateaus. cpl,rs was computed as tv/ (pel,rsi-pel,rse i) cpl,rs can be adequately estimated during psv using the airway occlusion method; ) during psv inspiratory plateaus are longer than the expiratory ones; ) the length of plateaus is negatively affected by the respiratory drive. foti g., de marchi l., *tagliabue m., gilardi p., giacomini m., sparacino me., pesenti a. inst.of anesth.and intensive care,-univ.of milan *dept.of radiology-sgh monza i we retrospectively compared ct scan and gas exchange findings between a group of patients successfully weaned from vcv to psv (group s = ii patients) and a group who failed the weaning (group f = patients). we selected ali patients (lis= . • in vcv mode who had available a chest ct scan performed within days from the weaning trial. a psv trial was began as soon as the patient reached hemodynamic stability and a pao > mmhg, irrespective of fie (peep < cmh ). maximum psv level was < (pel,rs-peep) measured during vcv, where pel,rs was the respiratory system elastic recoil pressure at end inspiration. psv ventilation was considered successful if a respiratory rate < bpm, an increase in fie lower than . compared to vcv, a pace increase < % of vcv value and hemodynamic stability were maintained during the next hours of psv. if any of these conditions was not met the trial was declared a failure. interdisciplinary critical care unit, regional hospital lugano-ch *surgical critical care unit, university hospital, geneva-ch objective: to assess the degree of correlation of cardiac output measured by thoracic electrical bioimpedance and thermodilution in mechanically ventilated patients with different levels of positive end-expiratory pressure (peep). methods: prospective study with ventilated patients, after head injury and with postoperative sepsis, with normal cardiac output: simultaneous determination of cardiac output by thermodilution and thoracic electrical bioimpedance performed with different levels of peep ( - - cm h ). results: cardiac output measured by thermodilution during sequential increment of peep did not vary: . + . for peep , . + . for peep and . + . l/rain for peep . simultaneously the bioimpedance device recorded a significant increase in cardiac output from . + . for peep to . + . l/mi for peep . (p < , ). conclusion: cardiac output measured by bioimpedance cannot replace the invasive thermodilution methods of cardiac measurement output during mechanical ventilation with peep. we also isolated a subset (h) of patients who had been hypercapnic (paco > mmhg) for at least days (range to days) before the end of cv. the psv trial was started as soon as pao was > mmhg, irrespective of fie and with peep < cmh and the psv level had to be < (pplateau-peep) as measured during cv. pace , pha, base excess (be) were collected before discontinuation of cv and on the ist day of psv: ) . ) weaning is more difficult in pts with head injury(p<o, ) and iss> (p<o,ol), in elderly(p<o, ) and in pts who need fi > , (p<o, ) and peep>io cm h (p<o, ). ) pts with iss> need longer duration of mv (p<o,ol). ) the mortality rate is higher in pts with iss> (p<o,o ), with head injury (p<o,ol) and in elderly (p<o, ). ) paradoxally, compliance was found to be higher in pts> years than in pts< years (p<o, ). s. crotti, p.pelosi, d.mascheroni, m.cerisara, a.lissoni, l.gattinoni. istituto di anestesia e rianimazione -irccs, milano, italia. obiectives. we investigated by ct scan the effects of extrinsic peep (peepe) on lung inflation, tidal volume distribution and regional compliance in sedated, paralyzed chronic obstructive pulmonary disease (copd) patients with dynamic hyperinflation (peep• methods. two ct section (end expiration, end inspiration) were obtained at lung base level in patients (individual analysis for each of the eight lungs) at peepe levels: peepe = cmh (zeep) and peepe amounting to %, % and % of the peep• at zeep ( . • cmh ). tidal volume was kept constant ( • . ml/kg). each lung section was divided into zones equally spaced along the vertical axis: upper (the most ventral), middle and lower (the most dorsal) zone. for each zone we computed: gas volume at end expiration (gase) and at end inspiration (gas• tidal volume (dgas=gasi-gase) and compliance (cpl=dgas/dp;.dp=plateau pressureactual peep• we also computed the lung inflation as the entire ct section gas volume at end expiration (total gase). results. at each peep level both gase and gas• were higher in the upper than in the lower zone ( we cone!ude that in copd patients with dynamic hyperinflation only the application of peepe higher than peepi produces a further increase of lung inflation, decreasing cpl of the upper zone (the more expanded one) probably by stretching phenomena, and causing dgas redistribution from the non dependent to the dependent lung zones. i. ravagnan, p. vicardi, f. valenza, d. tubiolo. p. pelosi l. gattinoni. st . anestesia e rianimazione, universita' di milano, ospedale maggiore -irccs, italy objectives: to investigate the effects of peep and ps on respiratory. pattern and inspiratory effort during ps ventilation in patients with acute lung injury or acute exacerbation of chronic lung disease. methods: pts with acute (a) (paco = • mmhg, pao /pao = . • and pts with chronic (c)(paco = • mmhg, pao /pao = . : . ) lung disease were studied in ps ventilation during the weaning phase. measuring airway pressure, esophageal pressure and gas flow we computed respiratory rate (ril bpm), tidal volume (tv, ml) and pressure time product (ptp, cmh *s/min), which is an index of oxygen muscle consumption. measurements were collected, after rain of stabilization, at and cmh ps and at two different peep levels ( and cndd conclusions: during psv: ) breathing pattern and ptp are different between a and c; ) in both groups peep does not modify breathing pattern; ) peep reduces ptp in c but not in a; ) ps modifies breathing pattern and reduces ptp in c but not in a. to test the performance of a new heat and moisture exchanger (hme): twistair, baxter. this is a hydrophobic-hydrophilic hme without hygroscopic salts. method: we evaluated the hme performance with a previously described method (i); it consists of a "lung simulator" connected to a mechanical ventilator and a flow divider, with a dry and a wet thermal probe inserted into inspiratory and expiratory limbs. on average ps level changes were associate( with opposed changes in both p . and rr. changes in p . were remarkable, homogeneous and highly significant (p<. ), while changes in rr were less pronounced, inhomogeneous and less significant (p<. ). it seems therefore that p . is a better index than rr for estimating a change in load for the respiratory muscles. recently, the lsf method has been described as an interesitng alternative to conventional tecniques used to measure total respiratory mechanics, providing, over these latter, advantages such as no need for hold maneuvers and no need for particular flow patterns. data on the reliability of the lsf method, however, are still few. methods. sets of measurements were obtained in each of ards patients, paralyzed and ventilated in cmv with constant inspiratory flow and an end-inspiratory pause equal to % of ttot. the lsf method provided data for total compliance (ctotlsf) and total resistance (rtotlsf) by multiple linear regression analysis of airway pressure, flow and volume change, applied over the entire breath. the lsf measurements were automatic and continuous, and each value used for analysis was the average of consecutive cycles. conventional measurements of dynamic.compliance (cdyn), quasistatic compliance (cqs), minimum inspiratory resistance (rmin) and maximum inspiratory resistance (rmax) were obtained by the constant flow, end-inspiratory occlusion method, each value being the average of measuremts. ctotlsf was compared with cdyn and cqs, while rtotlsf was compared with rmin and rmax. results. ctotlsf showed a high correlation both with cdyn (cdyn=. .ctotlsf+l. , r =. ), and with cqs (cqs =. .ctotlsf + . , r =. ). the average difference between ctotlsf and cqs, yet, was much lower (+. + . ml/cmh ) than the one between ctotlsf and cdyn (+ . + . ml/cmh ). rtotlsf correlated much better with rmax (rmax=. *rtotlso+. , r =. ) than with rmin (rmin =. .rtotlsf +. . , r =. ). the average difference between rtotlsf and rmin was + . + . cmh / /s, while the one between rtotlsf and rmax was +. + . , confirming the better agreement between rtotlsf and rrnax. conclusion.the agreement between rtotlsf and rmax suggests that rtotlsf takes into account both the airway and the tissue components of resistance, unlike rmin which only expresses airway resistance. the agreement between ctotlsf and cqs indicates that ctotlsf expresses the pure elastic components of the respiratory system. in the paralyzed patient, the expiratory time constant (rce) of the unit represented by total respiratory system and ventilator can be calculated from the slope of the expiratory flow-volume curve. recently it has been suggested that an estimate of this slope is provided by the ve/v'epeak ratio, ve being the exaled tidal volume and v'epeak the peak expiratory flow.the reliability of this method is still little known. methods. sets of measurements were obtained in mechanically ventilated ards patients during paralysis and cmv. measurements of ventilator expiratory resistance (rext) and of total respiratory mechanics were performed in order to calculate these reference measurements for ve/v'epeak: rcdyn=(rmin+rext)ocdyn, rcstat=(rmax+rext)ocqs, rclsf=(rlsf+rext)~ cdyn (dynamic compliance), cqs (quasistatic compliance), rmin (minimum inspiratory resistance), rmax (maximum inspiratory resistance) were obtained by the constant flow, end-inspiratory occlusion method. clsf and rlsf respectively corresponded to measurements of total respiratory system compliance and resistance by the least squares fitting method. ve/v'epeak showed a high correlation with all reference measurements, rcdyn (ve/v'epeak= . orcdyn-. , r =. ), rcstat (ve/v'epeak = . rcstat -. , r = . ), and especially rclsf (see fig the static pressure volume curve of the respiratory system (p/vst) provides valuable information, but the extensive data analysis prevents its use in clinical routine. dynamic p/vcurves are simpler to record. the objective of this study was to develop an automated method to obtain quasi-static p/v-curves (p/vqs) during low flow inflation as well as to measure resistance. preliminary tests were done in normal subjects and subjects with varying degree of lung disease. methods: a computer/ventilator interface (cvl) was constructed for control of a servo ventilator c during an automatic sequence: a) a normal breath, b) a prolonged expiration at zero peep, c) a low flow inflation of a predetermined volume, d) analysis of ventilator pressure and flow. pressure was corrected for tube resistance. airway resistance was calculated from a) and used to derive p/vqs from c). as reference, p/vst was obtained by the occlusion method. results: in non-obstructive diseases the method performed well at volume and pressure controlled ventilation. static and quasistatic compliance were virtually identical. p/vqs clearly reflected the lower inflection point and, when present, the upper inflection point (uip). in some patients uip was more evident in p/vqs, especially at an increased inflation flow rate. in obstructive diseases, the method for subtraction of resistive pressure was inadequate. conclusions: the system for automated computer controlled studies of lung mechanics based on a standard ventilator provides comprehensive information. differences between p/vst and p/vqs imply that the latter reflects also other factors than p/vst that contribute to impedance at hyperdistension. the clinical significance of the two curves remains to be shown. obiective: to evaluate the effect of combined high frequency jet ventilation (chfjv) in patients with severe respiratory insufficiency (sri). methods: in a retrospective study of the period - we analyzed patients suffering from sri who were refractory to conventional mechanical ventilation and were treated with combined high-frequency jet ventilation (chfjv). refractory to conventional mechanical ventilation was defined as a pao /fio < , despite maximal ventilator settings: peep > cm hz , fit > . . a total of patients matched these criteria, males and females with a median age of ( - ) years. seventeen suffered from severe trauma. chfjv was started following a median period of ( - ) days of conventional mechanical ventilation. prior to chfjv ventilation parameters expressed as median were the following: fit . , pao /fio , peep cm h peak airway pressure (pap) cm h . chfjv consisted of high frequency jet ventilation with a frequency of to breaths/minute, driving pressure of . to . arm, and inspiration time of to percent, superimposed on the whole cycle of conventional mechanical ventilation with a frequency of l to breaths/minute and tidal volumes of to ml. results: following two days of chfjv of patients showed an improvement of ventilatory parameters; peep could be reduced to < cm h in patients, the pap was decreased with > cm h:o in patients, fio could be reduced to < . in patients and finally the median pao /fio ratio changed from to . during chfjv patients died, of respiratory failure and due to multiple organ failure, died within two days of chfjv. the median duration of chfjv in survivors and nonsurvivors was days in both groups. conclusions: our data show that with chfjv in the majority of patients with sri who are refractory to conventional mechanical ventilatior" the ventilatory parameters can be improved. backeround and obiectives: although ventilation with peep above the inflection point (pinf) has been shown to reduce lung injury by recruiting previously closed alveolar regions, it carries the risk of hyperinflating the lungs. in the present study we set out to develop a new strategy to recruit the lung during ventilation with small vt, while maintaining peep levels as low as possible. we hypothesized that if the lung was recruited with a sustained inflation (si) to total lung capacity, recruitment would be maintained as long as the peep level was higher than the critical closing pressure of the lung, as observed on the deflation limb of the pv curve (ajrccm ; ( ) :a ). the purpose of this study was to examine the hypothesis that a strategy using si and a peep<pinf would induce less lung injury during small tidal ventilation than ventilation at the same low peep level without using a recmitment strategy in a model of respiratory distress syndrome. methods: we used a randomized protocol with groups to ventilate nonperfused, lavaged rat lungs with small vt ( to ml/kg) for hours and studied the effect on compliance and lung injury. group : peep>ping group : peep<pin f with an si, inflation to on h over sec, to recruit volume; group : peep<pinf without si; group : control group, lungs were inflated at peep<pin f, but not ventilated. results: in group and group static compliance did not change after ventilation (table) . in group static compliance fell significantly. group showed significant changes in the pv curve, which were less severe than in group . results from morphological examination are pending but preliminary results showed less lung injury in group , compared to group . conclusions: small tidal ventilation following a recruitment manoeuvre in a model of respiratory distress syndrome allows ventilation on the deflation limb of the pv curve at a peep below pinf. this strategy ( ) minimizes lung injury as well as, or better than using peep above pint" and ( ) ensures a lower peep to minimize the detrimental consequences of high lung volume ventilation. i~peep>pin~ _objectives-this report is presenting the results of the clinical study for using eeg examination as a method of the evaluation of patients ability for weaning. methods: the study inclljqles eeg examinations with fourier spectral analysis' of patients ~vith respiratory insufficiency and prolonged control mechanical ventilation (cmv). all patients have had a-rhythm of eeg before weaning. we have followed respiratory rate, tidal volume, respiratory pa{tern, end-tidal co and blood gases during weaning. results: patients had invariable eeg activity or short -waves period (till one hour). the weaning of this patients was fast arid sucsessful. other patients have had a decreasing of a-activity, an appearence of -waves for an hour and more, a short episodes of a-and e-activity. after that this patients had gas exchange and respiratory disorders with regression of the weaning right up to cmv. conclusion: eeg could be used as a method of the evaluation of patients ability for weaning from cmv. some eeg signs shows the overstrain of compensatory systems before the change to the worse of gas exchange and respiratory pattern. s. elatrous, p. aslanian, d. touchard, d. corsi, h. lorino, l. brochard. medical intensive care unit, inserm u , hopital henri mender, cr~teil, france. in vitro comparison of flow triggering (ft) systems demonstrated advantages compared to pressure triggering (pt) systems for some ventilators (puritan bennett ) but not others (siemens serve ). we studied the two types of systems in two groups of patients mechanically assisted with pressure support ventilation ( + cmh ). in the first group (pb ) the effort of breathing, assessed by the esophageal pressure time index, was significantly lower with the ft than with the pt ( + cmh .s/min - vs + , p< . ). by contrast no significant difference appeared in the second group (serve ), as predicted by the bench study despite marked interindividual differences ( + cmh .s/min - vs + , p = . ). we conclude that ) rigorously performed bench studies can predict in vivo effects, ) mild advantages can be found for the new triggering systems on some ventilators. objectives: pressore-volume curves (pv) of the respiratory system is of interest for the determination static compliance (cs , lower (lip) and upper (uip) inflection points which indicate zones of airway recruitment and overdistension. this study aimed to compare an "automated low flow inflation" method (alfi) to the reference occlusion (oc) method. the ability of the former method to identify cst, lip and uip was tested in icu patients. me,otis: ( arf and ards) sedated paralysed patients were studied using a serve c ventilator linked to a computer which automatically forced the ventilator to insufflate at a low constant flow a velum up to - ml or a maximum paw of cm h (alfi). the quasistatic elastic pressure (pel,qs was obtained by subtraction of the resistive pressure of tubing and patient and related to volume for calculation of compliance cqst. for oc tidal volumes (v from up to - ml were followed by a s post-inspiratury pause for determination of static pal (pel,st) in relation to volume. compliance was defined from the linear part of the p/v curves. lip and uip were defined from the consistent deviation of p/v data from extrapolated the linear part. ~,~ i~: in ards, mean cst was . + . and cqst . + . ml/cm h (us), lipst . + . and lipqst . + . cm h (us), uipst . + . and uipqst . + ~ cm h (us). nosocomial pneumonias (np) are frequent and often unsuspected during ards (bell, ! ). in the present study, we evaluated prospectively the onset of np during severe ards (group b of the european study). patients and methods: the charts of patients with severe ards have been prospectively recorded. a plugged telescopic catheter (ptc) specimen has been systematically performed every hours, for quantitative bacteriological analysis. the diagnosis of np was defined by a number > colony forming units / ml. results: for the patients studied, the mean saps score (+ sd) was +_ , the initial pao /fio ratio was -&-_ , the duration of mechanical ventilation (mv) was + days. the mean delay before the onset of the first np was . + . days ( - ), and the mean pao /fio ratio was +- . respiratory symptoms (purulent aspirates, new pulmonary infiltrates, or gazometric changes) were present in % of the patients studied. alteration of gas exchange was present in of the patients ( np) . a new pulmonary infiltrate was present in only np ( %). an increase of fever was noted in patients, an increase of leukocytosis > % in patients, an increase of volume and purulence of sputum in of the patients with np. the degree ofgazometric worsening (pao /fio before np minus pao /fio during np) during the first episode of np was + mmhg. excluding the bacteriological criteria of np, the number of criterias of np present was in / patients, ( / ), ( / ) or ( / ). two patients only had a pulmonary colonization (ptc: < cfu / ml) before the first episode of np. the incidence of np is high ( %) during severe ards. the first episode occurs in average:at the th day, and is the cause of a severe hypoxemia (pao /fio ) . the onset of a np may contribute to the high mortality rate observed in our patients ( %). each worsening of hypoxemia during severe ards should induce to suspect a np. respiratory system during mechanical ventilation. the me~hod quantifies the dissipative energy consumption of the respiratory system in terms of energy loss aek, inefficiency ~k~ and respiratory dissipative resistance rk~ over a given partition of the tidal volume. the method can be applied in intensive care units with no interference to ventilatory support. it allows for monitoring the combined effects of inhomogeneities, non-linearities and visco-elastic effects, that are subject to change in the respiratory system. the method is studied on pigs~ in the presence of a log-dose response curve of methacholine (mch) induced disease. in healthy pigs~ we find a mean value of energy loss, ae, of . • j/l, a mean value of inefflency, ~ of . ~= . and a mean value of resistance, ~, of . • cm h s/ . the respiratory resistance, rk, shows a variation over the partition of tidal volume with armax ---- . • . cm h s/l. during methacholine provocation~ ae rises more than five-fold up to . • j/l~ doubles to . • and t~ increases to a maximum of • cm h s/l, with armax : . • . cm h s/ . the variation in rk becomes more pronounced with higher doses of methacholine. methods: ards patients were prospectively studied. initially they were ventilated in the amv (assist mechanical ventilation) mode with the settings prescribed by their primary physician. after stabilization, ventilatory gas exchange and hemodynamic variables were determined. patients were then ventilated in the mrv (mandatory rate ventilation) mode with breaths as the target rate. in mrv the target rate is set and the ventilator autoregulates the pressure support level delivered ~o achieve this rate. after stabilization, the measurements done on amv were repeated. finally, patients were sedated and paralyzed and ventilated in cmv (control mechanical ventilation) with the ventilatory variables they had during mrv. measurements done in amv and mrv were repeated and respiratory mechanics were assessed with the constant flow end inspiratory occlusion method. results: two groups were recognized based on their response to mrv. tn group patients responded to mrv by decreasing their v and increasing the t/t t ratio. ve, vo , and aado decreased while paco increased and tda vo ume and co remained unchanged. on the contrary, in group v, vr and ve increased; ppeak and trr t remained unchanged, paco~ decreased while vo and aado increased with constant co, the pressure support level needed to achieve the target rate was much lower in group than in group ( , -+ . vs . _+ . ). obiectives : in the newly developed mode of ventilatory support ,,automatic tube compensation" (atc) the ventilator compensates for the flow-dependent pressure drop across the endetracheat tube (ett) thus allowing ,,e]ectronic extubation". the aim of the study is to investigate whether healthy subjects perceive atc in inspiration (atc-in) and in expiration (atc-in-ex) and whether atc provides an increase in subjective comfort compared with the conventional assisted spontaneous breathing mode (asb). methods : healthy volunteers (no preceding lung disease, non-smokers, male, - years)breathed spontaneously through an uncut ett of . mm id via a mouthpiece. the ett was connected with a prototype ventilator evita modified by the manufacturer (drfiger, lebeck) for atc. flow and airway pressure were measured at the outer end of the ett. three ventilatory modes, ( ) asb ( mbarover mbar peep), ( ) atcin, ( ) atc-in-ex were selected in random order. immediately following the transition from one mode to another the volunteers answered by hand sign how they perceived the new mode compared with the preceding mode: ,,better" (+ ), ,,equal" ( ) or ,,worse" (- ). inspiration and expiration were investigated separately by presenting mode transitions (in total; including ,,placebo" transitions). results : the difference between atc and conventional asb is perceived in inspiration and in expiration. atc is positively judged; asb is nega ively judged. the diagrams show mean values _+ sd of five volunteers investigated up to now. the new mode atc is perceived as an increase in subjective comfort. our explanation is that atc preserves the natural breathing pattern better than conventional asb. objectives: to determine the role of cerebral vasoconstriction in the delayed hypoperfusion phase in comatose patients after cardiac arrest. to correlate the results with indices of cerebral oxygenation and the levels of several vasoactive hormones in the jugular bulb. methods: in comatose patients after cardiac arrest we measured the pulsatility index (pi) of the medial cerebral artery by transcranial doppler sonography. the pi is a reliable indicator of cerebral vascular resistance. we also sampled blood from the jugular bulb and measured cerebral oxygen extraction ratio and jugular bulb levels of endothelin, nitrate and cgmp. the first measurement was done within hours after cardiac arrest and repeated , , , , and hours later. results: we studied patients, females, mean age , + , years. the pi decreased s!gnificantly between th~ first and the last measurement from . _+ . to . + . (p = . ). cerebral oxygen extraction ratio decreased also from . + . to . + . (.p = . ). endothelin levels were high, but didn't change during the studied period. nitrate levels varied in a wide range, but didn't change significantly. however, cgmp levels increased significantly from very low levels in the first measurement to very high levels hours later, rasp. . pmol/ml (median; th . - th . ) and . pmol/ml (median; th . - th . ) (p = . ). eighteen and hours after the first measurement we found a strong correlation between pi and cerebral oxygen extraction ratio ( r = . , p = . and r = . , p = . ). we.also found hours after the first measurement a significant correlation between pi and cgmp levels ( r = . , p = . ). we found no correlation between pi and endothelin or nitrate levels. conclusion.~; our results show a high cerebral vascular resistance in the first few hours after cardiac arrest, gradually decreasing during the next hours. this is accompanied by an initially high cerebral oxygen extraction ratio and low cgmp levels, suggesting that the cerebral vascular resistance is induced by active vasoconstriction because of insufficient cgmp levels, leading to a decrease in cerebral blood flow and a compensatory ~ncrease in cerebral oxygen extraction. objectives: sudden cardiac arrest is a major cause of mortality in western countries accounting for over half of all cardiovascular deaths. in most cases the mechanism of death is prolonged cardio-circulatory arrest due to ver:tricular fibrillation (vf) preceding final asystole. recurrent syncopes due to idiopathic vf with good neurological prognosis have been reported in patients with and without cardiac etiology ( , ). in the past measurements of cerebral hemodynamics have been repeatedly done in humans during cpr, but until today no studies of cerebral blood flow velocity (cbfv) have been reported during controlled cardiac arrest in humans not under-going cpr. it was the purpose of our study to evaluate the acute hemodynamic effects of untreated vf on cbfv. methods: after approval by the local university ethics comittee, five male patients aged - years without evidence of cerebral disease were investigated during vf while undergoing implantation of a pacer cardioverter defibrillator system (model d; medtronic| a standard anaesthetic regimen was used (propofol, fentanyl). after implantation of the automated cardiac defibrillator vf was induced by electrical countershock to test effective sensing, pacing, and defibrillation. to measure cerebral blood flow velocities (cbfvmca) the doppler probe was placed above the zygomatic arch between the lateral margin of the orbit and the ear and directed towards the m segment of the middle cerebral artery (mca). results: a total of phases of vf were investigated. duration of vf ranged from to seconds, with cbfvmc a (mean_+sd, cm sec - ) flow pattern changing from pulsatile to laminar flow immediately after onset of vf. conclusions: the underlying mechanism of the laminar cerebral blood flow observed during vf in our patients is uncertain, but it may provide insight into the prognosis of patients with idiopathic vf. theoretically, the laminar cerebral blood flow observed in our pulseless patients may provide a substantial amount of cerebral perfusion even during clinical cardiocirculatory arrest objective: to investigate whether the intensive care nursing staff can inflate more accurately a specific air volume with the laerdal resuscitation bag when they receive feedback after each inflation about the delivered volume compared to no feedback. method: icu nurses were asked to inflate a testlung model times with a specific air volume ( ml, ,ml or ml) under three different conditions (normal, decreased compliance and increased resistance) without and with feedback. we measured the mean absolute difference from the specific airvolume after each ten inflations. results: the largest absolute difference was found when icu nurses inflated ml ( ml). the mean inflated volume for this group was ml. when the icu nurses had to inflate ml the mean absolute volume difference was ml with a mean inflated volume of ml. inflating ml produced an absolute volume difference of ml with an mean inflated volume of ml. the absolute volume difference decreased when the compliance of the testlung was decreased and even more when the resistance of the used endotracheal tube was increased. when the icu nursing staff received volume feedback after each inflation the mean absolute volume difference was reduced between the ml and ml for all specific air volumes. % of the last inflations with feedback were significantly smaller than ml from the specific air volume (p < . ). conclusion: the majority of nurses overinflated the specific air volumes. the largest over inflation occurred when ml and the smallest when inflating ml. when nurses were provided with volume feedback the performed significantly better. we concluded that icu nurses are not able to inflate a specific air volume with the laerdal resuscitation bag without receiving volume feedback. feedback is desirable in order to reduce the volume trauma. objectives: a pro_found impairment in systolic and diastolic myocardial function following successful cardiopulmonary resuscitation (cpr) has been demonstrated by using langerdorff method in rats. in the present study we have investigated post resuscitation myocardial dysfunction in a porcine model of cpr. methods: ventricular fibrillation (vf) was electrically induced by alternating current applied to the ep{cardium of the right ventricle in domestic pigs. following rain of untreated vf, precordial compression and mechanical ventilation was initiated and maintained for min. electrical defibrillation was then attempted and of animals were successfully resuscitated. results: following successful cardiac resuscitation, stroke volume index (svi) decreased from prearrest value of . ml/kg to . ml/kg (p< . ), and left ventricular stroke work index (lvswi) from . to . mmhg,ml/kg (p< . ). both svi and lvswi remained depressed for another hours. these decreases were associated with increases in heart rate from bpm to bpm (p< . ). no significant changes from baseline in mean arterial pressure, mean pulmonary pressure, right atrial pressure and pulmonary artery wedge pressure were observed. prehospital resuscitation efforts c. k ppel. g. fahron, h. lufft, a. kruger, c. th(jrk, f. bertschat, f. martens dept, of nephrology add medical intensive care, virchow-klinikum, humboldt-universit~t, d- bedin, germany obiective: the success rate of prehospital resuscitation in patients with cardiocirculatory arrest in an emergency medical system (ems) may reach - % depending on the time of calling the ems, the distance to cover by the emergency ambulance and the training of the emergency physician and his staff. in the berlin ems, which is associated with the berlin fire brigade, the time between alarm and arrival at the scene ranges from - min, mean min. resuscftation is based on the advanced cardiac life support (acls) according to the guidelines of the american heart association. if resuscitation efforts fail to restore circulation, they are terminated after - min, depending on duration of cardiocirculatory arrest, pre-existing disease, age, absence of an even transient response to cpr. however, there is a lack of practical criteria for termination of cpr in individual decision making. patients: we report cases of prehospital cpr with primary asystolia terminated after - rain of frustraneous cpr efforts including highdose epinephrine and dopamine. results: after termination of cpr, the ecg monitor remained connected and showed permanent asystolia in all patients while the emergency physician completed his records. spontaneous resumption of respiration and circulation was observed in these patients after - min and cpr efforts were immediately resumed, nevertheless, of the patients died at the scene, while could be hospitalized with stable circulation. one of them died hours after admission to the icu, the other survived for weeks in a vegetative state. spontaneous resumption of circulation and respiration is most likely due to the development of extreme hypercapnia and acidosis, which -at least in some patients -seems to be a stronger stimulant of the circulatory and respiratory brainstem centers than cpr with high-dose catecholamines, conclusion: because of the legal and ethical implications of this rare phenomenon, emergency physicians should continue ecg monitoring for at least rain. after termination of cpr efforts. pulmonary artery catheterezation is used for patient's monitoring [ ]. we reported our results on such monitoring in [f.coaobbeb,r.fe enb~-kap~monorm~, ,n ,p. - ] .however not all of the received criteria assessments meet demands that are necessary for early diagnosis of critical states. here we report the data on po ,pco (mm rg),so ,ph levels in femoral [af) and pulmonary (ap) arteries blood, as well as on summary gas pressure (sgp) calculated from pe=(po +pco ) in mm hg in ap blood. these data were derived from:i) subjects free of cardiovascular pathology according to catheterization data during their spontaneous air breathing (n group in ap blood appears to be a measure of adequacy ratio between pc and sgp in ap blood during air breathing; partly its characteristics and variations ranges are presented earlier [ j. in control group it is equal to , • mm hg. tests on sgp neither exclude nor substitute conventional (pc and pco ) tests, but rather include them as a part choosing only additive characteristic -pressure. they appear to be a part of general system of human metabolism regulation by pressure (arterial,venous,intracardiac, tissue,liquor,onco-osmotic,etc ietraabdeminal pressure produces perturbations of cardiac, pulmonary, and renal physiology. this most often occurs fonowing eeliotomy for peritonitis or intestinal obstruction; bowel edema and distention prevent wound closure without unacceptable compromise of blood pressure or pulmonary compliance. a variety of temporizing measures have been reported for managing wounds that cannot be closed: ) using towel clips to reapproximate skin only, )i sewing silastic, marlex or other prosthetic grafts to the fascia to "enlarge" the peritoneal cavity, ) using loosely tied retention sutures for partial closure, ) simply packing the wound without attempts at c~osure. these techniques either traumatize the abdominal wall (complicating definitive closure), expose the bowel to damage, or allow excessive loss of fluid and heat. since we have evolved a suturelees technique which permits the abdomen to be partially closed in a quick, safe, sterile, sealed, atraumatic fashion -while providin! decompression of unphysiologic intraabdominal pressure. methods: whenever possible omentum is interposed between bowel and the open incision. viscera are covered by a layer of sterile, non-reactive plastic, placed deep to the fascia and extending we~t beneath the edges. sump tubes are placed above the plastic and covered in turn by two layers of an adhesive plastic drape which sticks to the skin and seals the wound in all directions, the patients remain intubated and paralyzed. results: we have used this technique in a total of patients, four of whom suffered from compartment syndrome. all of the latter were males and ranged in age from to . all four showed immediate physiologic improvement. all four incisions were eventually closed without complication. one compartment syndrome patient died t days later of multiple organ failure. there were no complications related to the closure technique in any of the patients. conclusions; . selected patients with abdominal compartment syndrome will benefit from decompression using this temporary sutureless technique. the technique a) is quick, safe, sterile, sealed, and atraumatic, b) minimizes loss of fluid and heat, c) facilitates eventual definitive abdomina| closure. although m. brunner m. mitllncr objectives: to determine incidence and predisposing factors for cardiac arrest occurring during the first hours after open heart surgery. methods: the study included patients who, following open heart surgery, had adequate cardiac function and in whom cardiac arrest was not anticipated. all data were prospectively recorded and analyzed. results: from / through / , pts underwent open heart surgery at our hospital. of th~se, pts ( %) (age _+ yrs) had a cardiac arrest during the first hours after transfer to icu. they were operated on for coronary artery bypass grafting (cabg) ( pts), valve replacement (vr) ( pts), cabg and vr ( pts) and aortic aneurysm ( pt). the preoperative ejection fraction was _+ % whereas bypass and aortic cross-clamp time were + and + rain, respectively. prior to arrest, they had a cardiac index of . _+ . l/min/m and were receiving . + inotropes. arrythmias leading to cardiac arrest were ventricular tachycardia/fibrilation ( pts) and bradyarrythmia ( pts). closed-chest cpr was initially performed on all pts and was followed by open-chest cpr in pts. eighteen pts ( %) survived to icu discharge. causes of arrest included perioperative myocardial infarct (t pts, %), tamponade ( pts, %), rupture of the proximal vein gra& anastomosis ( pt, %), graft occlusion ( pts, %); no cause was found in pts ( %). conclusions: postoperative cardiac arrest in stable cardiac surgery pts is relatively infrequent (- % incidence) and is associated with a high survival rate following successful cpr. perioperative myocardial infarct is the most common predisposing factor. group ~deptof anaesthesia and intensive care, semmelweis univ. medical school, buda military hospital intensive care unit, budapest background: when a cardiac arrest occurs in-hospital, the outcome can be improved by a higher quality of basic life support provided by the witnessing health care workers until the code team arrives. this basic life ~pport (bls) should include the best available method for airway management as well. since not all medical staff are ready for carrying out endatracheal intnbation, we investigated the effieacy of the use of different airway management methods during bls. methods: we have investigated the efficacy of airway management of doctors and nurses from different hospital wards: internal medicine, department of surgery, trauma, urology and gynaecolagy. comparing the bag-valve-mask, laryngeal mask and the endotracheal intubafion, we have measured the following parameters: time needs for correct application (sec.), number of incorrect applications (out of ten trial), efficacy of artificial ventilation provided by the device. we used a computerised als trainer manikin for the evaluation of the performance. total performance score was created after the measurement between - . after the first screening we held a x hours training. doctors and nurses were trained for the endotracheal intubation (group it , t ) , doctors and nurses were trained to use the laryngeal mask (group lm , lm ) . all respondent were trained to use the bag-valve-mask device. day, month and month after the training we have carried out retention study using the same method. results: we have found that the efficacy of the artificial ventilation using the above mentioned devices were poor before the training. the average after-training performance scores of the groups are presented in the table below. (bls) should be initiated by the witnessing health care professional. the cpr study introduced a multi level code system, which means bls included sophisticated airway management, early defibrillation and early epinephrine administration provided before the code team arrives. our previous studies confirmed a poor level of cpr performance and a high demand for cpr training among health care professionals. method: we established a cpr training course centre, where doctors and nurses are being trained for in-huspital basic and advanced life support. x hours of training were held. after the theoretical introduction a step-by-step training method ws used for trainees to be familiar with all sequences of basic and advanced life support. then we synthetised all separated sequences. afterwards, a r e play of rescue groups was taken in simulated situations. we also trained the multi level alarm system fur the in-hospital resuscitations. after the training all respondents had to sit for examination. the quality of performance was scored and compared to our previous results. semi-structured interviews were carried out before and aider the training among all respondents to collect information about the course. results: we have found a remarkably high interest among doctors and nurses in our cpr training courses. it was very important to use proper equipment for the training: audio-visual training facilities, computerised als trainer manikin, manual and automatic defibrillator units. the evaluation of the examination held immediately a~er the training course showed a significant higher quality of performance than before the training. the self.-eonfidence of the trainees for initiating and carrying out resuscitation had increased. their overall feeling about the course was positive and % responded the course "very useful". . % of doctors and . % of nurses claimed fur regular training facilities with als trainers, conclusion: the cpr training for health care werkers is mandatory including the training of sophisticated airway management and use of elad~l~ills~tt~r wlaa ~en ~r a~ti~atir ~nel r rm~a'*h*nr m~thnd for training will improve the efficacy, the satisfaction of trainees, therefore their compliance for further co-operation will also increase. s objectives: the effect of reinfusion in emergency surgery and gynecology. methods: we had an experience of autologous blood transfusion in patients whom was produce t an emergency surgical or gynecological interventions in occasion with break tubal pregnancies ( . %), penetrating abdominal wounds with injuries of mesenterial vessels ( . %), injuries of the liver ( . %), blunt abdominal trauma with lien ruption ( . %). in . % patients had the previous somatic pathology. blood loss volume was - ml, & the reihfuside blood volume was - ml, consisting - % of blood loss. it was needn't to fransuse donor blood in . % in further but - ml of contanined erythrocytes were frasfused for supporting of hb concentration on the g/l ( g/dl) rate at the other patients with isovolemie hemodiluttion. results: the arterial blood pressure fast stabilisation on the perfusion level had noted after reinfusion, excluding the case, when the volume of reinfused blood had conisted just % of blood loss at the patient with massive blood loss. complications have noted in two cases. one patient with slash wound, injury of arteria gastrica dextra and total blood loss of ml, has an episode of asystoly, dic (disseminated intravascular coagulation) syndrome, acute renal failure, and acute pancreatitis that we haven't connected to reinfusion. all the complications were successfully corrected and at thirty first day patient with subcapsular wound of the lien that has happened days before complicated with external rupture of the capsull & massive intraabdominal bleeding, has the hemolytical shock, dic syndrome, acute renal failure developed after reinfusion. he was died. all another have no complications. posthemorrhagic anemia had corrected rapidly than in case when hemorrange corrected exclusively by donor blood. conclusions: we consider that simplicity, accessibility, high effectiveness, quite well further results of blood reinfusion, except the case of blood reinfusing that was for time-expired out of blood vessels (more than days in our case) will promote to the wide spreading of this method, especially in emergency surgery, in massive injuries, & in disarters, all the cases of insufficiently of time for selection of lot of donor blood. objectives: study of a reaction of the oardioreepiratory system of pregnant women to i/v microperfusion of clophelinum which is known to eliminate hemodynsmic and endocrine nociceptive reactions and can be used for treating hypertensive syndrome in pregnancy and labor. methods: the following non-invasive methods were used: capnography, spirometry, oxygenography, indirect fick principle based on the circle breathing, plethysmography and integral rheography~ functional indices of cardiorespiratory function were evaluated. results: pregnant women with ~h-gestosis were examined before and after i/v infusion of i ml of . % clophelin solution, . mg/kg/hour. before the treatment intensification of carbohydrate metabolism, hyperventilation with moderate hypooapnia and complete respiratory compensation of metabolic acidosis~ increased alveolar ventilation, decreased alveolar volume, predomination of perfusion over ventilation, hypokinetio type of circulation with dominated load by peripheral vascular resistance to the blood flow was observed in this group of patients. microperfusion of clophelin imp~-oved the ventilation/perfusion ratio, ventilatory and gaseous exchange efficiency, resulted in a decrease of congestion in the pulmonary circulation, possibly owing to a decrease of peripheral vascular resistance by %, of the heart rate by io. %, of the oardial output index by . %. conclusionm: the resulted type of circulation with a decreased load on the heart both by resistance and volume allowed to improve the cardioreepiratory system function in pregnant patients. objectives: the injury severity score is a measure of severity of anatomic injuries. iss is a sum of squares of the highest degrees of the abbreviated injury scale (ais) for each of three most severity injured regions. the purpose of the study is to establish correlation between the iss values and mortality rate in older, polytraumatized patients. methods and results: iss was determined for patients. the mean iss value was . + . while the median value was . minor injuries were present in ( %) patients with iss less than , while ( %) patients with iss more than had severe injuries. increased mortality of the older patients was noted in the range - . all patients older than died while % of patients below yrs of age survived, indicationg correlation between iss and mortality rate in polytraumatized patients above yrs of age. conclusions: this mode of evaluating severity of injuries may help in triage, determining appropriate level of care and as an indicator of future outcome of polytraumatized patients. objectives : tissue hypoxia is a non exclusive cause of hyperlactatemia. other serious medical situations induce hyperlactatemia. therefore, lactatemia could be a non specific indicator of severity in patients admitted in emergency unit. the aims of this study were to examine the correlations between lactatemia with the short term survival course prognosis and the unit of hospitalisation; intensive care unit (icu) or medicine unit, in patients admitted in our emergency department. methods -lactatemia was measured as soon as the admittance, in arterial blood sample of patients which needed arterial blond gas. sixty-one patients were included during months. to assess the statistical performances of lactatemia, sensitivity (se), specificity (sp) and accuracy (ac) were calculated for the threshold determined by the youden's test (se+sp- ). results : fifteen patients were admitted in icu and in a medical unit. fifteen patients died. a group of patients had a lactatemia up to mmol.l" . in this group of patients, had acidocetosis, had asthma, had cerebral vascular ischemia, had neoplasia, had cardiogenic shock, was epileptic, had congestive heart failure, had acute respiratory failure, had septicaemia, had hyperosmolar status finally had medicinal intoxication. lactatemia was significantly higher in non survivor than survivor ( . • vs. . + . , p<o.oool, respectively), the cut point of lactatemia was . mmoll" se was %, sp was % and the accuracy was %. on the other hand lactatemia was higher in patients admitted in icu than in medical unit ( . + . vs. . • p<o. , respectively). conclusions : this preliminary results suggest that lactatemia is a good indicator of the short term survival course in patients admitted in an emergency unit. furthermore, hyperlaotemia is present in several pathology seen in emergency. objective: to determine the pattern of injury, total peripheral white cell, neutrophil, and lymphocyte responses, and the outcome of trauma patients who are leucopaenic on admission to the icu. design: prospective, descriptive study of consecutive admissions over months. setting: a -bed surgical intensive care in a teaching hospital. patients: thirty consecutive adults admitted to the icu following trauma. leucopaenia was defined as a total peripheral white cell count of < x , neulropaenia < x , and lymphopaenia < . x ~ cells per litre. measurement and results: the total peripheral white cell count was documented daily and the neurtophil and lymphocyte counts and differential percentages on days , , and . the incidence of leucopaenia was significanfiy higher in patients with gunshot wounds (p < . ) and hollow visceral intra-abdominal injury (p < . ). eight ( %) patients died. no significant differences were found in the initial mean total white cell, neutrophil, or lymphocyte counts nor in the differential percentages between survivors and non-survivors. the total peripheral white cell count increased significantly in survivors compared to those who died (p < . ) and significant differences were found in absolute neutrophil counts (p < . ) and differential percentages (p < . ) on days and . no significant differences were found in lymphocyte counts or differential percentages. conclusions: there is a signficant association between leucopaenia, neutrepaenia, and intra-abdominal hollow visceral injury and peritoneal contamination. survival is related significantly to resolution of these white cell deficiencies. these findings suggest a possible role of granulocyte colony stimulating factor in the trauma patient with intra-abdominal infection and persistent neutropaenia. amelioration of organization emergency care team in order to improve caring for urgent cases. objectives: emergency care team (ect), as integral part of health, respectively as activity of primary health protection care all acute difficult conditions which vitaly danger patients and distressed. the aim of our investigations was to indicate on the possibilities of amelioration of organization ect in order to improve caring for urgent cases. methods: in our investigations we used epidemiologicai and statistical metods all informations and matherials from terrain ects. results: the number of traumatized in traffic accidents etc. have a epidemic character. it is well known that . % traumatized died in first two hours. ect isn't enough qualified to care a traumatized on the place of accident and during transport. because that medical personnel must have an education with solid qualities. conclusions: . besides a physician specialized (vii --+ vii -~ viii gradus) in urgent medicine, it's necessary to introduce two ( ) medical technicians instead of one as is existing now ( ~ ). .the medical technicians can drive cars and one of them always, drives the ambulance car ("b" category). .the technicians have been specially educated for urgent cases (iv ~ v ~ vi). . such a new team structure should be more capable in professional sense. .we can see an importance of team-work in ects. . such teams should also be more economic in comparison with existing one. . we think, that on the general medical studies need to include a new discipline-urgent medicine! objectives: to detoxicate an organism since we have used biological sorbents: isolated from liver and spleen of human or animal cells or tissue fragments. methods: cellular dialysis ( dialyses) in patients with an acute hepatic insufficiency poisoning by hepatotropic poisons: cc , dichloethane, mushrooms -were conducted using "artificial kidney" apparatus, by means of arteriovenous shunt, formed at forearm with disposable dialyzers. hepatocytes suspension was poured into dializing circuit of dialyzer at - mg of cells per i kg of patients weight. dialysis was performed during - hrs. patients with purulent-septic states were treated as for hemoperfusion through prepared sorptional column: . ml of hemosorbent and . ml of fragmented spleen or hepatocytes suspension with the help of roller pump with the rate of - ml/min, during - min. average volume of hemoperfusion made . . conclusions: in all cases we used both native and cryopreserved biomaterial. analysis of the dynamics of clinical, biochemical, scintigraphic and us-investigastions allows to conclude that application of cellular-sorptional method of detoxication enable to prolong life and reduce lethal outcome in some severe category of patients with endotoxicosis. after infusion of diazepam ( . mg/kg),thiopentone ( mg/ kg) and vecuronium (lmg/kg),patient was intubated and mechanical ventilation was adjusted to mantain normoca= pnia. ct scan showed left parieto-occipital hypodensity with brain swelling. after admission in neurological icu,dexamethasone ( mg/ kg/die),mannitol ( .smg/kg) and diphenylydantoin ( mg/ kg/die) were administred. two hours later neurological status of the patien~ was normal and she was extubated. the risks of stereotactie radiosnkgety are related to: hemorrhgge during the latency interval;transient radia= tion effects;permanent radiatio~einduced complications; radiation induced neoplasia. no epilepsy is reported in pts with avm and no seizure before radiosurgery. in c nclusion, epilepsy can be a posmib~e delayed eompli c~t~n, ~fe~r rgdi surgery for cerebral avm. serum potassium deficiency is a frequently reported finding in the time course of acute myocardial infarction (ami). if this is also correct for patients with ami undergoing primary percutaneous transluminal coronary angioplasty (ptca) is unknown.for that reason we analysed in patients ( m., f., + y.) serum potassium concentrations on ad ,mission and directly after ptca.the reference intervall for potassium in our clinical laboratory is , - , mmol/l. although more than % of the patients with ami undergoing ptca showed normal potassium serum concentrations on admission a measurable decrease of potassium could be found in % of patients ( ) after ptca.therefore for the majority of patients with ami potassium supplementation prior to ptca can be justified to avoid electrolyte disturbances as a possible trigger factor for cardiac arrhythmias in the setting of ami and acute reperfusion due to primary ptca. objectives: diagnostic procedures are very important in the management of abdominal injures in trauma. the aim of this paper is to present our experience with peritoneal lavage as diagnostic procedure in abdominal injury. methods: we analyzed patients to whom peritoneal lavage was applied in the surgical emergency from st of january till st of december . results; in this four years period there were abdominal injures. there were blunt injures. among them in patients peritoneal lavage was applied. results were positive in patients and negative in patients. false positive finding were in patients and false negative in two patients. in patients with positive lavage there were more than , /mm red blood cells, more than , /mm white blood cells and the level of amylase was up to u/i. the gall, urine and stool were positive as well. in patients with negative lavage there were less than , /mm red blood cells, less than /mm white blood ceils and amylase were negative. conclusion: peritoneal lavage is useful and important diagnostic procedure in the management of abdominal injures in trauma. our experience with urgent surgical management of the acute gastric and duodenal bleeding s.se en md, z.milo~evi md, *s.srdi md, k.sar ev md. clinic for abdominal and endocrine surgery, institute for surgery; *clinic for cardiology, institute for cardiovascular diseases; school for medicine novi sad, university of novi sad, yugoslavia objectives: acute gastric and duodenal bleeding are very common complication of gastric and duodenal ulcer. the aim of this paper is to present efficiency of our surgical management of these acute conditions. methods: in this paper results of surgical management of patients with acute gastric and duodenal bleeding, treated in our clinic from the st of january till st of december , are presented. results: among operated patients there were females ( . %) and males ( . %). there were patients ( . %) with gastric bleeding and patients ( . %) with duodenal bleeding. bleeding from gastric ulcer were treated with the following methods: excision of ulcer with suture in patients ( . %), ulcer suture in patients ( . %), billroth i in patients ( . %) and billroth ii in patients ( . %). heinecke-mikulicz-weinberger pyloroplastica was the most frequently used in the treatment of the duodenal ulcer bleeding. bilateral truncal vagotomy was done in patients ( %), duodenotomy with ulcer suture was done in three patients ( . %) and three patients underwent billroth ii operation. we had three complicationsduodenal dehiscence in pyloroplasty. there were no lethal complications. conclusions: based on our own experience we conclude that acute bleeding from gastric and duodenal ulcer can be safely managed with urgent and modern operative techniques. dept. of anaesthesiology. emergency center of serbia, belgrade, srj b ectives and methods: cardiac contusion was evaluated by serial determination of cpk-mb fraction and co using non-invasive doppler technique in patients with blunt trauma of the chest. results: miooardial contusion was diagnosed in ( %) patients, while in ( %) it appeared unaffected. cpk-mb of % and more in comparison to total cpkwas . + , % in patients, and , + , % in pts (p< . ).c of . + . l/min in patients with cardiac contusion, measured hrs after admission at the latest, was significantly lower than in group of patients with co of . + . l/min (p< . ). conclusions: co values were significantly lower in patients with cardiac contusion and correlated with pathological values of cpk-mb fraction. objectives: extracorporal plasmophoresis (pph) was implemented by non-stop method in patients with obstructive jaundice. methods: changes of serotonin (s) and histamine (n) in arterial (ab) and venous (vb) blood were investigated before and after one-time session of pph. s and h in blood serum were determined with fluoremetric method. results: before pph the average s contents in ab and vb did not differ. after one-time pph session s in the blood outflowing lungs was % lower than in the blood inflowing. hours after extracorporal detoxication implemented the indices kept the same level. similar dynamics after pph was observed in h. before the session h contens was equal both in the blood inflowing and in the blood outflowing lunge. after pph h contents in ab was % lower compared to the vb and did not change for hours. conclusions: dynamics of changes of biogene amines shows that under the influence of pph there takes place not only mechanical removal of their surpluses together with plasm, but there sharply grows inactivating role of lungs towards ba substances, which is confirmed by reduction of their concentration in the blood outflowing lungs. this effect is probably connected with the "deplasmation" of the lung cellular elements. simultaneously lungs' cells are freed not only from plasm but from toxic adsorbents on their surface. as a result the lung cells activate absorption of the surplus number of amines from the vessel channel. it is not excluded that s inactivation is due to growing activity of monoaminooxidase resulting in deminishing of intoxication. acute pulmonary embolism: thrombolytic therapy or pulmonary embolectomy? i.lodiena md, phd, s.shevchenko md, pphd., medical university, kharkov, ukraine objectives: pulmonary embolism (pe) remains a challenging problem for diagnosis and management for the emergency physician. the aim of this work is to identify the best treatment for massive pulmonary thromboembolism. methods: during recent years patients with symptomatic acute serious pe were reffered to our observations. selective pulmonary arteriography confirmed this diagnosis in all cases. ( . %) underwent urgent surgery, of them ( . %) with cardiogenic shock and ( . %) with massive pulmonary embolism. patients ( . %) underwent thrombolytic therapy. results: in the patients who received thrombolytic therapy successful results were achieved in cases ( . %), in ( . %) patients subsequent pulmonary embolectomy was performed. mortality rate was ( . %) in the patients who underwent pulmonary embolectomy after unsuccessful thrombolytic therapy and ( %) in the patients with cardiogenic shock and massive pe. lower extremity deep venous thrombosis caused pe in % patients. pe mortality rate is repoted to be %, but all patients with cardiogenic shock and massive pe died. this very high mortality rate related to the clinical status of the patients whose condition became worse despite intensive treatment. conclusions: the results of this study show that in most cases thrombolytic therapy is an effective rapid method of treating pe. however, surgical treatment is preferable when the patients reveal adverse effects to drug therapy, when medical therapy is unsuccessful or when the patients are seriously ill with reccurent cardiac arrest. high-quality pulmonary angiography remains the most accurate and reliable means of diagnosing pe. this prospective study was conducted to investigate the serum thyroid hormone levels in traumatized critical ill patients. serum thyroid stimulating hormene(tsh),total thyroxine(tr ),free thyroxine(ft ),total tri-iodothyronine(tr ),free tri-iodothyronine (ff ) levels were measured within hr.of intensive care unit admission(first day) in multiple tratmmtized male patients(injury severity score higher than ). in fifth day the same hormone levels were repeated. tsh,et ,ft ,tr and ft levels were determined by radioimmueoassay(ria). the levels of 'i~sh,xt ,fr ,et and ft in survivors and nonsurvivors were compared with first and fifth days (table) . in conclusion, we are convinced that there was a high correlation between low ser~n thyroid hormone levels and mortality, serum thyroid hormone levels are prognostic value in traumatized critical ill patients. objectives: glucagon improves myokardium contractility, intensifies kidney blood flow, stops bronchospasm ( ), these were the reasons of investigating its effect on hemodynamics and metabolism in hypovolemic shock expermentally and in clinic. methods : in experiments on white rats with traumatic shock glucagon (novo nordisk) was injected intraperitoneally in dose rocg/kg. patients with hypovolemic shock (trauma, hemorrage) were injected glucagon intravenously in dose mg on the background of infusion therapy. results : glucagon hightened recovery indexes of glucose in rats and patients with shock, whereas in groups without glucagon hyperglycemia developed. rats with glucagon injection had double times urea content than the norm ( , + , mmol& norm - , + , mmol/i ), whereas rats without glucagon had urea content , + , mmol/ ( p < , ). osmolarity of blood plasma in rats having glucagon injections was , + , mosm/kg h , without the drug - , + , mosm/kg h ( p < , ), ldh activity - , + . and , + , ( p < , ) mcmol cec- accordingly. lethality in the group of rats without glucagon was % /n= /, with glucagon - (n = ). in patients with hypovolemic shock in minutes after glucagon administration stroke volume increased to % (sv), miocardium contractility -to % (mc), peripheral resistance of vessels ( vr ) decreased to %. in - , hrs sv increased to %, mc-to %, pvr -decreased to %. conclusion : the study provides the evidence that glucagon has an anti-shock effect, improves hemodynamics and metabolism indexes in cases of hypovolemic shock. reference : picazo j. glucagon in acute medicine : pharmacological, clinical and therapeutic implications (norwell, mass:kluwer publishing, ), p. n, vasina (t) ,n.sebbota hph ( ). dept; of acute endotoxicosis, n.v. s~lifosovslay scientific research institute of ermergency ltealth care, boscow, lk'ussia (i}. institute for problems of cryobiology and cryouledicine of the national academy of sciences of the f/maine, kl'k r-key, [ maine ( ), objectives: the investigation of isolated hepatocytes using's efficacy in patients with acute hepatic fai lln-e. methods: native and crioconsrved allo-and xeno-!mmatocztes fixed on semi;ermeable ~mbr~s were used. results: the artificial supporting system with isolated hepatocttes as metabolic ~its was. ap-plied in patients. during tl~ treatmen~ with this cytotherapz an improvement of clinic state and biochemical rates were observed even inthe first we~s. for instance, the kncreasing of glutamin acid's level in blood up to normal value and decreasing of ammontes -concentration from i ~ mmol/l down to # mol/l were discovered. ~ae diminution of ence,%halo;athy was noted too. general lethality was' equal z, there of i z concerned acute hepatic failure (/k~). conclusions: the using of sum~rting hepatic system is able to cope the ~nenomenon of ~i~. objectives: fat embolism (ee) is an important and insufficiently studied problem of the intensive care medicine. fe is clinically diagnosed in % and morphologically in - % of the victims with severe multiple skeletal injury that is accompanied by the shock. methods: during the last years patients with the cerebropulmonary form of fe were treated in the icu. the prognosis of the probability of fe development was done using the computer system "cite-prognosis" in which the triss-method, dynamic and statistical prognosis of the injury outcome~ and the values of the most severe injury in points were realized. the treatment consisted of the surgical stabilization of the fractures with the authors' design of the rod apparatus in the acute period, long-term mechanical ventilation with peep via tracheostomy, complex fluid therapy using perfluorochemical emulsions (pe), and electrochemically active solution of na hypochlorite (snh) via the central vein. results: the treatment of patients with fe gave a good result that was manifested by the complete regression of psychoneurologic and respiratory disorders. conclusions~taking into account the prognostic data~ it is necessary to provide early complex of intensive care to the polytraumatized patients. it includes the surgical stabilization of fractures, fluid therapy using pe and snh~ early long-term mechanical ventilation. dr. alexandr e. poladko, station of medical aid. kiev ukraine. objectives: the reason of investigation is to prove the necessity of beginning of the intravenous infusion of nitroglycerin before the hospitalisation. methods: from january i to june protocols of treatment myocardial infarction patients in kiev medical aid station. results: the results of treatment are better if infusion began early or prolonged during hospitalisation. conclusions: there were patients with acute myocardial infarction treated before hospital by the cardiological groups. patients were treated with the early infusion of nitroglycerin and without. the results of treatment were: in the group with infusion: mortality in the first hours % the full disappear of pain % in the group without infusions mortality in the first hours % the full disappear of pain % that's why our opinion is that infusion of n. is necessary in the treatment of the patients with myocardial infarction from the first minutes of illness. dr. gennady d. kyrzhner, station of medical aid, kiev, ukraine. object: looking for safe medication which is good for the treatment of arterial hypertension and is simple for use. we inspected the effect of prazosin in the conditions of the cail during the year. method: we used mg prazosin tablets sub lingua and measured blood pressure ones during hours. the parameters were registrated in protocol. result." one hundred patients took part in investigation. the middle age of patients was . the reason of hypertension was not taken into consideration. in two hours after beginning of the treatment we caught the reliable lowering of blood pressure in % of patients. the general condition of patients became better in - min. it was only one accident of complication during the treatment -the orthostatic hypoter~sion, conclusion: prazosin hydrochloride in the dose mg sub lingua is safe and simple for use in the urgent situations. objectives: hypomagnesemia is frequently observed in severely burned patients during the early period after injury. increased urinary excretion is insufficient to explain the magnitude of the magnesium (mg) depletion. the possibility of exudative cutaneous mg losses has been proposed, but not yet demonstrated. the study aimed at measuring the mg content of the cutaneous exudates and urine during the first week after major burns. methods: patients, aged _+ years (mean _+ sd), and burnt _+ % of body surface, were studied from day (d ) to d . serum samples were collected at do, serum and urine from d to d , and on dio, d , d . cutaneous exudates were extracted from the textiles surrounding the patients, collected over h periods from d to d , using bidistilled acidified water. mg supplements ( . to mmol/ h) were prescribed from d to dio according to the severity of hypomg (serum mg < . mmol/i). results: mg serum levels decreased below reference ranges in patients between d and d , and normalised thereafter during supplementation. urinary mg excretion was increased in patients, mean excretion being . _+ . mmol/ h until d . mean daily mg cutaneous losses, were mmol/ h, i.e. mmo; in days. large inter-& intra-patient variability: the daily exudative losses ranged from to mmol/ h. conclusions: the mean daily mg cutaneous losses after burns were larger than the urinary losses, and equivalent to the recommended intakes for mg; the addition of the exudative and urinary losses largely explained the increased mg requirements after burns. complex immunologic alterations occur following thermal injury. the activation and consumption of the complement and dotting systems are suggested to play an important role in the development of the capillary leak syndrome, tn burned patients, a generalized inflammatory reaction as well as the impairment of the host defense are considered to be the major reasons for complications, such as sepsis and multiple organ failure. in a pig model we investigated a possible protective effect of cl-inhibitor (berinert, behring). before conducting the animal experiments the human cl-inhibitor concentrate was analyzed for its inhibitory capacity on purified pig cls and its pharmacoldnetic behaviour in pigs (t / ,id ). pigs received anesthesia and analgesia and arterial, venous and pulmonary (swan ganz) katheters were placed into the carotis and jugular veins. the pigs were scalded for seconds with ~ hot water to achieve a % tbs partial-thickness burn. blood samples were taken prior and after surgery as well as , minutes, , , and every further hours after thermal trauma. two control groups (each n= ) included animals which were either scalded or not scalded and treated only by resuscitation of ringers lactat solution. besides various parameters blood samples were analyzed for complement. the pigs (n= ) received c -inhibitor concentrate at an initial dose of u/kg body weight either immediately or hours after thermal trauma (n- ), followed by three further applications (of reduced amounts) every hours. the clinical outcome as indicated by vital parameters and as well as demonstrated by reduced edema and inflammatory tissue destruction suggested a protective effect of cl-inhibitor. complement analysis revealed a faster recovery of the alternative and classical pathway activities in cl-inhibitor treated pigs as compared to the control group but only if the regulator was given immediately after thermal trauma. from these results a protective function of c -inhibitor on thermal trauma can be assumed. objectives: the aim of study was to characterise the pattern of secretion of interleukin- (il- ) and tumor necrosis factor alpha (tnf alpha) in multiply injured or not injured critically ill patients and to relate these results to their outcome. methods: blood samples of multiply injured ( ( ) ( ) ( ) il- (nis) ( ) ( ) ( ) il- (nin) ( ) ( ) ( ) conclusions: this data suggest tnf alpha is a better prognostic marker in patients with multiple injury, than in critically ill patients without injury. il- is not significantly higher in nonsurvivors of both groups. sepsis is associated with an increased production of nitric oxide (no), as reflected by a rise in plasma levels of nitrites (no ) and nitrates (no ). severe bum injury is associated with similar symptoms of inflammation like hypotension, high cardiac output low vascular resistance and increased vascular permeability so that an increased no production may be involved. the present study included patients admitted to the intensive care unit of the burn center of brussels (age : - years; burned surfaee area - %), and control (non-septic) patients hospitalized in the neurological rehabilitation unit of the erasme hospital (age - years). the patients in both groups were fed enterally with to kcal/kg/day of a standard solution. in the burn group, daring the study period, patients were mechanically ventilated, required vasopressor (dopamine) therapy, and received antibiotics; patients were discharged alive from the intensive care unit, and no patient died during the study period. plasma was sampled for no /no determinations (griess reaction) daily from day to day (n=ll) or to discharge (day , n= ; day , n= ) in the burn group, and once the control group. in the burn group, no /no levels were elevated at day ( _+ #moles/i), reached a maximal value on day ( - p.moles/l) and progressively decreased to day (fig). these values were higher than in the control group ( . :t: . #moles/l, all differences p< . ). however, no correlation was found between the plasma no /no levels and the age of the patient or the total burned surface area; burned patients who became infected tended to have higher plasma no /no levels than those who did not ( + vs - #moles/i, ns). the present study indicates that burn noa/noa (llmol**/l) injury is associated with increasedloo] [__ ] ~ no /no plasma levels, which are ao so consistent with an enhanced no pro- o duction, ao obiectives:urapidil has been recommanded for therapy of hypotension in neurosurgical patients, because it was found not to increase intracranial pressure in patients with compromised intracranial dynamics ( ). in contrast several authors reported an increase of icp after urapidil administration in patients with head injury ( ). our study was designed to determin the influence of urapidil on mean lumbar cerebrospinal fluid pressure (csfp) in awake humans with uncompromised intracranial compliance. methods: after approval by the local university ethics comittee, six volunteers (asa , male, female) were studied in the lateral decubitusposition with the vertebral column positioned horizontally. a gauge needle was inserted into the lumbar subarachnoid space at level l /l and connected to a trancducer (mx | medex inc.) for csfp measurements. the volunteers were advised to keep respiratory rate and etco constant. after a resting period of minutes basline measurements were performed including csfp, cvp, map, hr, etco (cardiocap | datex). then the volunteers were given urapidil . mg/kg over a period of minute. minutes later measurements were repeated. results: statistics: wilcoxon signed rank test, p< . significant; values: mean_+sd, si = mmhg. - _ " _+ * _+ * - _+ " _+ csfp: cerebrospinal fluid pressure, cpp: mean cerebral perfusion pressure. conclusions: during normoventilation urapidil leads to a decrease in map parallel by an increase in csfp and therefore in icp, most likley mediated by an autoregulatory dilatation of cerebral vessels. if this cerebrovascular response is suppressed by hyperventilation, no increase in icp is found despite a uredipil induced drop in map ( ). after longterm hyperventilation cerebralbloodflow is normalized. this might be the reason why the drop in bloodpressure following urapidil administration again led to an increase in icp ( background and objectives: the intestine is one of the most sensitive tissues to ischemia-reperfusion injury. reperfusion of the intestine is often associated with increase in mucosal permeability and ulceration. d(-)-iactate is produced by indigenous bacteria found in the gastrointestinal tract and mammals do not possess the enzyme systems to rapidly metabolize it. the present study was designed to determine the kinetics of plasma d(-)-iactate alteration in rats paused by acute intestinal ischemia-reperfusion injury. methods: following the anesthesia, the superior mesentcric artery (sma) was exposed and it was occluded by a micro-bulldog clamp applied at its origin from the aorta. after min of occlusion, the arterial clamp was removed and the supply area of the sma was allowed to be reperfused ( h). plasma d(-)lactate levels were measured by an enzymatic spectrophotometric assay. the endotoxin content of plasma sample was assayed by the limulus amebocyte lysate test with a kinetic modification of the test kit procedure. results: intestinal ischemia for min resulted in a significant increase in d(-)-lactate levels within the portal vein as compared to sham-operated animals. plasma d(-)-lactate levels had a tendency to further increase after reperfusion up to h. it was showed that significant elevation of endotoxin concentrations in portal vein was alread~r detected at the end of -min ischemia, reaching peak at . h post-reperfusion and decreasing gradually throughout the study period. at the end of ischemia, marked mncosai damaged such as edema, hemorrhage and necrosis was observed. there was evidence of injury to ti, e muscuiaris propria together with diffuse mucosal damage and destruction following reperfusion, particular at h postreperfusion. in addition, penetration of bacteria was commonly found in the intestinal wall at various time points following ischemia/reperfusion injury. conclusions: these data suggest that acute intestinal ischemia is associated with permeability change of mucosal barrier resulting in increased plasma d(-)qactate, which is also remarkably influenced by subsequent reperfusion. plasma d(-)-lactate may be a useful marker of intestinal injury following both ischemia and reperfusion insult. objective: to assess the clinical usefulness of two methods of intracranial pressatre (icp) monitoring: intraparonquimatous recording (icpc) and epidural recording (icpe), versus intraventricular icp (icpv) as a gold standard. we prospectively studied patients with severe head injury (glasgow coma score < ) admitted to our icu between fobmaly and december . icpv was monitored in all pativrats by means of a multipzffolated catheter connected to a water coinnm. six of patients were additionally icpc monitored by a fiber optic transducer (comma r~), and seven of patients were icpe monitored by a couplvd fluid system (plastimedr). icpc was placed homolatetal to the focal, lesion m two cases and contralateral in three; icpe was placed homolateral to the focal lesiou in two cases and contralateral in three. all three systems were calibrated at the ear level. stali~cs: correlation coefficient and lineal regression were done between icpe and icpv, and between icpe and icpv with the hourly p~rs of values obtained dttting assistontial period. results: of the six icpc -icpv studied pa~onts, we observed a correlation coef~cieut r = . (p < . ) with a regres~on line y = . + . x. four of ~hx lcpc -icpv patients had r > . when correlaliou eoet~dent was obtained indixddually. of the seven icpe -]cpv studied patients, we observed a cortelafiau ooeffioiont r = . (p < . ) with a regression line y = . + . x. corralalmu eoetfieiont was inwer than . in all seven patients. corrdation eoelfieients for levals of icpv > man hg, > mm hg and > tuna hg with icpe showed r = . , r = . and r = . respectively; and with icpe r = . , r = . and r = . . the obtained values did not change during the study. conclusdns: in our study icpe was considered a good type of icp monitoring. /cpe signiticantly infravalorates icp values. we observed a good correlatinn between icpc and icpv values in patients with high inttacramal presanre. objective: midazolam is a benzodiazepine agonist widely used for sedation in emergency medicine. few studies in animals and humans point to a direct analgesic effect of midazolam probably mediated by spinal antinociceptive receptors and/or peripheral benzodiazepine receptors ( , ). in our experience in the berlin emergency medical system (unpublished results) with anecdotal cases of extreme chest pain due to binge drinking but no evidence of acute myocardial infarction or extreme abdominal pain due to peritonitis, acute intermittent porphyria, peutz-jeghers syndrome or testicular torsion, we found that small doses of midazolam ( - mg i.v.) were much more effective in relieving pain than repeated administration of high doses of buprenorphine or morphine, which may be associated with a considerable respiratory depressant effect. the dose of midazolam required for pain relief in these patients is non-narcotic and allowed further communication on the character and localization of' the residual pain, which might be very important for the further diagnostic procedure. patients: ten patients with abdominal pain due to acute gastrointestinal bleeding, suspected pancreatitis, suspected acute porphyria, and chest pain with no evidence of acute myocardial infarction received first-line midazolam i.v. at an initial dose of mg and were asked how it affected the intensity and character of pain. results: at the chosen dose of midazolam ( - mg), all patients were responsive to detailed questioning on basic orientation, the character, intensity and localization of the pain, and medical history. none of the patients required an additional opiate. all patients stated that the pain was tolerable after midazolam alone. conclusion: our preliminary clinical observations suggest that low-dose midazolam might be an alternative to opiates in extreme pain of presumably visceral odgin. objectives: it is known that severe head injury in elderly patients is associated with higher mortality than in younger patients. it remains however to be clarified whether the preinjury pathology which is frequent among these patients, affects the outcome. methods: in an attempt to investigate this hypothesis, patients aged over years suffering from head injury, with glasgow coma scale (gcs) of or less, were studied retrospectively. twenty-six patients ( . %) had preinjury pathology i.e. diabetes mellitus, arterial hypertension, heart failure, alcoholism, parkinson's disease etc. (group a) and fifty-three ( . %) did not (group b). the following data were recorded: mortality in the i.c.u., duration of hospitalisation, incidence of infective complications and neurologic status at discharge. results: groups were comparable in terms of mean gcs ( . vs. . ) and median age ( . vs. ). the incidence of brain pathology in the two groups was the following: epidural haematoma . % vs. . %, acute subdural! haematoma . % vs. . %, intracerebral haematoma . % vs. . %, subarachnoid haemorrhage . % vs. . %, diffuse haemorrhage . % vs. . %, contusion . % vs. . % and non-visible pathology (normal ct) . % vs. . %. unilateral pupilary dilatation was found to be . % in group a and , % in group b. the mortality during hospitalisation in the i.c.u. was almost the same: % iu group a and . % in group b patients. however, group a patients had significantly more infective complications, required longer hospitalisation and had lower gcs at discharge. conclusions: the results show that the existence of preinjury pathology does not seem to affect the short-term outcome of elderly patients with severe head injury. it has however an impact on morbidity and perhaps long-term survival of these patients. the assessment of clinical development in intensive care patients with severe head injury still remains a problem. to optimize the monitoring of intracraniel prassure (icp) we rautlr~dly implant an eplduml measuring device in our hospital. the aim of this study was to prove the correlation of the icp-values with ct findings and clinical development. during a month period ( - r the icp was monitored in p~,tients ( male, female) with severe head injury by an eplclural measuring device (epldyn~/$plegelberg| the mean age was . years ( - ). the glasgow coma scale at admission was . ( - ). in all cases the device was placed wfihln the first hours after admission. the tcp was compared with physical examination, radioidglcal or intraoperatlve findings and cunlca! outcome. the average time of measuring was . days ( - ) . the traatment depended on the !cp values recorded. rising icp-valuea ~ed to radlologlcal c ntra!s by ct-scan. in case an intracranlai hemorrhage was detected and drained. the overall survival rate was . %. showed a complete resolutl n, in other . % psychological residuals like decreased mentatlon, in . % sensomotorlc residuals like cerebral nerve dysfunction and aphasia, and . % of the injured remained in a comatous status. in % of our cases the measured values correlated with clinical course and management. in cases ( . %) we observed a displacement of the icp-pevice. there was no icp induced infecllon. istituto di anestesiologia e rianimazione, universit& ,,la sapienza", rome, italy * istituto superiore di sanit& -servizio di epidemiologia e biostatistica, rome, italy objectives: acute renal failure (arf) can be a severe complication of trauma. the current incidence of post-traumatic arf is associated with high mortality . identification of risk factors and prevention of this complication could improve the outcome of trauma patients. methods: one hundred fifty three consecutive trauma patients (age . _+ . , injury severity score . + . ) admitted to icu were studied. incidence of arf was . % ( / ). arf was defined as persisteat plasma creatinine > mg/dl with or without oligoanuria . arf was defined as early when occurring within the first hours (earf) and late when the onset was after the first four days (larf). results: earf occurred in patients while larf developed in patients. age, iss, and incidence of rhabdomyolysis and acute respiratory failure were not different in the two groups. an higher incidence of multiple organ failure (mof) and sepsis ( . % for both) were observed in larf group, when compared to earf ( % and % respectively). abdominal trauma was more frequent in earf group ( % vs %). the gs for earf and larf were respectively _+ . and _+ . while in the group who not developed arf (narf) the gs was . • conclusions: gs score difference seems suggestive and can be that an abnormal cerebral activity (hipofisary hormones?) may play a crucial role on onset of arf in these patients. moreover the frequency of acute respiratory failure in the group of arf was higher ( . versus . ) than narf group. the early ipoxia in the early phase of trauma, then, may be another crucial point for development organ failure. these are preliminary data. a more exact statistical analysis must be perform to have definitive conclusions. to compare the active compression-decompression cardiopulmonary resuscitation (acd-cpr) with the standard cardiopulmonary resuscitation (s-cpr) in out of hospital cardiac arrest patients. is a controlled, randomized study. two groups of patients with cardiac arrest out of the hospitalwere formed. group i, (acd-cpr) and group ii (s-cpr). for the acd-cpr groupweusedthecardiopumpdeviceofambulnternational. asfortherest, the erc ( ) algorithms for acls were followed. the utstein style (for out of hospitat cardiac errest) was used for listing and evaluating all cases of the study. the cpr was contucted by the crew and the doctors of our mobile intensive care units (micu). we studied consequitive patients ( in group i) and ( in .group ii). demographics pre-cpr characteristics (e.g. ecg form of cardiac arrest) and procedures (eg bystanders or second tiers crew cpr, defibrillation, drugs) were quite similar for both groups. the mean arrival time of micu was min. in group i we recorded r.o.s.c. (return of spontaneous circulation) , %, death %, continuation of cpr efforts , %. while in group ii, %, %, and , % respectively (recorded percentage until the admission to the hospital). no significant difference was found in anyofthe short term outcome parameters. no complications related to the acd-cpr technique, were noted. not any significant difference between the two methods was proven (from this small evaluated sample). the results of previous clinical studies are controversial (i) . more sophisticated studies proved the superiority, in a certain number of parameters (e.g pressures, flow, etc) of the new technique although there are many difficulties for establishing clinical results. in the pre-hospital setting that is related to many parameters (speed of the intervention, effectiveness of bystanders cpr, education ofparamedics, etc.)the evaluation is even harder. the superiority ofthe acd-cpr can be proven when it is performed in almost times increased number of studied patients as w~ll as improvement of the technique could lead us to more established results. objectives; infectious morbidity is the major cause of mortality after burn injury, and is due to multiple factors. trace elements (te), which are involved in both humeral and cellular immunity, exhibit severely altered status after burns. te supplementation has been shown to be associated with increased leukocyte counts and shortened hospital stay. the trial aimed at studying the immune responses in severely burnt patients receiving normal te supplies or early large supplements. methods: patients, aged _+ yrs (mean_+sd), with burns covering + % of body surface were studied from day (d ) to d post-injury, were randomised in groups (g): g -control receiving recommended te supplies + placebo; g -receiving in addition large supplements of cu, se and zn from d to d . enteral nutrition was started within hours of injury in all patients. immunological parameters: peripheral leukocyte counts, proliferation of mononuclear cells to mitogens, cell surface molecule expression, and neutrophil chemotaxis at d and d . infectious episodes and micro-organisms were monitored until d . results: the patients' characteristics were similar g & g . the total leukocyte counts were higher in g between d and d , due to increased neutrophils (significant from d to d ). total cd + and cdlg+ cells did not differ, whereas cd + (monocytes) were significantly increased at d . proliferation to mitogens was significantly depressed in all patients. chimiotactism was not altered. the number of infectious episodes was significantly decreased in g with a mean of . _+ . infections during the first days versus . _+ . in the control group (p < . ). conclusions: the large te supplements for days was associated with a significant decrease of the number of infectious episodes. supplementation was associated with increases in total leukocyte, monoeyte and neutrophit numbers. further studies are required to determine the precise mechanism underlying the improved immune defences. objectives: evaluate the efficiency of local adsorption (la) with the use of carbon adsorbents in case of severe burns in expertment and clinic. methods: experimental studies on la were performed on a model of % body surface area iiib-iv burn in rats. a burn eschar was excised on the rd day after burn, the wounds were dressed with the gauze bandages (control) or with adsorptive dressings (la), dressings were regularly changed. clinical investigations were carried out in the course treatment of patients with severe thermal and radiation ilia-iv burn. in the dynamics of bum disease some indices of proteometabolism and intoyacation criteria were evaluated. results: the experiments have demonstrated that the application of la after early excision of a burn eschar exerts a pronounced normalizing effect on a protein electrophoregram and the activity of proteases and their inhibitors in burned tissues preserving vitality. thus, by the th day after burn infliction the activity of cathepsin d in injm'ed muscles is times lower under an adsorptive dressing than under a gauze bandage (control) (p< , ), the activity of trypsin-like proteases is . - . times lower and the antitryptie activity does not differ significantly from the normal level. the cytotoxicity of extracts of burned tissues after the adsorptive dressing application fn vivo and adsorption in vitro is - % and - %, respectively, of the toxicity of control extracts. a similar normalizing effect of la is ok~rved for an intact muscular tissue and blood serum. the dectron-spin-resonance studies have demonstrated that la allows to normalize antitoxic activity of liver and functional activity of kidneys. the application of la in the treatment of patients with severe burns have been shown to localize a region of irreversible tissue changes, accelerate rejection of a burn eschar, attenuate an endogenous intoxication level and, as a result, shorten the time for grafting of a burn wound and accelerate wound heating. conclusions: proceeding from the obtained results, we can consider la as an effective method of localization of a region of irreversible tissue changes as well as of correction of local and general metabolism failures and overcoming burn autointoxication during burn disease. c de deyne, t vandekerckhove*, j. decruyenaere, b. vaganee, v vandewalle*, f colardyn depts of intensive care and neurosurgery*-university hospital gent-belgium. jugular bulb oximetry is the first bedside available cerebral monitoring technique providing an estimation of the adequacy of cerebral perfusion. its routine use in all patients suffering from severe head injury admitted to our ic unit enabled an extensive analysis of all very early cerebral perfusion data in order to evaluate the incidence of abnormal sjo~ data (and their possible causes) in this very eady period after traumatic insult and to search for possible implications as to the emergency management. these very early data were defined as the first hours icu data and icu admission had to occur within h of traumatic insult. over the last years, pts with severe head injury (gcs< ) were monitored by jugular bulb oximetry, starting immediately after their arrival at the icu (mean of . h after trauma, range between - h). in a total of pts (= . %), jugular bulb desaturatiens (< %) were noticed during this early h period. in pts (= %), jugular bulb saturations higher than % were observed, whereas pts (= . %) revealed no abnormal sjo data ( - %) during these first h. concerning the periods with too low jugular bulb saturations (n: ), we found the following correlation ; in pts (= . %) cerebral perfusion pressure (cpp) was below mmng, in pts (= . %) paco~ was below mmhg and finally in pts (= %) we found primary intracranial hypertension. for the high jugular saturations (n: ) we found a primary intracraniaf hypertension in f pts (= %), and a pace level above mmhg in pts (= %). in all patients we could restore jugular bulb saturation within normal range ( - %) with the correct!on of the presumed causative factor. we can conclude that ultra early jugular bulb saturation data revealed a high incidence of abnormal values, with a predominance of jugular bulb desaturations, confirming once again the high incidence of disturbed and too low cerebral perfusion within the first hours after severe head injury. these jugular bulb desaturations were especially correlated to systemic causes, as a too low cpp (caused in the vast majority by primary map insufficiency, and not by intracranial hypertension) and hyperventilation were the major causes of the desaturation periods. as jugular bulb desaturatione are known to be significantly correlated to a worse neurological outcome after severe head injury, one might improve outcome by an emergency management avoiding these possible causes of jugular desaturation. therefore, extreme attention should be paid to the maintenance of an adequate mean arterial blood pressure (above mmhg?) even duhng the few time spent at the emergency department. one should be as attentive to the maintenance of normoventilation during this very early period of admission and hyperventilation without any knowledge of icp or sjo should be abandonned. recently, indomethacine has been proposed for the treatment of therapy refractory intracranial hypertension in pts suffedng from severe head injury ( ). indomethacine, a cyclo-oxygenase inhibitor, gives rise to a significant fall in cerebral blood flow by inducing cerebral vasoconstriction. therefore, its use could result in a drastic lowering of the intraeranial pressure (;cp) in pts suffering from intracranial hypertension secondary to cerebral hyperaemia and in whom the use of other cerebral vasoconstrictive drugs (barbiturates or hyperventilation) appears insufficient to control icp. for the last months, we included the use of indomethacine in our therapeutic flow chart for severe head injury management. pts revealing intracranial hypertension (icp> mmhg) and cerebral hyperaemia (sjo~> %) and in whom icp was not efficiently controlled by the combined use of hyperventilation and barbiturates were given indomethacine in a trial to control icp. a total of head injured pts received treatment for intracranial hypertension over the last months. six of them met the criteria set for the administration of indomethacine. in pts, no decrease in icp or in sjo was observed and both pts died due to therapy refractory intracranial hypertension. in the other pts, a significant fall in icp and in sjo was observed shortly after indomethacine administration. in pts we observed a catastrophic fall of sjo= even below %, indicating an extreme cerebral vasoconstriction with the possible risk of inducing cerebral ischaemia. in one of the pts, icp remained under control without further administration of indomethadne, but he died days later in multiple organ failure. the other pts, needed multiple indomethacine administrations (for pt even during consecutive days) to finally control icp. in all pts, icp was finally controlled, but only pt survived. both other pts died from systemic causes (multiple organ failure in pt, massive gut infarction in the other tat, possibly due to the systemic vasoconsttictive effects of the indomethacine administration). in conclusion, indornethacine might have a role in the treatment of intraoranial hypertension, especially when caused by cerebral hyperaemia. we observed however a poor final outcome and a threatening high incidence of systemic events (multiple organ failure, gut infarction) in those pts receiving indomethacine for icp control. therefore, indomethacine in the treatment of intracranial hypertension should be reevaluated in controlled study settings, before its routine use can be considered. untill recently, intracranial hypertension (ich) in pts suffering from severe head injury was managed in a staircase approach, with csf drainage as first therapeutic step, mannitol as second step, hyperventilation as third step, and finally, barbiturates as the last rescue step for therapy refractory ich. this staircase approach for the treatment of tch was only guided by the intracraniat pressure, and not by other parameters such as e.g. the actual state of cerebral perfusion of the concerned pt. jugular bulb oximetry provides us with the first, bedside and continuous available, estimation of cerebral perfueion. its implementation in a rigourous flow chart, based on as well icp-as jugular bulb oximetry-data might result in an altered strategy for ich management. we adopted a '~ugular bulb saturation (sjo~)-guided approach" for ich management in consecutive pts, suffering from severe head injury (gcs< ). we maintained csf drainage as first therapeutic step, but the decision for the second step was guided by sjo information. pts revealing ich and sjo=values above %, were treated with hyperventilation, and did not receive mannitol. if ich persisted, barbiturates were added as a third step. on the other hand, pts with ich and sjo= vales less than %, received mannitol administration as second step. hyperventilation and/or barbiturates were only added if ich persisted and if no cerebral hypoperfusion was discerned (sjo=> %). our objectives were to prospectively analyze this new therapeuticstrategy, as compared to the formerly used staircase approach of ich. we managed pts with ich, with an overall mortality of . % due to therapy refractory ich. all pts received standard primary care with head elevation, full sedation and normovenfilation. fer pts, csf drainage alone was sufficient to control ice of the remaining pts, pts received mannitol and pts were hyperventilated as second approach. in the third line, pts were managed with barbiturates, with mannitol and pts with hyperventilation. finally, barbiturates were used as the final rescue in pts. these results reveal a less frequent use of mannitol as only pts received mannitol, compared to the pts that would have received mannitol using the former staircase approach. hyperventilalien was used much earlier in the treatment course, as lots were already hyperventilated in the second line approach, were this was formerly exclusively reserved for the third line approach. finally, also barbiturates were used much eadier ( pts received barbiturates as third approach). we may therefore conclude to a important change in the management of ich, induced by a sjo -guided flowchart. however, future studies will have to elucidate if this new strategy for the intensive care management of severe head injury will also result in an improved outcome. obsectives: in a first series of experimental brain injury we investigated the course of brain po , icp and cerebral blood flow after traumatic brain injury (tbi), whilst accordingly there are very few data available and the mechanisms leading to secondary brain damage are poorly understood. methods: in piglets ( days old, , - kg) of either sex we produced a moderate brain injury ( , arm., msec.) using a lateral fluid percussion {fp) device. complete measurements were made before and min. after brain trauma and after , and hours including blood gases, cardiac output (htermodilution), heart rate, eeg, laser doppler flow probe (ldf} and icp values (camino), brain temp., po by a clake type oxygen electrode (licox) and coloured microspheres for regional blood flow. results: immediately after the trauma a typical "cushing"response to the icp peak up to mm hg being highly significant (before mean i mm hg, range - mm hg) could be observed: mean arterial blood pressure rose from appr. mm hg to ii mm hg for - min. in two animals this was followed by an ischemic period lasting min. accordingly icp values gradually returned to starting measures within hours; in the ischemic animals they remained at a level of about mm hg.-no secondary increase of icp could be observed, once icp dropped to starting values within hours. cerebral blood flow (ldf) fell from mean values being i before trauma to appr. zero and recovered to around . brain po started at mean values of mm hg (range - mm hg) and fell to around zero depending upon the severity of the ischemic reaction. on average values of mm hg were reached over the time course. conclusions: with our fp trauma model we can reproduce the well known "cushing"-response after brain injury; secondary icp elevations cannot be achieved, although local edema is observed. direct brain po measurement seems to be a very sensitive variable for detection of cerebral ischemia and anticipates eventually following icp elevations by far. pulmonary aspiration s,traoaras. v. sgountzos, p. agouridakis, m eforakopoulou, e. ioannidou. intensive care unit (tcu) of "kat" hospital, athens, greece ob!e=ives: the reported mortality rate after pulmonary aspiration is variable in several series. the purpose of this study was to find out the influence of preexisting disease or situation on morbidity and mortality of intensive care unit (icu) patients with pulmonary aspiration. methods: patients who were treated in icu and had pulmonary aspiration, were studied, entrance's criteria in the study, all of them obliged, were: ) suction of gastric contents from trachea during intubation, ) presense of a predisposing factor, e.g. coma. ) recent hypoxaemia or new infiltrates in xray. preexisting disease was recorded and correlated with complications and outcome. patients with glasgow coma scale , because of cerebral injury, and patients who died within days from cause other than aspiration, were excluded from the study. method of statistical analysis: chi-square test, results: one hundred forty five patients were studied. the trauma patients were and the non trauma patients . from the trauma patients, had cerebral injury and were polytreumatized without cerebral damage. from the non trauma patients, had malignant neoplasms, neurological diseases in terminal stage, old age, drug overdose, and several diseases. eighty seven from trauma patients ( %) and from non trauma patients ( %) manifested several complications (pneumonia, ards, etc), so there was no statistical difference in complications' frequency between the groups (p> , ). the severity of complications was also proportional in the groups. eighteen deaths were recorded in the trauma patients (mortality %). only deaths correlated directly or indirectly with the aspiration ( %). in non trauma patients, deaths were recorded ( %). twelve deaths were recorded in patients with neoplasms, deaths in patients with neurological diseases, deaths in aged patients, death in drug overdose patients, and death in patients with several diseases, the mortality difference in trauma and non trauma patients was statistically significant (p< , ). in patients with drug overdose the mortality was significantly lower from the other non trauma patients and the difference was statistically significant (p< , ). conclusion: the preexisting disease or situation plays a major role in the outcome of the patients with pulmonary aspiration. the mortality of patients with aspiration seems to be caused by severe preexisting situations rather, that lead to death, than from the pulmonary aspiration per se, which may be a final happening in a predetermined course. obiectives; the purpose of this study was to compare fluconazole and amfotericin-b in the treatment of fungal infections in severe trauma patients. methods: thirty five severe trauma patients who were treated in intensive care unit (icu), were studied prospectively. they all developed fungal infections, prooved with blood positive cultures and at least one of the following: fever, positive urine or bronchial secretions cultures, infiltrates in xrays. the patients were separated randomly in groups. the patients of group a ( patients) received fluconazole rag/day for days. and the patients of group ( patients) amfotericin-b rag/day for also days. compaiison's criteria were the clinical responce to treatment (fever etc), the fungal elimination (blood and other cultures), the relapses of the disease, the side effects of drug, and the outcome of the patients. as method of statistical analysis was used the chi-square test. results: nine patients from of the group a ( %), and from of the group b ( %), presented remission of fever (patients of group b had better clinical responce than patients of group a, and the difference was statistically significant, p< , ). all the patients before treatment had positive for fungi blood cultures. after days of treatment, patients of group a and none of group b had positive cultures. eight patients (from who had positive cultures of bronchial secretions before treatment) of group a. and (from ) of group . had positive cuttures of bronchial secretions after days of treatment, so positive bronchial secretions were fewer in group b than in group a, but this difference wasn't statistically significant, (p< , and p> , ): ten patients (from ) of group a and patients (from ) of group b had positive urine cultures, after days of treatment (positive urine cultures were fewer in group b than in group a and this difference was statistically significant. (p< , ). two patients of group a and none of group b had a relapse of fungal disease. in group a, no side effects were obsepced, while in group b were observed only minor side effects (small increase of serum creatinine in patients, chills and fever during infusion in patients, and hypokalemia in patients). three patients of group a and patient of group b died, because of sepsis. conclusion: amfotericin-b (even i~ short regimen of days), is superior to fluconazole in the clinical and laboratory responce and also in the relapse of fungal disease, fluconazole is superior to amfotericin-b as it has no side effects. ob!ectives: flail chest after thoracic trauma is a serious injury. it is controversial if flail chest by itself orthe concomitant intrathoracic injuries e.g. pulmonary contusion, is the cause of the reported significant morbidity and mortality. in this study we searched the influence of concomitant thoracic injuries in the course and outcome of patients with flail chest. methods: eighty five patients with flail chest after isolated chest injuries were studied, for the purpose of analysis, we separated the patients into groups, patients with isolated flail chest were included in group a, patients with flail chest and hemo-pneumothorax in group b, patients with flail chest and pulmonary contusion in group c, and patients with flail chest and hemo-pneumothorax and pulmonary contusion in group d. complications from the chest, duration of mechanical ventilation and mortality were compared in the groups. statistical comparison of results belween groups was made using chi-square and t-studend tests. results: the patients were . all patients received mechanical ventilation, twenty eight patients were ihcluded in group a, in group b, in group c. and in group d. seventy three patients manifested complications from the chest, especially pulmonary infections. there was no statistical difference among the groups as to number of complications ( twenty four patients had chest complications in group a, in group b, in group c, and in group d. p> , }. the duration of mechanical ventilation was not statistically different among the groups (the mean duration was , days in group a, , in group b, , in group c, and , in group d, p> , ). there was also no statistical difference in mortality among the groups (six patients died in group a. in group b, in group c, and in group d, p> , ). conclusion: flail chest by itself is a serious thoracic damage with many complications, regardless of the presense of other thoracic injuries, which don't contribute to greater morbidity and mortality. the present study investigated the correlation between blood lactate mortality and organ failure in trauma patients admitting between december , and july , in the icu. road traffic accidents were the most common cause of trauma in this studded population. brain damage was the main cause of mortality .nevertheless, of patients died from sepsis and multiple organ failure without significant brain damage and these deaths were potentially preventable. respiratory failure was the most common complication and was developed in ( %) of survivors and in ( %) of non survivors .we noted low fncidence of renal failure may be do to the early and aggressive ittv'asive hemodynamic monitoring and cardiopulmonary support. as part of our routine case protocol serial blood lactate levels were measured in each patient at least times a day until the valses returned within the normal range or until death. we analysed the blood lactate levels on admission, the highest value and the number of days until the first normal value (<l.smeq/i). initial lactate and highest lactate levels were significantly higher in patients with than without organ failure.( . vs . , . vs . meq/l, p< . )the duration of hyperlactemia also was higher in the patients with organ complications. ( . vs . days, p< . ). both initial lactate and highest lactate levels were higher in the non survivors than in the survivors.( . vs . , . vs . meq/l, p< . ) the present study demonstrated that not only the degree but also the duration of hyperlactemia can be related to the development of posttraumatic complications. serial blood lactate measurements are reliable indicators of morbidity and mortality following trauma. s current evidence suggest that peep only affects intracrenisl pressure (zcp)when it interferes with systemic arterial pressure, although the effects of peep over cerebral oxygenation remains unknown. objective: determine the effects of peep over icp and cerebral metabolism measured by sj , kjdo and ceo in severe head injured patients. meterial ~ methgds: patients with glasgow coma scale l at arrival, with difusse brain injury in the first gt, according to marshall criteria,lop and jo monitored, under analgesia and sedation, normoventilated with zeep, without mannitol treatment in the previous hours, normal x ray chest and within the first hours from injury, were considered candidates. peep value where increased fro~ zero to cmh , in three different steps, each one with min of duration. all date were analized on line, pmax p'p ..... p.._, from the ventilator, systemlc haemodynem]c data, cerebra perfuslon pressure (cpp), icp and sjo , in the case of hemodynamic deterioration, during the study, ppc ommhg, extra-volume were administered. if persistence, dopamine were begun or increased dosage. if no good response were obtained, patients were retired from the study. results. patients were candidates, of them were exc;uded due to haemodynamic instability, k total of completed all steps and were e~amined, men and women, age ! . yrs. at hospite; arrival, glasgow were < in patients, and > in the rest . patients mmhg at the beginning. zeep ob/ectives. critically ill patients are transpoded to an intensive care unit(icu), under conditions, which have not been systematically evaluated. therefore, we set suite investigate transportation and admission condition of these patients to our department. methods. we studied patients( females), aged (mean-..+-sd) . _ . yrs, which were consecutively (from august to march ) admitted to the icu, through the greek national emergency transporta~on service. apache ii severity score upon admission was . -+ . (range - ). the following data were evaluated: ) number of medical departments, where health care was provided until final admission to the icu, ) ambulance transportation conditions, ) catheters and tubes inserted before admission, ) vital signs upon admission ) information provided by referring physician (scored on a to scale: history, electrocardiogram, chest x-ray, laboratory data, drug therapy already administered), ) comparison of the state of the patient described by referring physicians, to the actual state u pen admission. resu/ts. one to four medical departments had provided health care before the palient was admitted the icu ( : . %, : . %, : . %, : %). thirty/ ( . %) patients were escorted by a physician. twenty-six/ ( . %) were transported on oxyge n, fio (mean__.sd): -+ %, pao : . -+ . mmhg. five of the remaining , for whom no oxygen was provided, had pao : . -+ mmhg. twelve/ ( . %) were intubated and ventilated during transportation. thirtyfour/ had a peripheral venous line, / had an arterial line, / had a nasogastdc tube, / had a urinary catheter. eleven/ were sedated and / were paralysed. three/ were on inotropes. vital signs upon admission were: arterial blood pressure, systolic . -+ mmhg, diastolic -+ mmhg, heart rate -+ bpm, temperature . -+ cc. patient information score was --. . . the actual state upon admission was found substantially different, as compared to the description of the referring physician, in / ( . %) patients. conclusions. we conclude that several aspects of the greek national emergency transportation service to an icu should be reevaluated and further improved, i. e. ventilatory support, adequacy of information provided and accuracy of prior description of the patient's state. a new perspective must be applied for critically ill patients transportation since . % of the patients were evaluated and treated in more than one, medical departments, mostly primary care, before they were finally admitted to our icu. dclhb is a human derived hemoglobin molecule that has been cross-linked to stabilize and permit heat pasteurization to remove residual proteins and inactivate viruses. dclhb is mixed with a lactated electrolyte solution to yield a total hemoglobin concentration of log/dl objective: to present an overview of four recently completed clinical safety studies of dclhb in the u.s. and europe, and to discuss the properties, actions and potential indications for dclhb. method: patient populations in the four studies included males and females ranging in age from to years. dosing ranged from mglkg to mg/kg. the controlled randomized safety studies were conducted in chronic renal failure patients, surgical patients undergoing total hip replacement or abdominal aorta repair and in hemorrhagic hypovolemic shock patients. these very diverse patient populations allowed safety evaluation of the product in patients who were generally elderly, often hypertensive with some degree of cardiovascular disease, and receiving medications for treatment of other conditions. results: over patients received dclhb in the four:studies. no product related sarious adverse events occurred during the clinical trials. conclusion: results from phase itll safety studies of dclhb in patients undergoing chronic renal dialysis, abdominal aorta repair, or total hip replacement and in patients in hemorrhagic hypovolemic shock, indicate that the product was well tolerated in these distinct populations. although these studies were designed to evaluate safety, the data suggest clinical benefit. follow-up efficacy trials are indicated. prehospital emergency services represent the extension of emergency care into the community and constitutes the manpower, communications, transportations and facilities used to provide care for patients outside hospital. one of the main points of the system is how to decide the hospitalization of patients and what kind of facilities to provide : emergency medical service, fire brigade, locat general praclitionner or ambulance officers. objectives : to realize guidelines for using the prehospital emergency medical service in case of patient'calls outside hospital. methods : from st june to july , all the calls for emergency care were analysed using a questionnaire of items (origin of the call, responses to the questions of an emergency practitionner, kind of emergency service provided and the issue of the patient). after taking account of the appropriatness of the decision, statistical method used was a logistic regression. results : calls were analysed. the criteria, for prehospital emergency medical service using, given by the logistic regression were as following : existence of a call for emergency, thoracic pain, dyspnea, seizures, cyanosis, drug intoxication, fall of the patient, fracture, age, the state of consciousness and the neurologic reactivity. the minimal and maximal predictive values of the model given by the logistic regression are respectively % and %. the performance of the model is %. conclusion : it seems possible to help medical decision of emergency medicine by using only some easy criteria and a predictive model. (italy) objective: to evaluate the incidence of blunt carotideal injury (bci) in patients admitted to our icu after head injury. methods: we reviewed the medical records of all patients diagnosed to have a bci. at admission, the severity of trauma was assessed either with glasgow coma scale (gcs) and with ct scan. bci was demostrated by doppler ultrasography (us) and by angiography (ang). results:since may to april , patients were admitted to our icu with bci ( m, f, age + ). a history of direct trauma was present in patients. admission gcs was in all patients, and was associated with hemiparesis in of them; the last became paretic hours thereafter. two patients had concomitant injuries (a homoiateral clavicular and a controlateral zygomatic fracture, respectively). the initial ct scan was negative in every patient, and showed signs of ischemia after a variable timespan ( - days) after the onset of the symptoms. the bci was diagnosed with us and ang, which demonstrated a thrombosis of the internal carotid artery (ic). in two patients, an intimai dissection was also present. three patients were treated with heparin associated with antiaggregating agents and were discharged alive. the last patient was referred to our icu after the development of a massive hemispheric infarction, and died three days after the admission. at necropsy, the ic thrombosis was associated to an extensive homolateral extra and intracranial venous thrombosis. conclusions:the presence of focal neurological signs despite a negative ct scan should address the diagnosis toward a bci, thus implementing the diagnostic workup with us and/or ang. tab i: distribution of l~tients (%) in the groups the outcome were monitorett results were sabmitted to statistical analysis using a continence table x in z test. res.cl~s: of patients were submitted to thrombolysts and died. the higher incidence of bracb, ar~lhmias (ii degree gg p t e and av block. i degree av block. avsb . <ii. sinus arrest) that requited the insertion of avsc . <cl temporar~ pace-maker was recorded in group a b vs c . ns in % of the cas ~. the rapid recognition of life tab li:;~ test. threatening conditions suggests the monitoring of v r-lead r b' in the course of inferior ami. the authors report their own expirience in application of ringer lact.(rl)and , %nacl/ %dextran . methods:in polytraumatized patients with developed hae morrhagic shock,injures of the chest(qg)and abdomen(q ) prevalid.replacement of the volume loss egan in institutions of general medicine,frequently by rl and dextran or haemaccel.after receiving necessary medical aid,polytraumatized patients wer transported to mma by helicopter,continuing replacing of circulation volume by the same solution. results:average quantities of the solution dministered ~o the injured wer~ - ml.a group of the inoured which,after the admission to mma was resuscitated by adml nistration of the , %nacl/ %dextran @(groupii)( -sml/kg observed to the final surgical management,had statistically higher map(increase of % in relation to admission), cvp(average increase of , cmh~o),diuresis(averagely omi after min.),better gas ~nalyses and acid-base status in relation to the group which was administered only rl(groupi)(increase of the map of q %,average increase c~ of cvp , cmh and average diuresis oml after omin). total quantity of the administered solutions was significantly smaller in the groupii( ml: ml).the only ob served complication was hyperhatr~f~a::and~moderat~hyper osmolerity of plasma which disappeared within hours. the quantity of replaced blodd was considerably smaller in the groupii(qooofnl: ooml).in the group i interstitial pulmonary oedema was observed in two patients(qo%),while: in the group ii no complication was observed.coagulation i disorder and hypotension were not recorded because of the fast infusion of hypertonic/hyperoncotic solution in the groupil as the infusion of , %nacl/ %dextran lasted minutes. w.scherzer(l~,k.lechner( ),r.simon( ). ll)dept.anaesthesiology,trauma hospital,vienna-meidlin !( )neurotraum.rehabilitation center,vienna-meidling both austrian workers'compensation board,vienna,austria what we usually call"unconcious" means only that the patient is not able to interact in any kind of way.experiences in intensive care medicine (robinson, ) ,in general anaesthesia (bennet, ) and with evoked potentials show,that unconcious patients are not automatically unhble to process and recall information.this implicates a challenge to start the efforts to restore the traumatica-!ly desintegrated brain function as early as possible in the acute phase ia (zieger, ) .we present a method to ~repare the patient for the rehabilitation goals of the postacute phase ib,the phase of stabilisation ii and of rehabilitation iii within one concept. to achieve sensory stimulation in phase la we use: attempt at some kind of dialogue by speaking to and tou-ching the patient,to make him experience his body limits, ~acio-oral therapy by ice application,tast and smell pro vocations and trials of feeding,early adaption to circadian rhythm and guiding the patient in every-day-life-ac-tivities e.g.:in the brushing of teeth,washing etc. to achieve motor stimulation and orientation in terms of ~-ace,time and gravity in phase la we use: avoidance of perceptual impairment as produced by air-:sack-beds or habituation to supine position,using method~ according to affolter( ) ,basal stimulation (bienstein, ) , bobath-therapy( ) and facilitation of a physiological moving pattern(pnf)according to knott( ) . conclusions: by integrating all these measures in the ~herapy and nursing even in the acute phase la one prei ares the comatose patient for multisensory stimulation nd motor training and all other rehabilitation activities in the following phases of therapy. s objectives." measurement of cardiac output (co) and stroke volume (sv) requires insertion of a central venous catheter and is associated with a considerable risk for complications. non-invasive methods for determination of hemodynamic parameters have been introduced recently. one of these methods is the aortic modelflow program, which provides co and sv continously using a three-element windkessel model. the aim of the study was to evaluate the accuracy of the aortic modelflow program in critically ill patients. methods: patients (mean age: ) admitted to the emergency department after cardiopuimonary resuscitation (n=ll) and for myocardial infaction (n= ), acute congestive heart failure (n= ) and anaphylactic shock (n=l) were included to the study protocol. after stabilization all patients received a swan-ganz catheter (baxter~; edwards swan-g-anz) via a central vein. co and sv were measured by the use of a cardiac output catheter and injection of rrd ice-cold glucose % non-invasive cardiac output measurement was done by using the continuous cardiac output software package modelflow (tno; portapres| results: overall, paired measurements were used for the evaluation of the accuracy of non-invasive obstained sv and co. mean values, standard deviation and range of co and sv evaluated by invasive and non-invasive methods are summarized in the aortic modelflow provides reliable hemodynamic data in critically ill patients. it may be a useful alternative due to its non-invasive approach and continous measurement. objectives: to determine the feasability and accuracy of a rapid urine screening test (triage tm, merck-diagnostika) in an emergency department. methods: prospective analysis of the triage tm testkit (tt) in patients admitted because of suspected drug abuse during an observational period of months. the tt is a eompetitve immunoassay which gives qualitative results within i minutes for methadone, benzodiazepines, cocaine, amphetamine, opiates, barbiturates and cannabis. urine samples were analysed with the tt and compared to the results of a enzyme-lihked tmmuno-assay (el.a) in our labratory (gold standard). results: during the study patients were enrolled, in of these patients substance abuse was proven by tt and verified by eia. we recorded no false positive or false negative results for benzodiazepines, barbiturates, opiates, cocaine and cannabis. two results were false positive and one false negative for methadone; one result was false negative for amphetamines. the triage-testldt had an overall sensitivity of . and specificity of . . conclusions: the triage tm testkit seems to be a a useful rapid test device in addition to quantitative tests for drugs of abuse in an emergency department. objectives: the purpose of this study was to evaluate the influence of several factors of the renin-angiotensin-aldosterone system on blood pressure response in patients with hypertensive crises. methods: patients with systolic blood pressure > rorohg and diastolic blood pressure > nunllg were included into the study. prior to treatment blood samples for determination of plasma renin activity (pra), angiotensin converting enzyme (ace), angiotensin ii (ang ii) and aldosterone (aldo) were collected. all patients received rog enalaprilat intravenously. success of treatroent was defined as a reduction of systolic blood pressure below mmi-ig and diastolic blood pressure below mmi-ig within minutes after start of treatment. results: patients were included in our study, ( %) patients responded successfully to treatment. mean arterial pressure decreased in responders by . mmhg and in non-respenders by . mmhg (p< . ). responders and non-respenders differed signii'icantly concerning pra (p= . ), ace (p= . ) and ang ii (p= . ). . . the extent of blood pressure reduction correlated positively with the pretreatment pra and ang ii concentrations (correlation coefficient for pra: r= . ; ang ii: r= . ). conclusion: our data confirm that in patients with hypertensive crises blood pressure response to ace inhibition is mainly determined by circulatory pra, ace and ang ii. as the extent of blood pressure reduction correlates with pra, ace-inhibitors in patients with suspected high renin status cannot be recommended, as excessive blood pressure reduction, which carries a considerable risk for further organ damage, may occur. f. staikowsky, n. grillon, f.pevirieri, c.jedrecy, c. zanker, f. michard, a. haft medical emergency department. hospital bichat, paris epidemiology of acute intentional self medications-poisoning (smp) in france is especially known by data of poison control centei,s and intensive care units (icu). the purpose of this study is pro~,ided characteristics of this problem in a med for adults. method: july to june , files of patients consulting to the ed for smp have been retrospectively analyzed. results: patients, women and men, . + years old (range - ) have been admitted for episodes of smp ( % of all consultations) whose relapses during the period of study. psychiatric disorders, drug addiction or hiv patients was found for respectively . %, . % and , % of patients. the interval of time between the ingestion and emergency consultation was noted for % of smp ( + min, ranges - ). the involved products name was known in totality in % of cases with an average number by episode of . + drugs (ranges - ). the most often, ( %) or ( %) different products were interfered. the nonbarbiturate psychotropic drugs accounted for . % of the products (benzodiazepines %, antidepressants . %, neuroleptics %, carbamates . %, imidazopyridines . %, cyclqpyrrol nes . %). analgesics and nonsteroidal antiinflammatories represented . % of all drugs, anticonvulsants . %, cardiovascular drugs %, antiinfective agents . %, drugs against cough . %, muscle relaxants . % and antihistamines h . %. the benzodiaz pines were present in episodes, alone in episodes. in . % of cases, there was a simultaneous intoxication with alcohol. the processing consisted of gastric lavage in . % of cases, activated charcoal in . % of cases, flumazenil in . % of cases, naloxone and acetylcysteine in . % of cases; orotracheal intubation was performed in patients. admission in hospital was effective for patients, in medical ward (n = ), psychiatry (n = ) or icu (n = ); no fatal case was recorded. conelusion: smp to ed are often benign. the benzodiaz pines are the most often incriminated but the new anxiolytics and hypnotics (imidazopyridines and cyclopyrrolones) take a growing place. the latsion burn center of athens. its planning constructive and functional refinements j. ioannovich, a. petalas-vourekus, d~ serbetis, h. carsin a bed burns unit is under construction following a donation to the general hospital of athens. the plan of the unit, covering a surface of approximately . m is based on the principle of three identical bed satelites which may function totally independent from each other. in the center of the unit the common facilities are installed, like operation theatres, storage rooms etc. this new modification in the plan of a burn unit is presented in this paper. the advantages from the fucntional, administrative and medical point of view are discussed. tiffs anisotropic conduodon could favour the ocenrence of a circular movement of the impulse that leads to tachyeardias by reentry. purposes of this work were to study, with the help of epicardial mapping, the influence of a trieyclie antidepressant, clomipramine (c), on the conduction velocity longitudinal (vl) and transverse (vt) to myocardial fiber orientation and on anisotropy (a = ratio vl/vt), and their modificutions by the sodium bicarbonate ( ). method: a plaque of electrodes, positioned on the left anterior ventricular wall of anesthetized dogs, allowed to deliver, thanks to central electrodes, programmed electrical stimulations inducing vcuttienlar complexes, and to collect them. each entailed unipolar dectrogram was processed by a computer system that drew the isochrones and a map of activation allowing the calculation of v. the c was infused ( . mg/kg/min iv) during rain; at t , dogs received the b until the retuni of qrs to its initial value fro). a lengthening of qrs of at least % of its value at to was demanded before the administration of b. results: dog was excluded because of an.~nsufficient prolongation of qrs before the administration of b. all values (map : mean arterial pressure, i-ir : heart rate, qrs andqt intervals, v) differed significatively ( < . ) compared to values control fro)except qrs at t . the b ( + ml/kg; ranges . and . ml/kg) modified no studied dements outside of the ( }rs. to ti t t t t t a , + , , + , , + , , + , , + , , + , , +- ,~ conclusion : the c slowed v l and v t without modify the anisotropy. the b did not modify the v of~conduction while the qrs prolongation was corrected. the c acts as a class i antiarrythmie drug on the inward sodium current during the phase of action potential; the gap junctions have shown to be important in the conduction and an action on the gap junctions such as a modulation of the junctional resistivity, can not be rule out. is the doctor a heroe ? p. t.schies~.he, t. bauer, m. seyr dept. of anaesthesiology and intensive care, aokh krems, austria objectives: helicopter emergency services (hes) are getting popular more and more. the results concerning outcome are encouraging. however, some recent accidents with dead or badly wounded hescrew-members have shown the relatively high risk for the crews. therefore we were interested to eval ate the motivation of physicians to participate in a hes. this survey was designed to investigate current concerns about safety and motivation of doctors on emergency call. methods: a questionnaire was sent to doctors of the austrian emergency system. the survey consisted of multiple choice questions and subjective scoring tables from (--full agreement) to (=disagreement). overall, "/. of the active emergency physicians participated in the survey. results: . % of the doctors assume the system is basically safe, experienced doctors tended to have less trust in safety. only % would not hesitate to go into action by dark. . % stdctly refuse night flights to accidents outdoors. although defibrillations are assumed to be safe dudng flight, only % would do it. . % of the doctors would rather stop flying. the most common reasons for ,uitting were wish of family and fear of an accident. . % conclusioq: short transportation times help to avoid trauma related stress, pain and shock-induced organ complications. therefore the physiologic and economic advantages of hes are undebatable. however, the survey data indicate a considerable concern about safety of the medical personal in a hes. crash landings within less than years with deadcases and badly wounded crew members in a small country like austda make desire for safe flying conditions understandable. obiectives: to evaluate the clinical usefulness of trachlight. methods: trachlight is a new device facilitating endotracheal intubation. a stylet with a lightprobe is inserted into the endotracheal tube. intubation is guided by the light glowing through the neck tissues, thus rendering direct laryngoscopy unnecessary. intubation using trachlight was studied in patients (age - years). the indication for intubation was elective surgery in patients (asa i-ii) and emergency intubation in patients. in the elective patients, anaesthesia was induced with thiopentone supplemented with fentanyl, and intubation was facilitated with vecuronium. the cause for intubation in the emergency patients was dyspnea in , cardiac arrest in , trauma in, and unconsciousness due to drug overdose or seizures in patients. intubation was facilitated with medication in patients. results: of the elective patients, ( %) were successfully intubated. six patients ( %) needed two attempts before successful intubation. the duration of intubation exceeded seconds in patients ( %). of the emergency patients, ( %) were successfully intubated. six patients ( %) needed two attempts, and the duration of intubation was more than seconds in patients ( %). in % of all patients, intubation was assessed as easy. no or insufficient glow, prolonging intubation or necessitating two attempts, was noted in patients ( %). oesophageal intubation occurred in patients. conclusions: trachlight may be a valuable adjunct for intubation in varoius settings provided that adequate training is provided. a learning curve was found to exist. objectives: to compare enoxaparin and standard heparin in cavhd and calculate the value of laboratory controls in the treaanent. patients and methods: twenty patients needing dialysis for acute renal failure participated in the study. the main exclusion criteria were massive bleeding or a thrombocyte level < x e /i. in each treatment the same type (av- , fresenius ag, germany) of a polysulfone capillary haemofilter was used. the study scheme consisted of two consecutive four-day cavhd treatments, one course for each type of heparin. the order of heparin administration was counterbalanced between patients. the standard heparin was given as a continuous infusion aiming at an activated coagulation time between and s. the initial enoxaparin dose was rag every :th hour intravenously, but was modified by any signs of coagulation in the dialysis blood lines or bleeding complications. results: the dialysis treatment was adequate in both treatment modes, with mean blood urea levels . and . mmol/l respectively (ns). the bleeding complications were moderate and similar in both treatment modes. the mean life-span of haemofilter using enoxaparin as an anticoagulant was some longer than using heparin ( . + . h versus . + h, ns). the mean aptt-levcl during heparin treatment was s and during enoxaparin treatment s (ref. - s). the mean daily dose of heparin was nag, that of enoxaparin lg mg. the mean anti-xa activities were . u/mi and . u/mi, respectively, reflecting a better bioavallability of enoxaparin. conclusions: both anticoagniation modes were equally effective and well tolerated. the amount of enoxaparin needed for a proper anticoagulation was, however, less than half of that of standard heparin. the changes in aptt level were too slight to make its use possible in controliing the dose of enoxaparin. the use of enoxaparin seems to be rather safe in cavhd even without laboratory controls. the adv~ucea in the management of computerized data of an intensive care unit have been petalled to the clinical advauces and the increasing sophistication of methods of diagnosis fop the clinical application an therapy. this has led our unit to design and develop a computational system called timbu which is used to help physicians assist patients. among its various uses, this system has a software for the hemodynsmic control of a critic patient. this program was carried out to get as fast as possible the hemodynamic data of the patients in an intensive care unit. as an example, we can mention that when we load data obtained through direct measurement from the monitors and the lab, the program calculates parameters that guide, intelligently, to the diagnosis and therapeutic behaviour of the hemodynamic problem through screen messages. the validation of this program in the unit of intensive care has demonstrated that its use allows a more efficient handling of the patient with serious hemodynamics and respiratory disorders. ohieetlve: traema is a heterogeneotm 'disease' that ecatr~ a~"o~s all age ~oupe with v~ying degrees of severity. this imerogeneity has made the di~e, trmma, diflkaflt to r the ehn of this stady wa~ to assr the fitaen of saps in ibis popeleties. methode: in order to compute the ~ probability, a model derived from logistic regression w~ developed. meam'e of calibration (goodaess-of-fit stetislj.r and di~'riminafion (roc ou~e) were adopted in developmm~ and validetlon set randomly taken from a database of pts eeeseemivety admitted in icu (arohidia). ~ witho= salm, p~ yom~ am is yam, with los ~horter thma hotam wore exr fa'om thi~ mmly~ir thi~ model v~s then evahmed on the ~per ~mbgro~ (i.e., trmma pts). if'it did t~t fit the data well ~, new model wm developed rer the logit only on trm=~apm. reims: data were availabte for pts during aperiod of three .y~m , treama pts were . %), teats of calibration iadioaled probability model did mot provide m adequate refle~on of the mortality ezperieace in pm with ireutae, being the observed mortality lower flma the expected (figm'o). a aew model was then variable. this oastomized model fit~ the de~t of trmara pts very well (g =- a p> . ; roc = , ). the di:lferencea between the two modele were evident. conclusion: this ltudy shows that mortality in iramna pts is over wcfe~d when ~se~ed by menm of saps. however the r mode! meets high standmcd in terms of calibration mid dil~'iminat'~o~ ']"he advaatage of ~imd models meaas the colleotion of the ~ set of variables for all pm admitted in icu e~einat the ase of diasma specific ~oring syatex~. ("sl"): effects on cardiovascular and hemostasis systems (cvs, hss) a.oborin~ph, ~.~yndiuk~ph, b.kondratsky ~pt. of'""su~gery and transfusiology, research institute of hematology, lvov, ukraine objectives: great interest has been shown recently in the use of hoss for the initial resuscitation of hypovolemic shock. methods: the study was carried out in dogs -~h hs was induced by jet momentary hemorrhage (h) from a. femoralls (the bloodloss volume made . + . ml/kg). the treatment was begun after .u+o. hrs of h. "sl", created on the basis of-sorblt and natrium lactate ( mosm/l) was injected into v. femofalls at the dose of io. ml/kg. results: it is established that before treatmen-~rterial blood and central venous pressures (abp, cvp) diminished to . mm hg and - . + . cm h (p .o ), while heart rate (hr)-increased to . + . per min (p<.o ). by this the indices of ~latelet counts (pic) and plasma fibrinogen (pf) lowered by . % (p<.i) and . % (p~. ), while fibrin degradation products (fdp) enlarged by . % (p~ . ). after - min of treatment termination abp and cvp increased to . + . mmhg and . +o. cm h (p<.o ), and ~[r diminished to t . + . per min (p>. ). at the same time the indtces of pic and pf enlarged by . % and . % (p>.i), while fdp diminished by . % (p>.i). one of dogs survived. life duration of the other dogs was . + . hrs. conclusions: the obtained data are ~he evidence of normalizing influence of "sl" on cvs and hss, and allow to recommend it as a mean of initial resuscitation of hs in clinic. oblectives: we prospectively studied icu patients with severe head injury (hi), which cerebral lesions monitorized with sjo through opljcal fiber and the cerebral flux with tcd. methods: since january until june , we collected ht admitted to the icu, and of them monitorized with optical fiber in the right jugular bulb and tcd. all patients needed mechanical ventilation related to gcs <__ , with ct in admission (classifing lesions according to marshall and al.) . we related the final results to the evolution of sjo and tcd, with other monitorizing methods like gcs, ct and icp. ~sults: conclusions: in patients with gcs _< , sjo is useful to evaluate the evolution towards vegetative state, still more in cases with ct type ii in admission and higher apache ill. elevation of icp implies an evolutive nsk to brain death and data of tcd is a good indicator of brain death, the complete monitorization of these patients can improve the therapeutic control of this neurologic problem, , ( m, f) , (m. age: + years), divided in two groups (a and b) under specific criteria(tremor and/or fever during admission in i.c.u., or not). the injury severity score was > in all studied patients. tbe group a ( m, ") had no tremor and/or fever on admisskm, while em group b (tin, the above criteria were ix)sitive. bhx~d samplings were taken - hours after accident and - rain. after admisskm in i.c.u. micro-eli~ method was used for measuring cytokinc-levcls. statistic analysis was performed by studcnt-t test. as control group, healthy people were examined. _resu!_ts-il-lct, il-ii~, il- and tnf-tt levels were similar to control group levels in both groups a and b. i!,- and g-csf levels were found increased in both groups (p< jxjl), while il- levels were statistically significant comparing to group a. in con_tin_skin, during immediate post raumatic period,proinflamatory cylokines il-i~, il-i~ and tnf.-ct, produced in an earlier stage than ,. , cannot be detected,whereas .- was increased significantly, especially in group b. g-csf was fimnd in increawal levels in both gr(mps, without statistically significant difference between gnmps a and i|. objectives-l~valantc proteolitic activity, disorders in" eariy, period after combined trauma and p(~.ssibilit, i' of their correction by injection of proteo[ysis inhibitors contrycal and s-fto~:nracil in combination with driving an isotonic snlu~ion of sodlum chloride and polig[ucine. methods: biochemicai studies of proteolitic activity in dogs with limited deep burn and acute bloodloss, . result:s: in case of deep % burn, cornplicated by bloodshed the of blood grows at - times. it; is the restdt of the pancreas glandischemi demage, caused by the centralised circulation of blood and intensifies the deviations of haemodiaamics and albumin exchange. the degree of endogene intoxication by mean mofecular peptides which are the products of albumin decay reses to %, and % in hours. in hours after the trauma the-process is accompanied b ! , % lower inhibitory activity of blood, where as at the peak of the trauma it was , ~ higher. that proves the nnfavuurahle process of the shock in case a combined trauma. conclusion: the vein injection of 'proteolysis inhihitotz cnntrycal and -fforuraei[ in cumbination with driving an isotonic solution of sodium chloride and p.dligh]cine to refill lhe loss of blood helps to lower at times the profeolitic activity of blood. but it still remains above the initial level. the degree of endogene intoxication lowers at times; [ emodinamics aml albumin exchange stahilised. objectives: nimodipine, a known calcium antagonist, has been shown to dispose a beneficial effect on patients with subarachnoid hemorrhage, but its efficacy on traumatic or spontaneous intracerebral hematoma has not been justified. therefore, we studied the effect of nimodipine on the histopathological changes following an experimental intracerebral haematoma in rabbits. methods: twenty-three new zealand albin rabbits of both sexes, weighing - , kgr and at age of - months were anesthetized and a small burr hold in the left parietal aerea was carried out under aseptic conditions. the dura was opened and . ml (this volume assuring a normal incranial pressure after kaufman ) of autologous blood was injected into a depth of mm via a needle of . mm bore. the wound was closed and the animals were left to recover. nimodipine, of , mg/kgr of by weight per day was given via a nasogastric tube to fifteen animals for a period of time of fifteen days (group b). six rabbits were given water and served as control (group a). both groups of animals weie sacrified on the fifteenth day, their brains were removed and immersed into % formalin solution. tissue sections of ~ were embedded into paraphin and stained with haematoxyline and eosin, mason and gfap stain for gliac cells. results: two animals died after the surgical procedure, because they developed large intracerebral bematoma. no animal developed neurological deficit except one of group a which manifested a right side hemiparesis. the results of the bistopathological changes are the following: i) the mean -+ sd diameter of the lesions in the group a was --. ~t while that of group b was + ~t (p< , ) ii) secondary ischaemic neural tissue changes, characterized by the extravasatlon of red cells, the presence of haemosiderin-containing macrophages and signs of low grade inflammation zpredominated in the specimens of group a and were totaly absent from those of group b. iii) a ring of gliac hyperplasia and a low grade local fibrosis was found, encircling the lesions in the specimens of group a in contrast to those of group b. conclusions: nimodipine when administered in rabbits following the development of a non increasing the icp experimental intracerebral haematoma, prevents the extention and the severity of the lesion. objectives: to study the efficacy and side effects of adding intramuscular clonidine (clophelinum) to analgesic regimen in early management of patients with serious burn injury. methods: pts with - % bsa second to third degree flame burns (respiratory tact injury excluded) to yrs of age were randomised to study (n= ) and control (n= ) groups. burn shock was treated with hypertonic saline -bicarbonate solutions ( mmol/l na +) ml/kg/%bsa for the first hours and ml/kg/%bsa for second day. analgesia in control group for the first hours was provided by regular hourly intramuscular administration of mg of morphine sulphate and mg of analgesic -antipyretic analgin with mg of diphenhydramine (dimedrol). from the rd day regular administration of morphine was finished. in the study group ixg of clonidine was added -hourly for hours and dose of morphine halved. vas, verbal rating scale for sedation (vrs, - ), sleeping time, spo , hr, bp, diuresis, vomiting and other complications were comparatively evaluated during patients' stay in icu. results: addition of ~g of intramuscular clonidine daily allowed to achieve better analgesia and sedation with halved consumption of morphine. mean vrs in study group for the first days was . - . vs . - . in control group with twice longer sleeping time. there was significantly less tachycardia in study group; dynamics of bp for the first hours did not differ considerably; later, there, was tendency for hypotension in study group without adverse effects on diuresis or other indices of tissue perfusion. because of high incidence of chronic ethanol abuse among study population pts of control group suffered from psychomotor agitation or delirium, probably as a sign of alcohol withdrawal syndrome (aws). this made regular evaluation of vas impossible. in the study group only pt showed sign of aws. mean vas score was in . - . range for first postburn days. pts appeared excessively drowsy due to clonidine, but it had no adverse effect on their overall clinical course. mean spo values in study group were in - % range, among controls - %; vomiting was absent in. cionidine group vs cases among controls conclusions: clonidine could be a valuable addition to analgesic -sedative regimen in burns, especially for prevention of aws and deserves further study in this regard. hemodialysis -hemoflltration modifications and/or intratracheal gas insuflation have been recently used for blood gas exchange in several models of respiratory failure. objectives: evaluate the combination of cavh-m and igi for respiratory support in experimental acute lung injury. methods: five mongrel dogs ( -+ kgr) were mechanically ventilated inroom air, paralysed, heparinized, connected with a cavh-m system (diafilter- polysulphone membrane) and remained stable for one hour (pao~= . • peco = -+ mmhg, ph= . -+ . , bp= -+ mmhg and pap= -+ mmhg). all was induced two hours after oleic acid infusion ( . ml/kgr) into the pulmonary artery (poo~= . _+ -p< . , paco~- . _+ -p< . , ph= . -+ . -p< . , bp= -+ -p=ns, and pap= _+ -p< . ). fio % for the next minutes did not significantly altered the b ood gas abnormalities. afterwards, pure oxygen applied simultaneously a) through the inlet of the filtrate's compartment of the hemofilter ( l/min) while filtrate and gas were removed from the outlet port (bypass flow ml/min) b) through a thin intratracheal catheter positioned cm above the carina ( l/min). the fio given through the ventilator readjusted to %. results replacement fluids/filtrate during the next four hours were not exceed . l/hour, whilst the blood gases and pressures were improved as follow: cavh-inlet:pao.= . objective. to compare the changes in humoral immunity in trauma patients following massive transfusion of autologous and homologous blood. methods. we studied randomised clinical groups of patients each containing patients with trauma and operation of large arterial vessels. the amount of autologous or homologous blood transfused to the patients was exceeding ml, while the patients in the control group did not recieve blood or blood products. results. we recorded most pronounced and characteristic changes on the -st and on the -th day in the group of patients recieving homologous blood transfusion, i.e. decreased amount of igg,iga,igm,c and c fractions of the complement system, haptoglobin and significant and sustained rise of circulating immune complexes up to the end of the study period. in the control group of patients the decrease was weaker and lasted only during the -st post-operative day; the dynamics of the circulating immune complexes level were almost the same as in the first group of patients. in the group of patients recieving autologous blood transfusion, the parameter values did not change significantly from preexisting levels after the -st day, while on the -th and on the -th day showed a tendency towards aslight rise. conclusions. autologous blood has a favourable effect upon humoral immunity and should be the transfusion medium of choice in cases where autologous blood reinfusion is technically possible. ivan petkov, m.d., rumen farashev, m.d. and dimitar terziiski, m. d. medicine, military medical academy, g. sofiiski str., sofia, bulgaria objective. the amount of blood lost during trauma and operation could hardly be forseen and donor blood supplies are not always available in sufficient amounts. rare blood group types and/or unexpected haemorrhage pose a great challenge to the transfusion therapy and the methods of intraoperative autologous blood transfusion. methods. we report a case of a -year old male patient with extremely massive intraabdominal haemorrhage ( m( blood loss ) during an abdominal aorta reconstruction following a traumatic injury of the abdominal aorta. we achieved a successful reinfusion of ml of autologous blood using an original autotransfusion system developed by us ( pat. no / . . ) . results and conclusions. the autotogous blood in the case reported here was the only and the most suitable transfusion medium for the rapid intraoperative compensation of the acute haemorrhage and the favourable outcome of the patient. the post-operative period was smooth and no significant disorders in the clinical course as well as in the laboratory tests ( morphological,biochemical,coagulation and immunological) were recorded. there were no complications during the postoperative period despite the fact that the amount of blood reinfused to the patient was slightly exceeding his own volume of circulating blood. objective. the haemoglobin concentration and the perfusion pressure value could not be the only criteria for the early signs of tissue and organ dysfunction. because of this, we employed the extensive monitoring of oxygen transport during severe trauma in order to. achieve dynamic evaluation of physiologic compensatory mechanisms and to assess the efficacy of intensive care management. methods. we conducted a prospective controlled trial on the blood oxygenation, oxygen transport and tissue perfusion during the first days after the trauma in patients with polytrauma. we used a swan -ganz pulmonary artery catheter (beckton -dickinson, u.s.a.), deseret cardiac output computer (medical inc., u.s.a.) and hewlett -packard monitor (hewlett -packard, germany) to measure and calculate all the parameter values. the severity of the injury was assessed using the apache ii score system. all the patients had scores over . results. the results show a significant decrease in the arterial blood oxygen content and in the arterio-venous difference, as well as an increase in alveolo-arterial oxygen difference and in the transpulmonary right-to-left shunt. the tissue oxygen supply and the tissue oxygen consumption reveal a tendency towards a decrease below the physiologic minimum of adeqate values. the erythrocyte current velocity and the ratio between oxygen transport and erythrocyte current velocity also decrease inspite of the optimal blood rheology. conclusions. the dynamics in the parameters values are most pronounced between the -nd and the -th hr after trauma, which predisposes patients to the risk of developing stable hypoxemia and characterizes this period as the most critical for tissue metabolism and organ dysfunction. posttraumatic changes in immune mechanisms in lung compartment in trauma were analyzed in ao and da inbred strains of rats which differ in their immunological reactivity: the former being low responder and lat-~er hiperresponsive. methods: the levels of tnf-alpha activity in the supernatants of cultured lung lobes and dynamics of cells migration from tissue explants in h lung cultures were assessed in ao and da rats subject ted to severe burn trauma. results: increased levels of tnf activity ( + pg/ml compared to + . pg/ml in control) were found od day following trauma in lung sups of ao rats while no changes in the levels of activity of this cytokine were found in lung-sups od da rats more pronounced extent and dynamics of cell emigration were noted in da rats, while almost unchanged in ao rats sharp rise in pmn percentages h following trauma ( - % compared to rare pmns in control), followed by increase in lymphocyte numbers at later time points among lung cell emigrants was detected in ao rats. slower but persistent increase ( %, h following trauma and % and % on days and after trauma infliction, respectively) in pmn numbers among da lung cell emigrants was detected, which appeared to be activated, as judged by their nbt reduction capacity. increased percentages of peripheral blood pmns and increased state of leukocyte aggregation/adhesion were detected in both strains, but different levels of plasma tnf: increased levels in ao rats on days and following trauma, and initially but persistently high levels of plasma tnf alpha in da rats ( - fold higher compared to initial levels in ao rats). conclusions:different patterns of local (lung) and systemic changes in cell numbers and cytokine levels implicate differential posttraumatic migratory capacity of pmns vs. lymphocytes in lungs in ao and da rats. early diagnosis of acute intestinal ischemia by color doppler sonography e. danse, b.van beers, p.goffette, f.hammer,aav.dardenne, f.thys, p-f.laterre, m,s. reynaert, .lpringot dept of radiology (profb.maldague) and dept of intensive care ( prof m,s.reynaert), st.luc univ.hospital, brussels, belgium ob emergeny medical squad service is the most important segment in the process of saving the people, in the cases of mass accidents, like industrial accidents caused by the: explosion, fire, chemical poisoning, traffic accident, elemental catastrophes and the war. because of that, each emergency medical squad service needs to have in its motor-pool vehicle for the mass accidents/ for provoding at least people, wounded as well as the people became ill/. objectives: presentation of such special vehicle, produced by "zastava-kamioni" and it's medical-technical equipment. methods: descriptive and comparative analysis of the medical and technical characteristics, based on the actual norms/din, , iso , yus.../ results: on the base of doctrinaired requirements of the emergency medical squad in the case of mass accidents, our researches resulted in the following medical and technical characteristics -the vehicles for mass accidents are gvw/with a payload off cca - t, with the fixed, closed body, type: universal van, -technical equipment aggregates, stretches, anti-fire device, equipment for pitching the tent and for maintaing technical conditions of the work -medical equipment: linen bags with complete sets of bandage material, means for the reanimation and immobilization, for the infusion, medical instruments and remedies as well as the tent for lodging at least wounded and sik people. in federal republic yugoslavia, it was proposed such vehicles for the emergency medical squad needs. conclusion: we suggest to introduce this vehicle in the production range of the ambulance vehicles for saving, especially in the circles where can occur serious accidents. introduction : carbon monoxide (co) poisoning commonly generates central nervous system abnormalities though an important cardiac morbidity and mortality must be considered. long-term exposure to co with cohb levels < % may be more dangerous than short-term levels of - %. we report a case of an adolescent who after prolonged exposure to co developed a severe reversible cardiac dysfunction with low levels of bloed cohe c a.ase history : a year old boy was found comatose at home. his mother in the neighbouring bathroom died severn hours earlier of what was later proven to be a co intoxication. on arrival the gcs was / and the patient was breathing spontaneously. a postictal status with eventual postanoxic encephalopathy was suspected. a coh'b level of % was objectivated. the cardiorespiratory situation quickly deteriorated requiring mechanical ventilation. chest x-ray showed diffuse bilateral patchy infiltrates. ecg revealed signs of ischemia. severe left ventricular dysfunction was evidenced by pulmonary artery catheterisation and echecardiography and later by isotopic angiography (lvef %). treatment was intensified with inotropic support, intta-aortic balloon counterpulsation and oxygen therapy. the clinical course was further complicated by a crush syndrome and renal failure. the patient's condition gradually improved and he fully recovered without any residual lesions (lwf %) conclusion : even after prolonged exposure cohb levels can be misleadingly low. high tissue levels of accumulated co can be associated with coma and fulminant cardiorespiratory failure requiring advanced life support facilities. introduction : both neuroleptics (nlp) and tricyclic antidepressive agents (tca) can induce arrhythmias, prolongation of the qt segment and the pr interval and hypotension. we report a case illustrating that combined overdose of these agents increases the toxicity of each compound and the risk for adverse cardiac events. .c, gse history : a year old male ingested mg doxepin (sinequanr), a tca and mg prothipendyl (dominalr), a potent nlp in an attempted suicide. upon arrival in the emergency department the patient was unconscious (gcs / ), breathing superficially, and presenting signs of recent vomiting. physical examination revealed a taehycardia of b.p.m., an arterial blood pressure of / mmh g. ecg showed a brood qrs complex tachycardia. a chest x-ray revealed the presence of an aspiration pneumonia. laboratory investigation demonstrated increased levels of crcatine phosphokinase, lactate dehydrogenase and aspartate transaminase ; hyperglycemia and leucocytosis were present. the plasma concentrations of doxepin and prothipendyl were respectively gg/l (toxic level #g/l) and i.tg/l (no reference). treatment consisted of mechanical ventilation, gaslric lavage and administration of activated charcoal and iv fluids and antibiotics. a hemodynamically well tolerated veatricular tachycardia developed / h later. nahco ( meq/ h) was administrated inducing an ectopic atrial tachycardia with a normal qrs complex and prolonged qt. h after admission a normal sinus rhythm was present; the prolongation of the qt segment persisted for days. the patient fully recovered. conclusion : the treatment with nahco~, alkalizing the blood and thus increasing the protein binding of the tricyclic antidepressant molecule, can readily correct the potentially life-threatening cardiac arrhythmias and therefore should be part of the routine treatment of combined tca-nlp overdose. ob/ectives: the development of diabetes insipidus (di) in patients with brain injury is a known negative prognostic sign. the aim of this study was to investigate whether this is also a reliable early prognostic sign of brain death. methods: this is a retrospective study of patients treated" during a two year period ( - - to - - ) in our i.c.u who meeted the following criteria: ( ) coma score _< gcs within the first hours, ( ) positive brain ct scan on admission classified according to marshall's diagnostic classification (classes - ), ( ) normal renal function during the entire icu stay. for the definition of di were used the usual di criteria plus hypematriaemia (serum na" >_ meq/l). survival was defined up to the th postadmission day. conclusions: according to the findings of this study, the development of diabetes insipidus in brain injured patients seems to be a highly specific index for brain death (positive predictive value = . ). however, further prospective studies are needed for the definitive evaluation of these findings in such patients. emergency care in italy, despite all efforts, is still lacking a nationwide organized prehospital care system and, until today, there are only different regional solutions. the majority of these realities imply rather simple ambulance first-aid services without attending emergency physicians and without resuscitation equipment. the emergency medical service (ems) system in falconara m., italy, was implemented in august by a collaboration between the school of anesthesiology and intensive care of the university of ancona and the, already existing, volunteer rescuer organisation "yellow cross". according to the guidelines pubblished in [ ] the pre-existing equipment of the volunteers was completed with type a ambulances and special equiped motorcar (patient monitor, defibrillator) for ambulance indipendent physician transpur[. a special data collecting schedule was created to memorise every emergency intervention in a computerised data-base. the intraining members of the school of anesthesiology and intensive care provide hour ready intervention. in this report the authors describe their experience concerning primary firstaid medical interventions. for a preliminary evaluation we considered, retrospectively, consecutive emergency interventions in the time period from novembre , to april , . the emergency physicians treated male ( %) and female ( %) patients, patients died before hospital admission and patients ( %) were treated at home by the ambulance indipendent physician and did not need any further medical treatment. in the same time period year earlier (november to april ) without attending physician the volunteer rescuers transferred all first-aid interventions to near-by hospitals. we conclude that the presence of an attending, iudipendently motorised physician in emergency interventions is essential for the establishment of precise priorities and may be helpful to reduce hospital admissions by ambulance intervention, though reducing primary" health care costs. we have developed the method of liquor filtration which allows to purify the cerebrospinal liquor from blood and its decay products in the subarachnoid bloodstroke. the hemipermeable dialysis membrane was used as a filter, which lets only in water, electrolytes and substances with small molecular weight. the liquor filtration was used for the treatment of patients with the subarachnoid bloodstrokes of different etiology. the perfusion of liquor was performed at the rate ml/min in the recirculatory mode. its duration was - min depending on the bloodstroke intensity. the filtration makes possible the most completely purifying of the hemorragic liquor, the reducing of the content of blood ceils and its decay products - times as less. the monitoring of the patient's state during the perfusion didn't revealed the departure from the norm of the main vital part. the liquor filtration technique compares favo-~ rsbly with the routine method of cleaning by the absence of toxical effect of heterogenous solutions on the central nervous system. the filtrstion of the cerebrospinal liquor in the subarachnoid bloodstroke sllows to provide the the early cleaning of liqour, the regression of meningeal syndrome and to improve the patient's state of health. e tabli~mczr bd ~ of rei~idnal medical first-aid zhoulittoing, ed., tan zi, m.d. dept. of sargery, the first teaching t[ospitat, yejin-l)a-l)ao, wuhan fltlna objectives: the medical first-aid is the most important task of the public hc atth department. in general, single hospital model couldn't fatty, effective ly rescue mony severe patients who need mergant treatment in the scene. bub establishing the medical first-aid network, the severe patients can be given the most timely und the most scientific emergent treatment. so that, the suc cessfut rate of the saving wilt be greatly increased. methods..; our hospital is a general big hospital. through developing and cons tructlng for more than ten years, the medical first-aid network distributed art over the area under our jurisdiction has been set up. it consists of thr ee units: the medical first-aid unib center comartd and mnagment unit, co m~nlcation and tiaison unit. the principle of the network operation is with oat having to far to mergoncy, specialized emergency and the best merge acy. results: the results of the network operation were notable. cmpari~ the to tat successful rate of the saving ( . ~), the successful rate of saving tra ma ( .~), the suscessfut rate of saving shock ( .~) and the successful rate of cardioputmonary resuscitation ( . ~) daring the three years after t he network operated with these before ( . ~), ( ]. ~), ( . ~) and ( ft. ~), the successful rates after operating were remrk~iy higher ( p<i).o ). conctusions~ the above results showed estabiishmnt and mena emont of region at medical first-aid network had very si~nificont action during mergentty s avin the severe patients. moreover, the regi~at medical first-aid network atso take an important part in prevention and health protection of the mass. objectives: se related mortality is due to the occurrence of both rv and lv dysfunction evoking cardiogenic shock and pulmonary edema (f. abroug, intensive care med ; s. nouira. chest ) . prospective evaluation of scorpion antivenon (sav), the only available specific treatment, is lacking. the aim of this study is to assess the effects of sav on the basis of a case-control study. methods:among consecutive victims of se presenting to the emergency department, were managed using sav (group sav+). these patients were matched to envenomated patients who did not receive sav (group sav- chest injuries are the cause of death in % of trauma fatalities and a major contributing factor in an additional %. pneumothorax is a major complication of chest injuries and can be initially overlooked bringing to additional morbidity and mortality especially in patients who require mechanical ventilation. the real incidence of pneumothorax in severe blunt chest trauma hasn't yet been established, as many pneumothorax can be missed on the initial chest x-ray (cxr) and computed tomography (ct) has not been routinely used. to evaluate the incidence of pneumothorax in patients with severe blunt chest trauma. methods: a prospective study. from january to december , all trauma patients with one of the followings conditions: fractures of four or more ribs, signs of lung contusion on the initial cxr or presenting a severe hypoxemia on admission (pao /fio < ) in the absence of pre-existing pathologies, were evaluated by initial cxr and subsequently submitted routinely to a ct. results: severe trauma patients were considered. of them, presenting a severe hypoxemia on admission, had no evidence of major chest trauma (ais< ). hypoxemia was the consequence of severe trauma in other districts; these patients were therefore excluded. patients with severe thoracic trauma (ais>= ) were admitted into the study. the mean iss was . ( - ). thirty-six patients required artificial ventilation for at least hours during the icu slay. three of them, who had a tension pneumothorax, were submitted to an emergency thoracic decompression on the field by the emergency helicopter team. in cases pneumothorax was diagnosed an the initial cxr more patients had a pnx which was identified only on the ct. in cases a large pnx with lung collapse was missed on the cxr. in our group of severe blunt trauma patients, % ( / ) presented a pnx that required the insertion of a thoracic drainage. only one third ( / ) of the pneumothorax could be recognised on the initial cxr, while other were decompressed before performing the cxr. as many as % of the cases of clinically significant pnx were missed on the cxr, and a ct performed soon after admission allowed an early diagnosis bringing to changes in the treatment. (as the patients were mechanically ventilated a chest tube was inserted in all these cases). in cases, the initial cxr overlooked a huge tended pnx which was the cause of hemodynamie instability. conclusion: in patients with severe blunt chest trauma even large pnx can be missed on the initial cxr. moreover due to the non compliant compressible lung, a % pneumothorax which can be recegnised only on a ct, can bring to high intrapleural pressure altering eardiopulmonary function. n. andoeli , .~osid, m.zesevid, m.risovid, d.stepi , d.djokid b~rga~yc~qterclinicalcaqterafserbia, belgrade cb~ctives:~lis study ~ the use of ~rq]ofol earbired with k~t~ine (aq a~sjgh~ic s@~qt widn inirjrsic armlgesic pro~mities) or with fsqtmtyl,with psrtial azgmsis an hgenxlyn-a~ic ~ durirg ~ ~ re:~ver~ f~m ~ in hxh ~ of ~ti~. ~: yali~mial and ~bod: a~it p~tie~ts a~ i-ii were included in ibis shxly. patients were rsrd]nly dieided in two ~ns. all d~tie~ts ~me given - prcpofol bolus doses (o, ~gkg) for ird~iqn of ~. ~ia ~s m~sjn~ with an infusion ~ ~ropafol. as sdflitianal were given fan-i~l (o, n]g) ~tely before ~ anj trad~e~ irfojoation followad by feasted bolus of o,i mg in ~ro o l.patients in gr~ o received i~ (an initial bolus dose of rg slowly intcavax~ rd mg as infusion over ~ rain) .infusions of pro~fol or imcpofol with kg~mine ~ stopfsj - rain ]:~o~ extuhation.arterial blood ~ (sistolic arterial blood preassu-re~zap,mean ~rterial blood pr~,d~lic arterial preassure-[zp a~ h~art rate-~) ~ m~ before induction of a~ io, snd rain aftem ~ intutation. results: arterial blood preasstre ~s decreases duri~ irn~ction of sn~wd~sia in hy~ ~n~s,tnt mare in th~ ~ who r~eived fsqtanyl.~ere w~s statisticslly sifnific~ntly difemerme dmir~ m~ of an~ia. arterial blood r~easatre and heart rate were stable in the t-..e~min -~a ~. all th~,fl-e keta'nire grcqo hsd e~rly :~e~y time. ctrmlusi~s: ~e ombiretion of protxfol wilh keta/ne for irduorion a~d ~ of sn~sd~esis w~s yell accept~ by p~tierfcs anj coald he ~ as an alterrstive ~o ccnva~icrsl a~es -d~sia. objectives : assess the relation between cytokine or endotoxin release and indices of splanchnic malperfasion after hemorragic shock in multiple trauma patients. ]~r study was approved by the local ethical committee. trauma patients admitted to the emergency room who met the entrance criteria of more than hour map < mmhg or use of vasoactive agents or blood lactates > mmol/ were selected for study. a nasogastric tonometer (tonometrics, inc, plastimed, france) and a swan ganz catheter were placed on admission. phi, lactates, hemodynamics, plasma cytokine and endotoxin concentrations were measured on admission and at . , , , hrs. an immunoradiometric assay was used to determine plasma concentrations of il (n< . ng/ml) and tnfc~ (n< pg/ml). plasma endotoxin concentrations were measured using a chromogenic limulus assay (n< . eu/ml)( endotoxine unit= pg). results : severe multiple trauma patients (age = _+ yrs, iss = -!-_ , saps = +'~, mean-+sd) were studied. they received + packed red cells during the first h. mean duration of collapsus before inclusion was . _+ . hrs. death occm'red in ~tients. ~ pglml, *: ng/ml, etox : endotoxin(eu/ml), lact: lactate (retool/l) a significant correlation between initial il level and saps was observed. in the early post-injury period phi, sao , svo , vo were significantly associated with ;il release (p< . at ho, h , h ). later a significant correlation existed between lactates and ii (h , h ). a peak of tnf was detected at and hrs. it was associated with low phi and low arterial ph of the early post-injury period (p< . iat ho, h , h ,h , h ) and with high lactate levels of later period (_>h ). only the late release of endotoxins (i{ ) was correlated significantly with initial !oxygea-delivered parameters. iconclusion : there was a marked increase in il in the early phase of trauma . i and tnf release after major trauma iwith hemorragic shock is associated with splanchnic malperfusion, as assess by the ivery low values of phi. lactates seem to be a later indice. toxic effects are a well-known complication of an overdosage of prescription theophylline. what is less known is that over-the-counter (otc) asthma medications contain theophylline, and that in some cases this might cause toxic effects. a case seen by us involved toxic effects from theophylline in an otc medication and to date is the only published case in the english literaturet the rationale for this study was to delineate the otc products containing theophylline from whatever data sources available. hyperthermia frequently occurs in intensive care treated patients and intentional application of whole body hyperthermia together with chemotherapy is a therapeutical access to treatment of malignant disorders. anaesthetic support is required in either condition. due to the marked decrease in systemic vascular resistance seen in hyperthermia an additional vasodilatory effect of the anaesthetic is unwanted. the vascular effects of anaesthetics in hypertherm organisms is not known in detail. therefore, we performed an experimental study to detect the effects of inhalational anaesthetics in whole body hyperthermia. in sprague-dawley-rats katheters were inserted into trachea, jugular vein, and carotid artery. for continuous monitoring of cardiac output a flow probe was placed around the aortic arch. the rats were mechanically ventilated with different concentrations of inhalational agents in oxygen. we compared the effects of enflurane, isoflurane, and halothane in stepwise increased body temperature by submerging in a temperature controlled water bath. results: isoflurane lowers arterial pressure more than halothane or enflurane. the inhalational anaesthetics lower the cardiac output similarily and independently of temperature. isoflurane decreases systemic vascular resistance independently of core temperature and the decreasing effect of halothane on the resistance is completely abolished in hyperthermia. conclusions: the influence of hyperthermia on the systemic vascular resistance is dangerous. this allows no additional effect of the anaesthetic management. in spite of the vasodilating effect of inhalational agents in normotherm subjects, this effect is abolished in hypertherms using halothane. the condition of management of analgosedation in hyperthermia is different from normothermia. objectives: to evaluate a bedside computer processed cerebral function monitor for assessment of brain wave activity when clinical/visual clues are not present. methods: ten icu patients undergoing neuromuscular blockade monitored with the aspect brain wave monitor from january to june , . results: time to onset and depth of sedation were readily apparent to icu physicians not specifically trained in eeg reading. objectives: to determine whether non-depolarising neuromuscular blockade reduces oxygen consumption (vo ) in sedated, apnoeic patients. methods: haemedynamic. metabolic and oxygen transport variables were determined in sedated, apnoeic patients with severe acute lung injury. all patients were ventilated using a puritan-bennett ae ventilator with integrated metabolic monitor. inclusion criteria were; ) stable cardiorespirator s" status; ) systemic and pulmonary artery catheters already in situ; ) inspired oxygen < %. patients were sedated with midazolam or propofol to abolish response to verbal stimuli, and sufficient morphine or alfentanil to abolish all spontaneous respiratory efforts. following baseline measurements, neuromuscular blockade was induced with intravenous vecuronium, ug/kg, followed by an infusion of ug/kg/h to maintain the train-of-four ratio at . a further four sets of measured and calculated variables were obtained at min intervals. results: statistical analysis was by repeated measures anova. there were no significant changes in any variable over time. the changes in calculated oxygen consumption (vo fick) , and measured oxygen consumption (vo gas), and in energy expenditure (ee), are shown in the table. objetive: to study the effects on coronary hemodyrtamics and myocardiai metabolism of administering propofol during postoperation sedation of patients with normal coronary circulation and good ventricular function undergoing cardiac surgery. patients and methods: patients ( women and men) undergoing aortic and/or mi~-a/ valvular cardiac surgery were selected, with an ejection fraction greater than . and normal coronary circulation. for postoperation sedation propofol was administered in . mg/kg i.v. bolus, followed by a . mg/kgth perfusion. all data were registered before administering propofol and after minutes, the patients being hemodynamically stable and a rectal temperature of _+ . -~ systemic and pulmonary hemodynamics, and global, as well as regional myocardial blood flow, and metabofic variables were measured. results: the patients studied were about years old, and the average period of aortic cross-clamp was . min. the adminstering of propofol caused a decrease in the coronary blood flow (- %), great curonary vein flow (- %), myocardial oxygen consumption (- %), regional myocardial oxygen constanption (- %), myocardial oxygen extraction (- %), regional myocardial ooxygen extraction (- %), while coronary vascular resistances and global coronary vascular resistances did not change. oxygen saturation increased in the coronary sinus (+ %) as well as in the great cardiac vein (+ %). in no patient were significant changes suggestive of myocardial ischemia objectified. there was also found a decrease in systolic (- %), diastolic (- %) and mean (- %) arterial pressure, systemic vascular resistance (- %), and cardiac output (- %). conclusions: in accordance with the clinical conditions of this study, the administering of propofol is not likely to cause changes in coronary autoregulation, oxygenation and myocardial metabolism. obietive: analyse the effects of . % "end tidal" isoflurane (sedative dosage) on the metabolism and coronary hemodynamics during the postoperation period of patients undergoing cardiac surgery. patients and methods: patients ( women and men) undergoing aortic and/or mitral valvular cardiac surgery, with an ejection fraction greater than . and normal coronary anatomy, were selected. after the surgical operation, . "end tidal" isoflurane was administered for postoperadon sedation. the determination of variables to be studied was carried out before and minutes after administering isoflurane, die patients being hemodynamically stable and a rectal temperature of _+ . -+c. systemic and pulmonary hemodynamics, and global, as well as regional myocardial blood flow, and metabolic variables were measured. results: the average age of the patients studied was -+ . years. during surgical operation the period of aortic cross-clamp was . _+ . rain. the administering of isoflurane was followed by a statistically significant drop in coronary perfusion pressure (- %), coronary vascular resistance (- %), regional coronary vascular resistance (- %), regional myocardial oxygen consumption (- %), regional myocardial oxygen extraction (- %) and accompanied by a significant rise in oxygen saturation in the coronary sinus (+ %) and in the great cardiac vein (+ %). myocardial oxygen consumption, myocardial exu'action of lactate and regional myocardial lactate extraction did not change. in no patient were enzyme or electrocardiograph changes objectified. systolic (- %), diastolic (- %), mean (- % ) arterial pressure, and systemic vascular resistances (- %) decreased, while cardiac output did not. discussion: the administering of . % "end ddal" isoflurane, in the clinical conditions of this study, produced a decrease in systemic arterial pressure due to a reduction of systemic vascular resistance without deteriorate cardiac output. at coronary circulation level, has and effect on coronary autoregulation but had no effect on oxygenation and myocardial metabolism. the idea of tiva implies the realisation of major anesthesia components (los of consciousness, neurovegetative inhibition, analgesia, myorelaxatiou, providing the adequate gas-exchange) through i.v. introduction of drugs exclasively. aim: providing for the main tiva components with minimal side effects of the drugs used, taking into consideration the patients characteristics and the surgery specific character. methods: anaesthesias have been conducted in patients aged years ( females, males), undergoing planned and urgent operations with the pathology of lower, extremities, perinaeum, small pelvis, hypogastrium and with reserved spontaneus respiration against a background of % insnffladon through mask. operations lasted from . - . h. anaesthesia adequacy was assested by constant monitoring: "cardiocap" (nibr hr, rr, sao , t), through glykhaemia level and mimicry reactions. standart premedicatioo of m-cholinolytics ( . mg/kg) and h -blockers ( . mg/kg) on the operational table was sumplemented by administration of . - . mg/kg of lidocaine, . . mkg/kg of clonidine, . - . mg/kg of pentamidine by the tachifilaxia method. the premedication adequacy was assessed through haemodynamics characteristics. sedation: . - . mg/kg of droperidoi, .l- . mglkg of diazepam and analgesia: - mkg/kg of phentanyl, . -- . mg/kg of ketamine were introduced fractionally according to indications. infusion rate of ringer-lactat solution was - ml/kg/h and depended on the intraoperational blood loss volume and on the patients preoperational condition. the duration of postoperative analgesia was registered. results: clinical assessment of analgesia according to this techniques allowed to decrease the anaigetics dosage to the subauaesthetic levels. smooth stabilisation of haemodynamics (bp) at proper age norms in patients with the initial hypertension by the -th min. of anaesthesia as well as the absence of its increase in response to the additional introduction of anaesthetic have been achieved. (hr) had no abrupt changes and remained in the range of - per rain. adequate external breathing: decrease (rr) by - per rain., with sao increase from % to - %. hypoventilation was avoided by respirate ventilator. according to unauthentic data the glykhaemia level had been lowered by -t % to the end of the operation with the initial moderate hyperglykhaemia of up to mmol/l the cutaneous covering grew warm and got pink colouring. no mimicry reactions. in the postoperative period patients were in the superficial sleep state ( - ) and analgesia lasted - b. there were no complications due to anaesthesia. conclusion: combined using of bz, opiates, neuroleptics potentiate the i.v. anaesthetics effects allowing lowering of each tiva component dosage and, as a consequence avoiding their negative influence on respiratory and heart vascular systems. complex application of adrenergetics (therapeutic doses of cionidine and pentamini with using of taehfilaxy effects) permitted to provide for analgetic and neurovegetative components of general anaesthesia under subanacsthetic doses of tiva main components, and manifestation of hyperdynamic reactions of haemodynamics decreased while using of lidocaine -the economicai activity of heart-vascular system. good level of muscle relaxation was achieved allowing for widening of surgical intervention extent without respirator ventilators and inhalation anaesthetics application. anaesthesia is easily controlled due to fractional introduction of drugs with quick recovery of cns functions after anaesthesia. postanaesthetic analgesia is increased while concurrent opiates doses are decreased. absence of marced haemodynamic, endocrine and metabolic reactions during the operation and after it resulted in shortening the period of patients staying in hospital. a yo white man was admitted to hospital for dyspnea and a productive cough. he had cabg in past, but no recent cardiac ischemia. physical exam: decreased breath sounds over right lung. chest xray: consolidation of right lung. admission medications included diltiazem, furosemide (both were continued) and trazodone (which was discontinued). admission ecg: sinus rhythm, qt . /qtc . sec, with st and t wave abnormalities similar to prior tracings. he required intubation and mechanical ventilation for progressive hypoventilation and hypoxemia. between icu days and he received haloperidol, - mg/d (cumulative dose rag) for agitation and delirium. icu day : qt . /qtc . sec. icu day : for better control of delirium, trazodone " mg q hs was added. icu day : he developed frequent nonsustained ventdcular ectopy. icu day : qt . /qtc . sec, pha . , paco mm hg, pao mm hg, k . meq/l, mg . meq/l. later in icu day the patient had brief episodes of torsades de pointes, each responding to precordial thump, and finally rhythm stabilized with i.v. lidocaine and magnesium. haloperidor and trazodone were discontinued. ecg was unchanged and myocardial infarction was ruled out. next day, icu day : qt . /qtc . sec. torsades de pointes, a form of ventricular tachycardia characterized by a twisting qrs axis, is commonly associated with qt prolongation. haloperidol is used frequently in icu for control of agitation and delirium, with reported doses up to mg/day. over past decade, cases of torsades de pointes with prolonged qt related to haloperidol have been reported. trazodone may also prolong qt and cause ventricular arrhythmias, especially in patients with pre-existing cardiac disease. in this patient, trazodone likely exacerbated qt prolongation from halopeddol leading to torsades de pointes. critical care physicians must be aware of this interaction. it is imperative to follow the qt interval for patients receiving halopeddol, especially when another drug also known to prolong qt is added. one must consider discontinuing the drug when qt/qtc becomes prolonged. objectives: analgesics and intravenous anesthetic drugs are routinely used in critically fll patients, who often suffer from a secondary impairment of the immune system. previous in vitro studies have demonstrated inhibitory effects of these drugs on polymorpho nuclear cells (pmn). the potentially important role of endothelial cells (ec), however, was not investigated, since suitable test systems were not available until recently. therefore a physiologically more relevant in vitro migration assay through cultured human endothelial cell monolayers (ecm) we established. using this assay system, the comparative effects of fenlanyl, sufentanil, propofol and the known pmn inhibitor thiopontal were tested. methods: human umbilical vein endothelial cells (huvec) were isolated and cultured on microporous membranes (cyclopererm) until an ecm was grown. pmn from male and female volunteers were separated by standard procedures. ecm and pmn were preincubated with clinically relevant concentratious of thiopental ( m), propofol ( p_g/ml), the solvent of propoful (intralipid), fentanyl ( ng/ml) and sufentanil (sng/ml). after preincubatiun (ecm minutes, pmn minutes) with the reslx~tive drug, leukocyte migration towards the chemoatfractant fmlp ( o - m) was measured in a two chamber well system for hours. the migration rate of untreated (untr.) and treated (treat.) pmn through untreated and treated ecm were determined. as a control untreated pmn and untreated ecm were used. results are given as means from independent duplicate determinations and expressed as a percentage of control (table) . statistical analysis was done with student's t-test. results: clinical concentrations of fentanyl, sufentanil and prupofol showed similar inhibitor~ effects as the known pivin inhibitor thit e ). % conclusions: for the first time we could show that analgesics and anesthetics exert their inhibitory effects not only on pmn, but mainly on the interaction of pmn with endothelial cells. moreover, we could shmv a significant suppressive effect of the opinids fentanyl and sufentanil on both ec and pmn. the known inhibitory effect of thiopental obtained in ec-free test systems were also confirmed in our physiologically more relevant assay system. objectives: to investigate when and how sedation is used in a consecutive cohort of patients admitted in a large sample of italian intensive care units (icus), gathered in a network named giviti, representative of the italian icus system. methods; the study called for a recruitment period of one month, from january to february , , data collection included age and other demographic variables, acute diagnostic broad profiles, severity of illness scores, treatments, lenght of stay and vital status at icu discharge. as concerned sedation, each patient was observed until discharge or for a maximum period of seven days. information on all the drugs used for analgesia/sedation, the route and modalities of administration, the timing, dosages and purpose of the administration have been recorded. results: the study involved the cooperation of icus, of which enrolled at least one case. the total sample included patients. overall, . % of patients analyzed (t / ) received at least one prescription of sedative during their stay. globally, at least one sedative drug was prescribed to these patients in days in icu. although over drugs were reported to be used, pharmacological principles accounted alone for % of all prescriptions. opioids were actually used in % of prescriptions; propofol in % and benzodiazepine in . %. as regards the way of administration, intravenous administration was applied in % of cases and, followed by intramuscular in . %. moreover, non-steroidal anti-inflammatory drugs (nsald) were used in % of patients and neuromuscular blockade agents (nmba) in %. detailed analysis on certain subgroups (surgical, trauma, ventilated patients etc.) have been also carried out in order to describe the practice of sedation in these peculiar subgroups. findings will be widely discussed during the presentation. conclusions: these results should be interpreted keeping in mind how peculiar is the intensive care setting compared to many other less complex settings of hospital care. in conclusion we thought it was important to present the data currently available in the most neutral form, to start moving in a direction which will enable us -by means of more specific and detailed studies, and with the cooperation and involvement of all those participating in the project -to shed light on one of the many aspects of medical practice in the field of intensive care which deserve closer attention. introduction: the aged run perilously high risks in cardiac surgery: among others, of haemodynamic fluctuations, respiratory depresskm and organ failure. response to anaesthetics is a crucial determinant for post<)perative complications, none the less being reintubation due to mechanical ventilation difficulties which increase morbidity, mortality and intensive cdre unit (icu) stay. objective: we wanted to assess our a,aesthesia window (selection, and a view of the induction -extubation period) for predicting safe and swift awaking, thus: icu dismissal for the aged. methods: in , selected patients (pts) (> y, f) followed a regular elective cardiac surgery protocol (propofol given at precisely designated time intervals). upon cu arrival, they were subjected to an admission protocol. our predictive criteria for early extubation at h included: a) alertness and ready response to commands; b) adequate gag reflex and sufficient protection for respirak)ry tract; c) pao > mmhg with flu < . ; d) stable ph> . with spontaneous respiration; d) stable haemodynamics without dysrhythmias; e) adequate perfusion and diuresis (> .(i ml/kg/h); f) mediastinal bfeeding< ml/h for at least h; g) normothermia (core temp> ~ and no shivering). subsequent reintubation was for: ) rr> /min; ) spontancx)us ventilation for rain with paco > mmhg; ) pao < mmhg with fio > . ; ) ph> . ; ) heart rate>] bm; and/or ) non mental alertness; and ) other medical disorders, after which adequate weaning therapy was necessary. then, successful weaning after h was considered: ) spontaneous breathing without any forrn of mechanical assistance; ) stability in haemodynamics; and ) elimination of fever threat. results: pts ( %) were extubated at h without complication; other pts ( %) at h but had to be reintubated because they were hypoxic and began weaning therapy; finally, they were all re-extubated by h. only pts ( %) proved problematic. conclusion: a,aesthesia wimhlw options (selectkm, extubation, reintubation and weaning) predicted quick (times propofol administration) and safe (rigid criteria) extubation ( %= h and %= h), exempting pts with developed post-operative complications ( %=extubation< h) unrelated to al~aesthesia window or icu protocol. dismissal and recovery then became an abbreviated question of time. fifisetll p, domeneg~i ~, sforzini i., veronesi i~, maconi a.g. *, breg~ massone p.p h [] ic+pca request conclusions:using e~aprenorphine, a synthetic,long-acting, ago-antagemist opinid drug as analgesic, in the major surgery we obtained the best clinic results with association of conttheus infusion of haft dose drug with bohts of pca in the first - hours and just pca in the secmad day after surgery when the patient is less sleepy. in this way we dent have a great sav~g of suppled drug but the major well-belng of patient without ~erious side-effects and quick mobilization; the dosage used don't compromise a good awake of patient: all patients are sleepy but ready for answer, no allueinatian, bradipnea but not less than b/m without ipoxia. also the patient proffered this kind of truit meut than the traditional at demand. the ward staff feel it useful] and rehabl~ the negative feed-back technology of the electronic infuser system makes possible to use it safe in the ward with high drug's concentration too. the infusion rate of low dose of drug assure a continuative analgesic covering ~n the first postoperative periad; the pca mode involves the patient him-self in the managemenl of therapy and enables him to choose the best way to confront the dll~icuity of postoperative period without call medical stall using pca-device we have had no probicm~ no accident. analgesia during extracorporeal shook wave lithot ripsy a .levit, b.grinbezg regional hospital, ekaterinbu~g, russia b~ectives: our task was to compare ~he analgetic effect of norphin and tramel. methods: study was made of two groups of uro-li~patients aged - . group a ( patients) received baprenorphine hydrochloride (norphin) at dosages of #. • mg/kg. group b ( patients) received tramadel hydrochloride (t~aasl) st dosages of . z . mg/kg. before the procedure diazepam was administrated i.v. ( . ! . mg/kg). blood saturation (spoz), hemodynamics incides (bp, hr,sv,co,sap,svr) were examined and the patients' subjective assessments of snsesthesis quality were analyzed. the hospital ethics committee approved the investigation. results: when using norphin hr increased by . % on the onset of the procedure while sap and sv decreased by .%% and . %, respectively (p< . ). however, there were no reliable co chsnges. spoz ~educed by @. % (p< . ) and remained lower than the initial one after the procedure was oyez. when administrating tramsl min. after ste~ting the procedure sap and svr increased by ~ . % and . % respectively. sv and co decreased insignificantly. nine patients in group b saffeting some dlscomfo~t needed additional tm~msl in~ection. in the course of the whole p~oced~e spo, was constant and was highez than that in ~he case of nozphin (p<o. ). conclusion: respiratory suppression by no~phin can limit its use in case of lithotzip@y while the advantages of tremsl a~e: lack of dyspnoe and good contact with the patti-b_ the role of uncommon agents of antinociception, placed epidurally, in the control of pain transmission in the spinal cord is well documented. somatostatin as an inhibitory agent is found in the dorsal horn of the gray matter of spinal cord. the effects of somatostatin is similar to the opiates if injected into the epidural space (ref. i, , , ) . the aim of the present study is to present the clinical effect of above mentioned palliative therapy in the case of cancer and non cancer pain. informed consent had been obtained from every patient before treatment was initiated under the legalisation of the local ethics committee. using the permanent epidural catheter and continuous infusion pump, we administer somatostatin in the dose of up to microgram per hour for one up to three days. the patients paln feeling was evaluated using a vas (visual analogue scale). we found that the opiate resistant pain level decreased by %. the rest pain could be controlled by pereral opiate analgetics or by combination of somatostatin and an opiate plus a local anaesthetic agent epidurally. theoretically, it seems that epidural administration of somatostatin and local anaesthetic agent is useful in the control of acute pancreatic pain and systemic effect of somatostatin is beneficial in the treatment of acute pancreatitis. few studies have been performed in the critically ill (ci) and because of the complex pharmacodynamic and pharmacokinetic changes that occur, it is difficult to predict accurately drug responses and interactions in individual patients. midazolam (m) is a watersoluble benzodiazepine with a rapid onset and short duration of action in normal individuals; however its metabolism is decreased in the ci. critical care physicians every day experience suggest that among the ci there is a different pattern of response to m; here it is hypothized that such a response could be due to different metabolic behaviours of ci. m (i.v.infusion rate - rag/h) and antipyrine ( rag p.o.) were infused to ci with ( patients -group ) and without ( patientsgroup ) endotracheal intubation. blood samples were collected at fixed times during and after m infusion. total urine nitrogen and antipyrine elimination half-life demonstrated different metabolic characteristics in group chypermetabolizer") as compared to group chypometabolizer"). results follow. the results of our study are preliminary and more kinetic data in critically ill patients are needed to statistically confirm our approach of "hypometabolizer" and "hypermetabolizer". objectives: some clinieai and experimental studies suggest that propofol decreases myocardial contractility in coronary artery bypass grafting (cabg) patients. the intrinsic negative inotropic action of the drug may be independent from changes of preload and afterload, whereas the myocardial depression induced by propofol may depend on changes in ca ++ ious availability. aim of this study was to verify the hemodynamic effects of propofoi in a group of cabg patients with uneompromised contractile function and to minimize myocardial depression, during induction of anesthesia, choosing to associate calcium chloride (cac ) in an other group and to define its role in producing this effect. methods: fourty patients, randomly divided in two groups ( pts a and pts b), undergone elective cabg, were enrolled in this study. anesthesia was induced by senior anesthetist in both groups by means of fentanyl mg• kg < in ", pancuronium . mg x kg - and propofol mg • kg - in ". a blind investigator administered via another venous way at same speed ( ") saline in patients of group a and mg x kg - cac in patients of group b. hemodynamic data (heart rate hr, mean arterial pressure map, mean pulmonary artery pressure pap, pulmonary capillary wedge pressure pcwp, central venous pressure cvp, cardiac index ci, stroke volume index svi, systemic and pulmonary vascular resistances indexed svri and pvri) were obtained at baseline (to), ' after anesthesia induction (t ) and " after tracheal intubation (t ). results: hr decreased moderately in both groups (p = ns). map decreased as well, more markeatly in group a at ti and t . this trend was statistical significant (p < . ) in both groups. pap, pcwp and cvp unchanged following induction in any of two groups. ci decreased in group a (p < . ) dramatically dropping at t , while in group b it showed a negligible decrease at t (p = ns) and value similar to baseline at t . svri and pvri did not exhibit statistically significant changes. conclusions: the use of propofol as a starter of anesthesia in cabg patients allows to reduce total dose of fentanyi, dramatically reducing post-operative intubation time. propofol-fentanyl association prevented the hyperdynamic response to stress (i. e. laryngoscopy, intubation) but severely depressed ci and svi. cac simoultaneous administration effectively minimized the negative inotropic effect of propofol. moreover no unwanted side-effects due to cac were observed. diseoordinated labor activity (dla) is one of most serious in obstetric pathology.medication sleep effect to a woman in birth is of limitation of pathologic impulsation from the central nervous system, but it does not influence processes resulting in and supporting dla on the organ level. constant discoordinated uterine contractions during medication sleep (ms) imerease disturbances of uterine blood flow, organ hepoxemia connected with hyperergia of vascular wall and myometrium to catecholamines, that reduces efficiency of anesthesiologic protection. we applied hexoprenalin in complex with ms to primaparas. efficiency control was being accomlished through hysterograms, grade scale of analgetic effect where hemodynamics reaction to pain, significance of emotional reactions and their vegetative manifestations before and during ms were registered, and also through hydrocortisone level in plasma before and during ms. the examined women's average duration of ms was hours more than in control and made hours, though their average labor duration and in control was the same and made hours. % of the examined women experienced independant labor, as for the control group -only %. supplementary methods of anesthesia were required in % in control,that increased medicamentous depression of the fetus, but for the examined group -only in %. the examinees's newborns experienced favourable terminations, in control newborns were in state of light rate asphixia. analgetic effect was complete in control just in i %, and of examinees -in %. on the women's hysterogrnms before ms discoordinated contractions were registered on the background of increased tonus. during ms in control similar uterine contractions were constantly registered, but for the examinees they were not marked at all or had the proper character. the examinees' hydrocortisone level was reduced for % and in control only for %. the study has allowed to make a conclusion that hexoprenalin applications in complex with ms in case of dla make anesthesiologic care safer and more effective, promote simultaneous removal of patholgic processes in the central nervous system and uterus, which lead to dla development. objectives: the aim of report is to present a clinical study results for the evaluation of anesthesia for a patients in unconscious states by eeg examination: methods: the study was conducted on patients after a severe abdominal operations on stomach and intestine and on one patient after traumaticat exarticulation of upper extremity. all patients have different unconscious state levels from somnolence to coma ]. also thay hav~ had respiratory insufficiency and control mandatory ventilation have been used. we were used intrave,~ous injections of morphine hydrochloride from initial dose of mg. than we were increased the dose to mg i.v. by drops with an accompanying eeg monitoring and fourier spectral analysis. initially all patients have had -rhythm with slow and a-activity. an c~-activity index have been lower than %. results: after anesthesia the o-and a-activity have disappeared. the or-activity index have increased to %. also we have noted decreasing of the unconscious state levels to a possibility of a verbal contacts ( - points gcs). conclusion: the quality of anesthesia could be evaluated by eeg monitoring. a regaining consciousness afte~ the injections of high doses of morphine, probably, may be a result of an opiate deficiency (full or partial). anesthesia with the following drugs: clonidine, l-arginine, l-n-arginine-methyl-ester (l-name), and their combinations. tail-flick latency (tfl) was determined at time zero and min after each treatment. clonidine (i- ~g/mouse) prolonged tfl in a dose-dependent manner. at a clonidine concentration of gg/mouse, tfl increased . -fold compared to time zero. l-arginine increased tfl at a high concentration (i gg/mouse). l-name suppressed tfl . -fold compared to time zero at a concentration of gg/mouse. conclusion: we have found that no is involved in clonidine spinal analgesia. studies are currently being undertaken to assess the effect of combination treatment of the above drugs on clonidine supraspinal analgesia. objectives: hemodynamic changes ussualy accompany laryngoscopy and tracheal intubation.the aim of this prospective study vas tu present the effectivness sufentanil in preventing reflex circulatory respone of laryngoscopy and tracheal intubation and provides stable hemodynamio condition for induction of balanced anaesthesia. methods: adult asa i-ii patients sheduled for elective surgery.they were allocated randomly and divided into two groups:fifteen patients received single bolus of placebo prior to laryngoscopy.and tracheal intubation. anaesthesia induction was then performed with thiopental (smg/kg) and atracurium ( , mg/kg), followed by neurolept anaesthesia. mean arterial preassure (map) and heart rate (hr) were measured before, during and min after intubation. althought ecg was recorded at the same time intervals. results: the groups were compared with regard to the changes in arterial blood pressure,heart rate and electrocardiogram. mean arterial preassure and heart rate were incresed significantly during and after laryngoscopy and tracheal intubation in control group but remained unohange the baselin values in the group who received sufentanil. control group ecg suggested more changes than sufentanil group. conclusions: the results indicated that , mg/kg sufentanil for anaesthesia induction reducing the pressor response to laryngoscopy and tracheal intubation. obiective: the aim of this experimental study was to evaluate morphologically the influence of anesthesia in liver as well as in isolated hepatocytes (hc). methods: a total of female swine ( - kg) were used as liver donors and were randomly assigned to one of two groups regarding anesthesia protocol: group a (n= ): induction with intravenous sodium thiopental in a dose of mg/kg, group b (n= ): propophol in a dose of mg/kg for induction'and mg/kg/h for maintainance of general anesthesia. both groups were under the same preanesthetic regimen (diazepam, ketalar and atropin) and received standarised doses of fentanyl and pancuronium during anesthesia. in beth groups total hepatectomy was performed and the liver was perfused with - it of cold ( oc) university of wisconsin solution for a period of hours before hepatocyte isolation. liver tissue samples were fixed in formalin for the morphological study and stained with hematoxylin-eosin and pas. for hc isolation collagenase solution (type v, sigma, c- , . mg/ml) was infused via portal vein and the liver was incubated at oc for rain. cells were filtered and then washed in cold hanks' solution. isolated hc were fixed and prepared for staining or simply cryopreserved (- oc). results: histology was completed in / experiments ( in group a and in group b). mononuclear infiltration (halothane type injury) was found in all specimens ( / ). focal zonal necrosis and acidophilic bodies were found in specimens from group a ( / and / respectively) while portal inflammation with piece meal necrosis was found in one specimen from group b ( / ). smears of hepatocytes -beth formalin fixed and cryopreserved at - oc -were stained with hematoxylin-eosin and showed morphologically intact hepatocytes. conclusion: pre[imineq~' study of our material reveals mote frequent livet injury in the thiopental group, but this finding has yet to be established by increasing the number of observations. objectives: in this paper we present our experience and point out the importance of sedation of those patients who suffered severe head injuries/contusic cerebri with signs of decelebration/and who were put on ventilatory machine to obtain better mechanical ventilation in neurosurgical intensive care unit of emergency center in belgrade. methods: patients were treated from jan. till dec. results: in patients we successed to reduce agitation and decelebration. conclusions: the major demand of sedation in neurosurgical patients are that the patient should be calm, comfortable and painless. references we compared three analogous groups of patients in each, with orthopedic operations on lower extremities. in each group we applied different kind of analgesia: first group rece-ned local anesthetic ( ml . ~ bupivacaine) when analgesia was first demanded; second group received ml fentanyl via peridurai catheter when analgesia was first demanded, and third group received m] fentanyi intravenously on demand for analgesia. we used visual analog scale (vas) for estimation of pain intensity and success of applied therapy. statistical data analysis was performed using student's t-test. tha best vas had group in which local anesthetic was applied periduraly. second was the group with periduraly applied fentanyl, and third was the group with intravenously applied fentanyl the longest duration of the effect of analgesia ~/as in the second group. there were no adverse effects and complications, apart from mild sedation in the third group. for orthopedic operations on lower extremities, application of peridural catheter for anesthesia and successful postoperative analgesia with local anesthetic, or opioid analgesics is acceptable. background and obiective : tympanic temperature measurement (ttm) is a relatively new procedure which provides accurate estimates of core temperature in stable postoperative patients and in patients admitted to the emergency ward. however, experience with this technique in hemodynamically unstable adult icu patients is limited. design : prospective study with patients serving as their own control. se:ttijlg : adult medico-surgical icu in a tertiary care hospital. patients : consecutively enrolled patients with thermodilution catheters in place and without contraindieation for rectal temperature measurement and without hypothermia (core temperature < ~ interventions : tympanic temperature, measured by a commercialized tympanic thermometer (genius a first temp) was compared with pulmonary artery (pat) and rectal temperature (rt). within minutes, core temperature was assessed in each patient by the same trained individual using the three techniques in random order. measurements and main results : mean bias (ix~ and its variability (sdr of method comparison pairs were calculated using the methods comparison analysis as described by bland and alunan (lancet, ; i : - objective: to measure the prevalence of pressure sores on the intensive care unit and the effect of risk calculation on pressure score prevalence and pressure score severity. method:in during a period of months a series of prevalence studies was carried out on the sicu to investigate how many patient days with pressure sores were taken care for by the icu nursing staff, what kind of preventive interventions were done and what the patient risk was on developing a pressure score. results: on average there was a prevalence of pressure sores on the buttocks of . % on the short stay unit and . % on the long stay unit; preventive interventions increased when the pressure score was visible for the nurse and the majority of patients had a high risk or extra high risk for developing pressure sores according the pressure score risk calculator. as a result of these findings the nursing management decided in that nurses had to complete daily on every patient the pressure score risk assessment in order to be able to take adequate preventive interventions. when the results of the two studies were compared, the most important results were that: ) there is no indication of a reduction in patient days with pressure sores (short stay unit: %, long stay unit: %); ) there is a reduction in severity of pressure sores (grade iv decreased from . % to . %) because adequate preventive measurements are initiated by icu nurses in an earlier stage. possible explanations for the increase of patient days with pressure sores on the long stay unit are that the average pressure score risk score was increased from . to . and the mean frequency of nursing patients on alternate sides decreased from . to . times each day. conclusion: daily assessment of the individual patient risk on developing pressure sores does not decrease development but reduces the severity of the developed pressure sores. method: the conventional woundcare method of patients with an open abdomen is to change frequently the saline soaked ribbon gauze pads ( - times each day). this method is labour intensive for the itu nursing staff and rather uncomfortable for the patient. problems that often occur are: insufficient hygiene, frequent nursing interventions, a progressive risk for deterioration of the skin and underlying tissues, difficult to measure abdominal output and in case of bowel fistulas enteral feeding is hardly possible. in using the new method, stomahesive is applied on the skin surrounding the wound. a large size of surgical film dressing, opsite | is applied over the abdomen. two or more foley ~ catheters ch are inserted through an opening in the surgical filmdressing. the application of several lateral openings in the catheters is advised. the drains are held firmly in place between two layers of sterile opsite | "flexigrid ~'' x with the so called bookend method and are connected to a low vacuum suction system ( - kph) results: we studied the frequency of dressings changes on patients in the itu. in total they had days with an open abdomen. during this period there were dressing changes. this means one dressing change every days. further advantages of the new method are; more hygiene in patient care, no maceration and irritation of the skin, nursing on alternate sides is possible, output can be measured, in case of bowl fistula, enteral feeding is possible and an abdominal lavage is possible on the sicu by opening the opsite | conclusion: this new method of woundcare management is more patient friendly, prevents several nursing complications and decreases the work load for the nursing staff. objective: to investigate the interaction between the ventilator and the closed suction system related to possible induced negative pressure by this sytem. method: we studied in a testlung model what the effect was of the different ventilators (evita: dr~iger; servo c: siemens), ventilation modes ppv, simv) and ventilator settings (tv, trigger level, frequency, peep level) on the induced negative pressure by the closed suction system during suctioning. results: when using the evita the magnitude of the negative pressure increased when the ventilation frequency and tv decreased.. the largest negative pressure (- cm h ) was produced with a tv of ml and a frequency of /min in the ippv mode with trigger level on - cm h and a peep level of + cm h . the servo c showed a complete different pattern. the largest negative pressures were measured when no trigger level was set ( cm h ), the induced negative pressure was not depended on the tv or ventilation frequency but more depending on the ventilator mode. the simv mode produced the smallest negative pressures ( - cm h ) and the ippv mode the largest ( - cm h ). conclusion: the effectiveness of the closed suction system is very much depending on the kind of ventilator, ventilator mode and ventilator setting that is used. if the interaction with the used ventilator is not known by the icu nurse this piece of equipment can cause damage to the patient by inducing large negative pressure in the comprimised lung and there fore creating the possibility of alveolar collaps and atelectasis or oedema. introduction : ethical problems are daily and disturbing preoccupations for the icu staff. objectives : improving ethical management in icu by creation of an intelprofessional group of ethical reflexion ( iger ). methods : existing was evaluated by a preliminary formulated series of questions sended to the whole staff (q , n = , items) after two training sessions (laws, deontology, professional values, culture, norms of quality) followed by persons ( %) and directed by an external chairman, satisfaction and needs of the staff were evaluated by a second series of questions (q , n = , items). at the issue, iger was created. results : q : responders ( %). law's misinformation : to %, disagreement for limitation of intensive cares : %, hope to improve collective decisions and creation of iger : % q : responders ( %) : satisfied by the training sessions : %, non satisfied : %, want to continue the project : %, refuse : %. iger was constitute by physicians, nurses, secretary, dietetic (n = ). the first working theme was {< therapeutic's withholding and withdrawing decisions in icu ~>. four subgroups of iger's members (having access to an ethical library) worked independautly and submitted their reflexions in a tdmestrial plenary session of iger in the presence of an external chairman, allowing a synthesis. at the issue a report was writted to be used as a reference for bedside and individual decisions. conclusions : constitution of iger seems to improve ethical management in icu. the first result of iger is that it is now possible to began collectively a reflexion concerning therapeutic's withholding and withdrawing in icu. the work is going on and further subjects will be studied. objectives: ) to compare the value of heat-moisture exchangers with bacterial filters (hmef) and without bacterial filters (hme) in the prevention of colonization of ventilator tubing and ventilator-associated respiratory infections. ) to asses the temperature and relative humidity of inspired all using both types of heat-moisture exchangers. methods: mechanically ventilated patients were randomized, to either hmef or hme. endotraeheal aspirates, pharyngeal swabs and samples from tubing were collected for bacterial cultures on the st, nd day mechanically ventilation and weekly thereafter. temperature and relative humidity were measured in patients ( hmef and hme) h and h after placing the hme or the hmef. results: both groups were comparable as regards age, mechanical ventilation period, severity score (saps ii), leukocyte count, and number of patients with prior antibiotic treatment. from the hmef group, ( %) ventilator tubing yielded microorganisms in, at least, one sample as compared to ( %) of the hme group; p=ns. the incidence of respiratory infection was similar in both groups ( % vs %, p:ns, for hmef and hme respectively). among the bacterial species isolated from ventilator tubing in the hmef group, ( %) were not isolated from pharyngeal swabs. a similar ratio was shown in the hme group ( / , %). both heat-moisture exchangers were efficacious in keeping a good relative humidity of inspired air ( % • vs % • .%; p=ns, for hmef and hme respectively). relative humidity was significantly higher after h of mechanical ventilation in the hme group as compared to hme group ( . % • vs . % • %; p= . ). conclusions: both types of heat-moisture exchangers have the same effect on the prevention of colonization of ventilator tubing. similar relative humidities are achieved when using either type of heat-moisture exchanger. results: tumor and nontumer enhrgements of the thyroidea were present in ~ of the operated, surgicel adrenal disease in io!, hyperplssle or persthyroid gland tumor in ~ end endocrine pancreatic tumors in %. in the intensive oere unit, these patients wore screened by noninwsive monitoring in ~ of cases: and invasive monitoring was applied in % of ceses.the basic noninvesive methods included: electrocardiogram with standard end precerdial leeds, percutaneous eutomotlc measurement of systolic, diastolic and mean arterial pressure, measurement of hourly diuresis and body temperature, frequency, hearing capacity and rhythm of one s own breathbng bs well as pulse oxymetry. a special plece in monitoring and control of vital parameters in postoperative period belonged to the nurse, thoroughly trained for enelysis end interpretation of the observed parameters which would be discussed in the paper. it has been believed that the leader sits at the pinnacle of power. over the years, this has proven to produce frustruation and anguish instead of the expected results. leaders have not been able to produce the changes they know are essential to their organization's survival with this command-and-control paradigm. through literature reviews and evaluating leadership styles, one can clearly see the most effective form is that of empowering people to a new level of performance -not ordering it. changing the leadership paradigm to a manner/style that has been shown to be effective and one of people empowerment shifts the focus to personal responsibility for performance. removing obstae}es~ stimulating self-directed actions, and determining focus and direction are just a few elements used to create the successful environment of empowerment. with increasing pressure in the health care arena, it becomes critical that a leader's job is to get the people to be responsible for their own performance. developing ownership, creating an environment where people want to be responsible, being a mentor or coach, and learning faster while encouraging others to do so demonstrates the commitment to effective leadership. this presentation will illustrate the critical components that are achieved when every person in the institution is empowered to perform at a level that is directed toward positive, effective results. herrera m. (md) . icu. hospital regional. malaga. spain. the systems of veno-vanous continuous haemofiltration (wchf) have a high cost and a limited life span. in an attempt of lengthening their mean life it has been proposed to accomplish programmed washes of the ~-stems. this practice supposes an increase in nursing workload. in order to evaluate the real efficiency of this practice we have accomplished this study. material: prospective randomized study of all the filters of vvchf used during the last year in our icu. we have determined two groups of filters, in the first (group a) we accomplished washed in a programmed way, and in the other (group b) only when the alarms of the system suggested a clotting of the filter. for the statistical analysis we used the kaplan-meier test for survival analysis. results: we have studied a total of patient submitted to wchf during the last year. we used a total of filters with this results. objectives. sounding out the nurses about the need to inform patients" relatives and the rigth kind of such information, like a preliminary approach to an information cuality assessment, methods: we inquired all the nurses of the intensive care unit of an regional hospital by an semiestructurated questionary which included personal data: age, sex, contractual relation, professional experience.., and opinion data: do you think to inform relatives is a nurse task?. which of the next informafions do you think is more important?, please, write others topics about information you think are relevant. we process the data on epi-info estatistical program and use x test to compare the results. results" from nurses of staff refused to flu the quetionary, and were not available. of the remaining, %were v~men and % men. the mean age were . % had an svable contract and ( eventual, the mean professional experience were of years and % worked in the unit since more than years. the % answered that offer information to relatives is part of the nurse activities. we did not find differences with nurses who answered negatively comparing by sex, age, contractual relation or proffesional experience. the three information topics found out like more important were: ) to inform about patient mood. ) to inform about happenings from the last visit. ) to inform about dressing instrument required by the patient, nurses who answered negatively think that to inform is a doctors task or that nurses are not competent. conclusion~ intensive care unit teams (nurses, doctors and auxiliar personnel) should get accord on who and how to inform relatives, we consider the nurses' role on information as unquestionable. objective: investigate the respiratory and cardiovascular response after discontinuing oxygen therapy durir~ intr~/]o~pital transport. desiqn: fifty-one patients ( male and female, aged + , and , , years respectively, ~+sym) being on therapy were studied prospectively in two consecutive intrahospital transports. oxygen therapy was continued in the first transport while the second one was performed as usually, i,e, without . during transport each patient was monitored by pulse oxymeter and holter whereas arterlal blood gases were tested just before a~xl aft~-trar~portation. results: compared to daseline, pa and sa were signif~canthy decreased in the case of oxygen discontinuation (p< , i). paco was significantly inur~ds~i only in the subgroup of patients with obstructive lun[ disease (p< , ) . heart rate increased in all phases of the transport when administratlon was discontinued. blood pressure remained stable in either case. the percentage of supraventricu!ar extrasysto!es, ectopic v~r[hicui~r contractions and st-s ~ment depression was progressively increasing and became very high at the end of transport in the case of therapy discontinuation. other arrhythmias did not change significantly. conclusion: discontinuation of oxygen therapy during intrahospital transport causes severe drop of pao and sa , increases the heart rate and contributes to the appearance of arrhythmias which were not present before. methods:for evaluation of the functional state of brain the complex of methods was used,whieh included electro encephalngraphy ( brain mapping ), rheoencephalography, tetrapolar transtorax rheography. for the estimation of humoral status the level of histamine and serotonine, products of free-radical oxidation,enzimatic markers of ishemic damage of brain and of endogenous intoxication was investigated. results: patients with encephalopathies after resuscitation were observed.asystolia was as a result of:shock, trauma, asphyxia,poisonings,appiication of drugs, eclamp sia,injury of the heart,diseases of fhe cardiac vessels. all patients with postasystolic syndrome entranced in comafose condition.in the group (reconvalescents) the depth of coma by glasgo~ pittsburg"s scale was , +- , . the duration of coma was from rain. to hour,average , +- ,sh.ln the group (the deads) the depth of come was , +- , .the artificial lung ventilation was used in all patients:in the group , +- , days,in the ~ , +- , days.apallish syndrome developed in cases,in patients diagnozed <<brain death~. the th degree encephalopathy (coma) was found to be associated with disorganized eeg-pattern with predomi nant alpha-( group) and slow ( group) activity. the relative power of delta-and theta-ranges was about - per cent.the patients with apallich syndrome demonstrated greatly elevated theta-range power, that of beta -range was essentially decreased. the degree of disturbances of circulation in the both groups was different.cardiac index (ci) and stroke index (si) in the group decreased on , % and , z, in the -on , % and (; , %.general peripheral resistance (gpr) increased in the group on , %,in the -on (; , %.in the group brain blood flow was increased, in the -decreased on , n.in the group there was the normotonic type of reg-curve,in the -atonic or hypotonic type (without reaction on pharmacologic influ ence).disturbances of permeability of cellular membranes (enhanced free-radical oxidation with an increase in di enic conjugates on and n) and vascular ones (an increase of serotonin concentration on and %, hi stamine content on and %) resulted in hyperfermentia (an increase of concentration of creaune phosphoki nase in the group on %,in the - %.the level of middle molecules (mm) increased in the group on , %, in the -on , %,of polyamines (p) (spermidin,spermin,putrescin).coefficient of correlation between mm and p was , . immediate correction of neurocyte metabolism is impossible due to marked brain oedemaswelling,severe dis ordes of cerebral circulation, disturbances of membrane permeability.thus,the following treatment algorythm might be advisable: . protective inhibition of brain and reduction of its energy requirements. . recovery of cell and vascular membrane fuoctions. .recovery of blood circulatiom .oxygenation of nervous tissue and drainage of brain disintegration products.in patientshbo carried out. .active methods of detoxication (homo-and enterosorption). .correction of cerebral metabolism. . dehydrative therapy must be very cautious. conclusion: the activation of energetic metabolism in neurocytes must be carred out only under neurophysiolngical control and after definite preparation. ( ). the clinical estimation of acute cerebral failure carried out by olasgo-pittsburg"s classification of coma. we studied such groups of patients: poisoning co ( ), asystolia ( ), eclampsia ( ), acute renal failure ( ), control group ( ). methods: electroeneephalngraphy (brain mapping) was fulfilled by means of encephalograph <,medicor ~ in monopolar channels. the estimation of eeg patterns by zhirmunskaya method ( ) was carried out. there were analyzed and changes of eegb by results of rapid transformation furie after rhythmical photostimulation(phts) , , , hz. we studied also the figures of absolute and relative power in such diapason : delta ( - hz), theta ( - hz), alpha ( - hz), betal( - hz), beta ( hz and more). results: all eeo changes were divided into following types: -organized ( , groups); iii -asynehronic ( group); iv -disorganized with prevalence of alpha-waves ( , , groups); v -disorganized with prevalence of delta-and theta activity ( , , ; , groups) akkording to our model of hormonal-metabolic disbalance in pregnancy , one of the main reason of destosis is hypoergos , reesults in endotoxemia and neuro-endokrine disregulation. so, stady of central nervous system function give us the information about adaptation processes. the aim of our work is to study the degree of neurologikal deficit in destosis when olifen and lipin were used. there were studied pregnants with different severe forms of gestosis. the degree of neurologikal deficit was valued by datas of neurologikal status . eeg with ,~brain mappinng~, , electrikal resistance of the skin. the komplex inteensive therapy with applikation of olifen and lipin allowed to improve the patient,s condition , decrease the degree of neurolodikal deficit and mortality . kostenko v.phd,kabanko t.,phd,galalu s.md,beliavtseva n. ,shramenko k. phd intensive care department,donetsk medical university, donetsk, ukraine plasmapheresis (pph),as other extracorporal methods of detoxicatin,has a great importance in contemporary medicine. the role of pph is special,because of its simplicity, effectiveness, atraumatic.we incalcated pph in obstetrical clinic. there are rough changes in agregate condition of blood in more of pregnants.these changes lead to disturbances of central,organ and peripheral hemodynamic.the therapeutic effect of pph is due to influence on agregate condition of blood. we considered that application of pph is very effective in pregnants with:bronchial asthma, severe forms of diabet, psorias, gestosis, allergic diseases. we used the membrane pph in pregnants: apparatus-,,hemos-pf>,, plasmofllter pmf- ,with effective area- cm,the volume of extracorporal contour- ml.such pph has no the ~ agressive effect,,, as in cases of application another extracorporal methods. this method was incalcated in our practice recently, so results will be reported in further publications. ( ). post-operative cerebral neoplasm ( ), post-operative subdural hematoma ( ). icp was monitored via a catheter inserted in the lateral ventricle and values were continuously digitally recorded by means of a bedside computer data acquisition system (maclab). the fiberoptic tracheobroucosenpe, which guided the procedure, was passed between the nasotracheal tube and the trachea in order to avoid hypoventilalion. the patients had stable baseline hemodynaimcs. propofol infusion and fentanyl boli were administered to mantain stable mean arterial pressure values. peak (mean(sd)) icp duping the minutes pre-ciaglia procedure (baseline values) were compared with values during ciaglia procedure, and the minutes p st-ciaglia procedure. data were compared with repeated measures anova. results: ciaglia procedure duration was (mean(sd)) ( ) objectives: transient global amnesia (tga) is a syndrome caracterized by impairment of short-term memory, inability to form new memories, retrograde amnesia and repetitive queries, without other neurological signs and symptoms. the pathophysiology of tga is unknown; thromboembolic, epileptic, migrainous and metabolic mechanisms have been suggested. to address some of these issues, we undertook a study of cases of tga in whom we examined clinical, laboratory data, electroencephalogram, ct of the head, ultrasonography ecodoppler. methods: patients were included in this study: men and women. the mean age was years. all cases underwent a standard clinical examination, electrocardiogram, routinary humoral tests and x-ray, electroencephalogram (eeg), ct scan of the head, ultrasonography ecodoppler. results': the mean duration of amnesia was h. m. +/- h. m. hypertension was found in patients ( %), ischemic heart disease in patients ( %), hypercholesterolemia in patients ( %), hypertrigliceridemia in patients ( %), smoking in patients ( %), atrial fibrillation in patient ( %), history of epilepsy in patient ( %), migraine history was not recorded. ct scans of the head showed multiple small deep infarcts in patients ( %), a single hypodense lesion in patients ( %). in patients electroencephalogram was normal ( %), in patients there were widespread nonspecific electrical changes ( %), in patients there were focal nonspecific eeg abnormalities ( %). conclusion: in our study tga was more common in women ( %). we showed a prevalence of hypertension, hypercholesterolemia and cerebral infarcts compared to normal controls. we have demonstrated a higher incidence of nonspecific electrical changes in tga of lower length, while ischemic lesions in ct of the head were more frequent in tga of greater length. these data seem to be in agreement with the hypothesis that tga is a heterogeneous clinical syndrome, consisting of pure, epileptic, and ischemic types. however we did not find any correlation useful in discriminating pure from associated tga forms. from our study it is tempting to speculate that pure tga is a rare event, underlying still unknown mechanisms wich differ from ischemic, epileptic, migraineous causes. objectives: aneurysmal subarachnoid haemorrhage (sah) is special condition increasing intracranial pressure (icp) in various ways. at the other hand cerebral vasospasm and related delayed ischaemic deficit (did) could answer for the poor outcome. triple h therapy seems today a basic option to prevent did, but it may increase the icp worsening the altered intracranial pressure condition and thereby the cerebral perfusion pressure (cpp). is there any way to individualise the triple h therapy when it is necessary? methods: between sept. march thirty-seven patients with intracranial aneurysms were operated on within hours following sah. five patients were in hunt-hess iv at admission. all patients received triple h therapy in a preventive fashion following surgery and were monitored by daily transcranial doppler ultrasonography (tcd). icp and cpp was measured in twenty-four cases. twenty-two of them received lumbar liquor drainage (lld) and nineteen were administered induced hypertension. the other group was treated by basic triple h therapy. results: in group with monitored icp the outcome was twenty-one excellent, one poor, two died (one of them died from extracranial decease). in the other group four had excellent, six moderate, two poor outcome, and one died. conclusion: according to our recent observation the patients can be divided into two groups of therapy. in group i, the patients with elevated tcd values and either low or high icp reacted to lld. we are concerned that haemodilution and slight hypervolaemia should dominate in the triple h therapy. in group ii patients having high icp with tcd and/or symptomatic vasospasm should be managed by the induced hypertensionhypervolaemia dominated therapy focusing on cpp (icp) and focal neurological signs. air emboli were detected in lo% (n= ) of natients undergoing coronary srtery bypass craftin~ (cabg). central nervous system ~ysfunction occured in ~$ of the nstients with air embnli and in none of those ~ithhout air embo!i. hvtothermia is the classic form of oro-tect~on used dur~nc ~"~" " ~ ~ ca~.,~modu] :r, on~_,_. bj/oass. the surf~eon sho,;,ed thorough!~: evecnnte air from the heart, but the onesthesio!o[[ist can signifieamt!y influence the outcome by emt!oyin ~ methods to detect and treat air emboli. the changes in head rate are primarily due to alterations of autonomic tone. the heart rate variability (hrv), that express the degree of heart rate fluctuation around the mean heart rate, reflects somehow the condition of central nervous system. hrv may be measured by a number of techniques. short-term time-domain variables of hrv are reflect generally the vegal activity. in this study the changes in hrv variables of patients with brain damage, and in addition the changes in hrv measurements in comparison with the clinical evolution were evaluated. eight patient with brain damage and six normal individuals as control group were studied. a elecrocardiographer with availability of computation the sequence of beat-to-beat intervals for one minute was used. the following variables of hrv were measured: ) standard deviation (sd) of beat to beat r-r interval differences that reflects the respiratory control, )the maximum/minimum (max/rain) interval that reflect variability related to baroreflex and thermoregulation and ) the coel~cient of variation (cv), the results are shown in the in the patients with brain death and in vegetate state there were virtually no hrv. increased hrv pattern was found with clinical improvement, the changes of hrv precede of the changes of gcs, we conclude that time-domain hrv could reflects the degree of brain damage, it is good prognostic index of the brain damage and may change earlier than the gcs. objectives: cerebral co vasoreactivity is an important determinant of cerebral blood flow (cbf) and has been shown to be of prognostic value in head trauma (acta anaesthesiol. scand. ; : - ) . we wondered whether co vasoreactivity could be selectively altered in one hemisphere in comatose patients. methods: patients ( m/ f, age - yrs, glasgow - ) in coma due an acute brain lesion (trauma, hemorrhage, or infection) were studied. cbf was measured bilaterally using jugular thermodilution at paco , , , and mmhg by increasing pico with mechanical ventilation kept constant. normal co vasoreactivity was defined as an increase in cbf of at least i ml/min. g per mmhg paco . results: patients had normal co vasoreactivity bilaterally, patients had altered co vasoreactivity at both sides, and patients had a normal response at one side (left or right) with an altered response on the other side (dght or left). for the patients left cbf was in mean ! ml/min. g lower than right cbf (figure methods: following institutional approval piglets (body weight :tl . ) were anaesthetized by % fluothane. a catheter was placed in the right femoral artery for blood pressure monitoring and a fiberoptic catheter (oxymetncs- abbott) was advanced via the right internal jugular vein to the jugular bulb for sjo determinations. another catheter with a balloon on the tip was advanced in the right atrium via the right femoral vein. a mean arterial pressure (bp) at mmhg was achieved by appropriate balloon inflation for rain and two groups were cleated: i) the hypoxemic group by respirator disconnection (*) and it) the hyperoxemic group by fio =l on respirator (o). samples were obtained at time ( ), ' min at hypoperfusion ( ) arid at reperfijsion at ' ( ), ' ( ) and ' ( ). pao , pjo and oxidative brain stress evaluation was performed from jugular bulb blood. the latter included: i) no synthase (nos) and xanthine oxidase (xo) activities by a method based on the oxidation of scopoletin detected fluorometrically, it) no levels estimated as onoo-by luminol enhanced chemiluminescence in the presence of ~tm hydrogen peroxide (h ). resul'~s: the mean pao was mmt-ig for group i and methods: we retrospectively reviewed all upper gi-endoscopies, performed in the period january -july in patients ( men and women) admitted at the icu's of our hospital. results: it concerned surgical, medical, eardiological and neurological patients with a mean age of . yrs (range: - ). in %, the endoscopy was performed at the icu and in % at the endoscopy department. in % of the cases, the endoscopy was primarily diagnostic, of which % was performed for localization of upper gi blood loss. in % the endoscopy was primarily thempentic, of which % was performed for placement of a duodenal feeding canula. location of the upper gi bleeding was: variees ( %), duodenal ulcer ( %), oesophagitis ( %), gastric ulcer ( %), others ( %) and none ( %). as coincidental findings were noted: cesophagitis ( %), gastritis ( %), gastric deer ( %), duodenal ulcer ( %), duodenitis ( %), oesophageal ulcer ( %) and others ( %). conclusions: there were marked differences in indications and findings of endoscopy at the different icu's. these differences reflect an admission bias and differences in populations and treatment preferences. compared with cardiological and neurological icu's, substantially more endoscopies were performed at surgical and medical icu's. in a considerable number of cases, no source of upper gi blood loss could be found endoscopicaiiy. when upper gi blood loss was the icu admission diagnosis, the main cause was needing varices, which could be controlled endoscopically in the vast majority of cases. when upper gi blood loss was ndt the icu admission diagnosis, peigie ulcer and oesophagifis were the main causes of bleeding. because of the considerable number of coincidental almom~adities found at endoscopy, there is still room for debate whether antacid medication and/or motility stimulating agents should be given prophylactically at icu's. many studies have shown that blood lactate levels in survivors and nonsmvivors of traumatic and septic shock are significantly different. the degree of multiple organ failure is related to the duration of lactic acidosis ( ). the aim of this study was to evaluate blood lactate level as a prognostic marker of high risk postoperative patients who may benefit from invasive hemodynamic monitoring and aggressive fluids administration and early inotropic support based on oxygen transport parameters. methods: patients undergoing elective long term vascular and abdominal surgery (asa i-bi) were studied. blood lactate levels were measured after icu admission. in the case of blood lactate level above mmoltl, measurement was repeated every hours for hours or until normaiisation (blood lactate level less than mmol/ ). type of surgery, length of surgery, amount of fluids delivered intraoperatively and postoperatively, hemoglobin levels, hemodynamic variables, diuresis, postoperative complications, length of icu stay and clinical outcome were recorded. because no attempts were made to randomisr therapy or change our standard therapy protocol institutional approval was not required. rebuts: the frequency of postoperative complications was , % and mortafity was , % in a group of patients with blood lactate level less than , mmol/l (n = ). frequency of complications ( , %) was significantly increased in a group of patients with blood lactate levels , - mmol/l (n = ), mortality was , %. mortality ( %) and frequency of complications ( %) were significantly increased in a group of patients with blood lactate levels above mmol/l (n = ). conclusion: blood lactate levels can serve as early marker of high risk postoperalivr patients and may predict increased risk of postoperative complications mad ~e death. objective.~: investigated practicability and clinical value of the routine measurement of hepatic venous oxygen saturation (shvo ) after major liver surgery, as shvo is considered an indirect parameter for splanchthc and hepatic blood flow. methods: consecutive patients were included in this study after liver resections for primary or secondary liver tumors. patients suffered from liver cirrhosis (childs a). immediately after post-operative admission on the icu a pa-catheter ,was inserted under fluoroscopy via the right jugular internal vein into the hepatic vein contralateral to the resection area. hepatic venous and arterial blood samples were drawn every two hours. shvo was correlated to the clinical course, macro hemedynamics, abgs aug other established lab parameters. results: in out of attempts the catheter could be placed correctly. in four cases after right hemihepatectomy the left hepatic vein could not be intubated due to a dorso-lateral tilting of the left liver. this is also reflected in a significantly longer time of fluoroscopy for catheterization of the left hepatic vein ( . _+ % rain vs. . + . rain; p < . ). the procedure requires a total of between and minutes. relevant clinical complications were not observed except for short term supraventricular arrhythmias during passage of the catheter through the right atrium. hemodynamics and pulmonary function could be considered normal in all individuals at time of measurement. shvo showed a span from . % to . % with a mean of . % -+ . %. the following statistically significant findings could be obtained: (a) patients with liver cirrhosis showed a significantly lower shvq than patients without ( . % • . % vs. . % • . %; p < . ). (b) a negative correlation between shvo immediately after operation and the duration of intraoperative hepatic vascular occlusion could be observed (r = - . ; p < . ). this correlation could also be seen for the first post-operative hours (r = - . ; p < . ). (c) a negative correlation between shvo and the difference between arterial and hepatic venous lactate levels was found (r = - . ; p < . ). conclusions: the routine measurement of shvo appears to be a promising extension of post-operative monitoring after major liver surgery. it is a safe method easily feasible on any major surgical icu though relatively time consuming. a further validation of this method is necessary in larger studies. therapeutic recommendations on the basis of shvo findings cannot be given yet. methods: in cases after major liver resection, in which abnormally low readings of shvo suggested an impaired hepatic blood flow, pgi was applied at a dose rate of ng/kg/min. as shvo can be considered an indirect parameter for hepatic blood flow, the effect of pgi infusion on shvo was measured. moreover, the changes of macro hemodynamics and pulmonary function were monitored. results: before the application of pgi z mean shvo for all patients .was . % ( - - - ). in three cases without major structural alteration of the remaining liver tissue the continuous intravenous administration of pgi lead to a sustained increase of shvo z to an average of . % ( . - , ). the postoperative course in these three cases was uneventful. in two cases with compensated liver cirrhosis after hepatitis c no change in shvoz under pgi infusion could be observed. both patients died and days respectively after operation in protracted liver failure. side effects of pgi included a slight decrease of systemic and pulmonary vascular resistances. consequently map decreased by up to % as did intrapuimonary right-left shunt increase. in none of the observed patients did these side effects posed a limitation of continuous application of pgi z. conclusions: in patients without structural alteration of the liver the systemic application of prostacyclin at a dose rate of ng/kg/min could significantly increase an abnormally low hepatic venous oxygen saturation after major liver resections, tn two cases of severe liver cirrhosis a similar increase could not be observed. after first clinical investigations and with the results of recent studies in animal further controlled clinical studies of prostacyclin in the postoperative management after liver surgery appear justified. any delay in gastric emptying can promote micro-aspiration and give rise to ventilator associated nosoarnnial pneumonia. h -receptor antagonists have been suspected of promoting pneumonia by changing the gastric ph. in a few tri',ds on humans ranitidine was noted to delay gastric emptying. the aim of this prospective, randomised, blinded study was to evaluate in a ventilated icu population if there was a difference between cimetidine (c) and ranitidine (r) on the gastric filling index (gfi conclusion: in this population there was no difference in gfi between c and r; however the age and creatinine were significantly different and could have favoured the c group. also the very long t/ could have hidden smaller differences between c and r as has been described in volunteers. between april , and april , , patients with severe acute pancreatitis were admitted to participating hospitals. patients were entered into the study if severe acute pancreatitis was indicated, on admission, by multiple laboratory criteria (imrie score >_ ) and/or computed tomography criteria (balthazar grade d or e). patients were randomly assigned to receive standard treatment (control group) or standard treatment plus selective decontamination (norfloxacin, colistin, amphotericin; selective decontamination group). all patients received furl supportive treatment, and surveillance cultures were taken in both groups. results: fifty patients were assigned to the selective decontamination group and were assigned to the control group. there were deaths in the control group ( %), compared with deaths ( %) in the selective decontamination group. (adjusted for imrie score and balthazar grade: p = . ). this difference was mainly caused by a reduction of late mortality (> weeks) due to significant reduction of gram-negative panreatic infection (p = . ). the average number of laparotomies per patient was reduced in patients treated with selective decontamination (p < . ). failure of selective decontamination to prevent secondary gram-negative pancreatic infection with subsequent death was seen in only three patients ( %) and transient gramnegative pancreatic infection was seen in one ( %). in both groups of patients, all gram-negative aerobic pancreatic infection was preceded by colonization of the digestive tract by the same bacteria. reduction of gram-negative colonization of the digestive tract, preventing subsequent pancreatic infection by means of selective decontamination, significantly reduces morbidity and mortality in patients with severe acute necrotizing pancreatitis. ieco by sodium hypochlorite (nacio) infusion is considered to be a model of microsomal oxidation in liver on cytochrome p- . active c provides oxidation of toxic metabolic products in the blood and exfused during plasmapheresis plasma, and also hydrophobic to hydrofilic transformation of substanses. sterile nacio in necessery concentrations was obtained by electrolysis of saline ( , - , % naci solution) in electrochemical set e~io- (russin,moscow). methods: . the nacio in concentration ragfl ( - ml/ h ) was administred into central veins in patients with extensive peritonitis and endotoxicosis - /t. erytrocytes resistance to nacio, circulating blood volume glycemia and hemostasis were initially estimated. . after plasmapheresis exfused toxic plasma was mixed with nacio conccantration of i mg/t in : ratio in sterile "hemacons".the effectiveness of plasma detoxication and possibility of its reinfusion were evaluated by determination of albumin effective concentration (eca g/l), the concanlration of medium molecular oligopeptides (mm , ) and other biochemical tests (bilimbin, creatinine, carbomide and so on). results: . the intravenous administration of nac excels detoxicative effect of hemosortion by - % provides effictive presentation of protein components and blood cells and improves the transport function of albumin by %. . the return of exfused plasma after its purification ieco was - %. only the remaning - % of deficient plasma were compensated by fresh cryoplasma and albumin solutions. ischemic hepatitis (ih) is a severe complication in critically ill patients. acute circulatory failure of multiple etiology can lead to splachnic hypoperfusion and cause acute and reversible anoxic damage. over a period of mos pts, m and f, mean age + . yrs developed liver disease compatible with ih. eight pts had a documented hypotensive episode (six pts with septic shock and two hypovolemic shock), while cardiogenic pulmonary edema in the absence of hypotension was responsible for ih in the remaining four pts. all the pts had a rapid striking elevation of ast, &lt and ldh with equally rapid resolution of these parameters to near normal wimin days (mean . ). the mean peak level of ast, alt and ldh was iu/l (range to ), iu/l (range to ) and iu/l (range to ) respectively. serum total bilirubin levels rose transiently with a moan t:eak level of . mg/dl (range . to . ), while altered coagulation paran-,ete's (pt> . times normal) was observed in four pts and clinically significant coagulopathy with fibrin degradation products occurred in one pt ( . %). renal impairment (cr> . mg/dl) was manifest in all pts; six pts developed non-oliguric renal failure ( %) while two pts required hemodialysis. ten lots required vasoconstrictor inotropes [dobutamine (range - pg/kg/min) and dopamine (range - pg/kg/min), while replacement of circulatory blood volume was performed in two pts with hypovolemic shock. eight lots expired ( . %), but none died as a direct result of hepatic damage. the mortality rate was higher among pts with concurrent renal failure ( %). it is concluded that: ) ih is not uncommon complication in the icu with the prognosis depending on the underlying disease. ) clinically significant coagulopathy is uncommon complication of ih. ) titration of inotropes is required to obtain optimal cardiac output support and subsequently liver blood flow. it is difficult to ascertain the perfusion of free flaps such as jejunal loops after surgery. objectives: to assess ischaemia as evidenced by intramural ph of jejunal free flaps used for reconstructive surgery following total pharyngolaryngectomy. methods: the sigmoid ph tonometer ( tonometrics inc.,usa ) was used to monitor intramural ph of the jejunal free microvascular flaps ( phig ) in patients who underwent total pharyngolaryngectomy. a standard general anaesthetic was given and all patients were admitted to the icu for controlled ventilation and monitoring. all had similar postoperative care. phig was measured pre, post-revascularization of the flap and on icu admission, , and hours postrevascularization. objectives: to classificate the wide spectrum of itc of anp into distinct pathophysiological patterns according to presentation and course. patients (pts) and methods: pts, ~( , %), ( , %) were admitted in the icu because of anp and acute respiratory failure(arf), ilean age: , • years. hean stay in icu: , • days. pts were operated, of them twice. hean value of ranson's scale: , • ( - ). we analyzed hemodynamic measurements,arterial blood gases(abg), x-ray findings(xrf), ct-scans and operative records. results: patterns of pleuropulmonary complications were identified: a)early hypoxia without xrf - pts. b)early ards with typical xrf - pts( died), c)early arf with xrf(atelectasis,infiltrates)- pts( died). d)late ards with typical xrf- pts( died), e)pleural effusions in various combinations with the above patterns - pts. overall mortality rate: / = , %. conclusions: l)frequent x-rays and abg are important for the classification of itc of anp. )even though patterns of classification in anp are not clearly distinguishable,they facilitate an anticipatory management. )deterioration of abg and xrf indicates that preventive measures for arf must be intensified and agressive surgical therapy is required. )delay of surgical therapy is related to worse prognosis(p<o, ) and prolonged hospitalisation time(p<o,ol). in the contrary, the early timing of the operation before infection and extrapancreatic necrosis is essential for a better prognosis (p<o, ). objectives: the development of cholestasis during intensive care treatment is allied with an inferior prognosis. this study investigates the sensivity, specification and also the positive and negative forecast of patients survival -exemplary for bilirubine. methods: during a period of months all surgical patients on icu were registrated prospectively (n = ). for documentation of disease development a standard foldcrform based on a computer-data-system was used. the results of this kind of documentation showed aapprax. , m!ll. of single patient informatioas. results: of patients didn't suffer from liver disease or have any operation on liver or bile tract. of them have had a normal scale of bilimbine in the postoperative period, a higher scale of bilirabine was regisu'atcd by patients. at a ,,cut-off" of rag% (total-bilirubine) the sensitivity was , , specivity , , positive predicive level , , negative predictive level , for survival criteria of a patient. the max. reached level of bilirubine, also the daily increase turned out almost the same results as they were found for other parameters nf cholcstasis like ap or gamma-gt. objective: the aim of this experimental protocol was to evaluate the morphological and functional parameters of isolated pig liver grafts, perfused in an extracorporeal liver support system. methods: the system consists of the graft liver, a membrane oxygenator (oxygenation surface . cm z, flow= itlmin), a heater, a centrifuge pump and a fluid reservoir. twenty pig livers, mean weight gr (min , max gr) were obtained and after a mean cold ischemia time of min were perfused fo} a mean of . h (min . , max h). perfusion solution consisted of r/l and hemacell. inflow to the graft liver was performed through the portal vein at a mean pressure of ( - ) mmhg at ~ while outflow was secured through the suprahepatic inferior vena cava. during perfusion the following parameters were evaluated through pre-and posthepatic hourly (to-t ) sampling: po , " + + " pco , ph, hco -, na , k , ca , osmolality, glucose, lactate, ast, alt, alp, u bilirubin and coagulation factors i, v and viii. biopsies were obtained periodicallty. b_p_~: results were as follows: mean ph fell from . at t o to . at t while mean output po fell from at t o to at t . mean output ast values increased from at t o to > at t while mean output alp values increased from . at t o to at t . mean output k + values increased from . at t o to > at t . histology revealed lesions of ischemic necrosis, more prominent after t . conclusion: results show that the isolated liver graft presents satisfactory function and morphology at least for a five hour perfusion period in the described extracorporeal circuit. correction of ph contributed to an increase in bile flow. between and the practice of transplantation has changed drasticaily in switzerland -besides kidneys also hearts, heart and lung, lung, iiver and pancreas transplantation has started in several centers. major information efforts have been made, organ exchange rules were set up and a national coordination center was initiated. the aim of this retrospective single center study was to assess the influence of transplantation on organ donation. in the past eleven years organs were donated from potential donors i single, multi organ donations) analysis of refusal was evaluated categorized into medical and/or familiar reasons. the number of potential donors increased from ( ) ,to ( ) with a concomitant drastic reduction of donations from % in to % in ; amounting to a net unchanged number of donations over the last years ( = ; = ) . the import and export of donor organs was balanced since the introduction of the national coordination center. in contrast multi organ donation increased from % in to % in despite of the more stringeant selection criteria, in conc]usion the introduction of a full range of transplantation procedures at several new university programs and the increase of multi organ donation has not had the forecasted impact on organ donation despite a sustained informative and promotional campaign, objective: monitoring hepatic venous oxygen saturation (svho ) provides online information about hepatic-splanchnic oxygen supply-demand ratio [ ]. previously, x~ reported hepatic venous catheterization in patients undergoing orthotopic liver traru~lantation (olt) [ ] . in the present study, we assessed the effects of nitroglycerin (ng), a vasudilator that affects the venous capacitance vessels more than arterial vessels and prostaeyclin (pgi , flolan r~, wellcome, uk), an arterial and splanchnic vasodilator on hemodynamies and hepatic venous oxygen saturation (svho ) in human liver transplantation. methods: with institutional approval and informed consent, consecutive patients, mean age - -_ years, were studied following olt. postoperatively, fiberoptic pulmonary artery catheter was inserted into the right hepatic vein. timed infusions of ng at a rate of . gg/kg/min and pgi at ng/kg/min were initiated for a rain period. each sequence was followed by baseline therapy for rain. results are expressed as mean=tsd. statistical analysis was performed using friedman's-two-way-anova-test, significance was accepted at p< , . results: ng at . gg/kg/min induced a decrease of mean arterial pressure (map) ( _ [baseline] vs. + mmhg) and pulmonary artery wedge pressure (pcwp) ( j: [baseline] vs. : mmhg). cardiac index (ci) ( - vs. + l/rain/m ), oxygen delivery index (do i) ( -+ vs. + mgnfin) and svho ( _~ vs. -l-_ %) were decreased (p< . ). pgi at ng/kg/min induced a reduction in map ( • nm~. _g) and pcwp ( + mmhg). ci ( _+ l/rain/m ), do i ( : ml/min) and svhoz ( + %) were increased (!o< . ). vasedilatation induced by ng decreased systemic oxygen supply and impaired splanclmie oxygenation. pgi increased systemic oxygen delivery in parallel with svho , suggesting a corresponding improvement of hepatic-splanchnic okygenation. thus, if vasedilator therapy is indicated in th orient receiving liver grafting, pgi appears to be advantageous. however, due to its platelct aggregation inhibiting properties, the usefulness and safety of pgi in olt patients has still to be determined. objectives: to analyze the effect of steroid treatment given to donor on the early function of transplanted kidney. methods: from january, until now donors were involved into this prospective study. every other donor was treated with mg/kg solu-medrol one hour before organ retrieval. according to the steroid treatment of the donor the recipients were divided into two groups: group -steroid pretreatment goup (y~= ), and group -control group (n= ). the donors and the recipients were treated using the same kidney transplantation protocol onl~r the adults, and the first cadaver kidney transplanted patients were involved into the study. the daily routine parameters were analyzed pre-and intraoperafive, and on the - th, th and th postoperative days. results: we could not show any clinically important differences between the two groups in respect of donor parameters. preoperative, the patients in group had slightly lower ereatinin level ( -+ g.,non vs. -+ gmol/ ) which persisted into the early postoperative phase. the values of the other examined pre-and intmoperativc parameters were almost the same. during the first postoperative days the patients in group i needed less diuretics (furosemide and renal dose of dopamine) and their sodium excretion was closer to the physiological range than in group . the other parameters did not differ significantly. the less furosemide need in group ! pe~isted to the end of the first month. conclusions: according to our data the steroid treatment of the donors improves the early function of the transplanted kidney in some respects. to prove the real benefit of the donor steroid treatment needs more data and further analysis. objectives: severe infections may compromize the outcome of liver transplantation..determination of new parameters may increase the knowledge of pathophysiologic mechanisms and may lead to changes in postoperative therapeutic management of patients at risk. methods: between august and september , patients with transplants were monitored for cytokines and extracellular matrix pammeters on a daily basis. serious infections (n= ) included microbiologic evidence and more than secondary organ failures. patients with cholangitis (n=ll) or uneventful postoperative course (n= ) referred as control groups. results: -year patient survival was . % ( / ): patients died due to serious infections, while died for other reasons. mean bilimbin, stnf-rii-, ifn- -, il- -, il- -, il- -, laminin-and neopterin levels were significantly elevated in patients with serious infections compared with patients experiencing mild cholangitis or with an uneventful postoperative course. a further increase of all parameters was observed in patients who subsequently died; tnf-ri/: _+ pg/ml vs • pg/ml; ifn- : _+ pg/ml vs . -+ . pg/ml; il- : -+ pg/ml vs -+ pg/ml; il- : -+ pg/ml vs _+ pg/ml; il- : _+ pg/ml vs • pg/ml; laminin: -+ ng/ml vs -+ ng/ml; neopterin: _+ nmol/ vs _+ nmolb for non surviving vs-surviving patients. a significant decrease of sialic acid yeas observed in patients with serious infections; and a further decrease occurred in patients who subsequently died: -+ mg/l vs • mg/ . conclusions: the increase or decrease of various cytokines and extracellular matrix parameters may be indicative for severity of infectiolx routine monitoring of these parameters may improve current diagnostic tools and poss~ly lead to changes in therapeutic management of patients at ~k. objectives: evaluation of the cytokine network after liver transplantation may give some insight in pathophysiologic mechanisms of rejection and may lead to detection of patients at high risk. methods: patients with transplants were monitored for various cytokines on a daily basis between august and september . rejection was assessed by histology in combination with clinical signs of rejection and laboratory investigations. results: during the first postoperative month, patients ( . %) developed rejection; patients were successfully treated with methylprednisolone (steroid-sensible rejection), while further patients required additional treatment with fk or okt (steroid-resistant rejection). patients subsequently developed chronic rejection. mean levels of various cytokines and extracellular matrix parameters including tnf-rii, ifn- , il-ib, il- r, il- , il- , il- , hyaluronic acid and neopterin were significantly higher in patients with steroid-resistant than in patients with steroid-sensible rejection. a further increase of some parameters was observed in patients who subsequently developed chronic rejection; bilirubin: . -+ . mg/dl vs . -+ . rag/all; tnf-rii: -+ pg/ml vs _+ pg/ml; il- : +- pg/ml vs -+ pg/ml; neopterin _+ nmol/ vs -+ nmol/ ; hyaluronic acid: _+ ~tg/l vs _+ ~tg/l for patients with chronic versus patients with acute steroid-resistant ~ejection. sialic acid levels decreased in patients with acute steroidresistant rejection; and a further decrease was observed in patients who tieveloped chronic rejection: _+ mg/l vs _+ mg/ . ~onclusions: various cytokines and extraeeuular matrix parameters were indicative of severity of rejction. the extensive increase of bilirubin, tnf-ii, il- , hyaluronic acid and neopterin may indicate subsequent chronic ection. monitoring of these parameters may, therefore, lead to changes in immunologic management after liver transplantation. background : combined kidney and pancreatic transplantation is being performed with increasing frequency in patients with diabetes mellitus and renal failure, as it offers more chances of success and better results than kidney transplantation alone. mycotic arterial aneurysm constitutes a devastating complication following pancreatic transplantation. all cases of mycotic arterial aneurysms have been however reported with exocrine pancreatic drainage into the gastrointestinal tract. intervention : we describe a series of consecutive whole kidney-pancreas transplantation performed at the university of geneva hospitals ( beds) between december and may . exocrine pancreatic drainage into the bladder (epdb) was performed to improve early detection of rejection episodes. epdb was hypothesized to reduce the risk of contamination from the gastrointestinal tract and the subsequent possible occurrence of potentially fatal infectious complication. in all patients the dual transplantation was performed through a median incision according to the procedure described by nghiem. results : two out of the patients who received kidney-pancreatic transplant developed arterial mycotic aneurysms and days following surgery. aneurysms developed at the site of the arterial anastomosis used to rearterialize the homograft. both patients had peritonitis caused by candida albicans requiring surgical drainage and intravenous antifungal therapy. rupture with hemorragic shock occured in both patients leading to graft removal in one patient, and three episodes of lffetreateniug hemorragic shock followed by graft failure and removal days after transplantation in the other. conclusion : arterial mycotic aneurysm constitutes an early, lifetreatening complication of kidney-pancreatic transplantation; it mandates graft removal. although exocrine pancreatic drainage into the bladder consitutes a definitive advantage for caller diagnosis of graft rejection, it does not eliminate the risk for retrograde colonization and subsequent severe infection in our experience. s. bocharov, i. teterina, regional clinical hospital, irkutsk, russia acute profound loss of blood can result from the very different injuries and hepato-pancreato-duodenai operations enter such a rank. ill-timed and inadeguate correction of operation hemorrage is one of the reasons for postoperation complications, including polyorganic insufficiency. the pathogenesis seems to be very complex. in early stages of bleeding the liquid enters the vessel bed, followed by hypoproteinosis and hematocrit fall. however, as decompensation develops, the fluid leaves the vessel system in the result of increasing postcapillary resistance and lowering col-ioidnooncotic blood pressure (cop). the resulting hypovolemia causes primarily acute disturbance of central hemodynamics and then of microcirculations and transcapillary exchange. central hemodynamic failure after acute loss of blood manifests itself through cardiac output lowering and capillary blood flow deceleration. taking into consideration, that % is critical value for cpv loss and for cev it is %, we consider arising the level of cop to the immediate task. cop raising allows to normalize transcapillary exchange, which we assess through cop and mcp (mean capilary pressure) gradient. the next task is to make up for globular volume till homeostasis providing level. considerable attention is given to catabolism inhibition and maximum possible enegry provision. control over high proteolitic activity of blood and callicreinkinin system activity implies direct proteases inhibitors. reologic, membrane stabilizing, antihypoxanthine and anticoagulant therapies are obligatory. virehow clinic, dept. of surgery, humboldt university berlin, germany regarding a high mortality up to % of fulminant hepatic failure orthotopic liver transplantation seems to be the only promising therapeutic approach in many cases. this study shows experiences from a transplantation center. between june and april patients suffering fulminant hepatic failure were admitted to our surgical intensive care unit all patients showed severe liver dysfunction with grade ii to iv encephalopathy. after a period of diagnostics and conservative treatment ranging from few hours to days (mean . days) we reported of these patients as possible organ recipients to eurotransplant. all of these patients were transplanted within hours, ( %) of them even within hours. the principal aetiologies were hepatitis b ( ), hepatitis c ( ), nanb hepatitis ( ), mushroom poisoning (amanita phalloides ). after transplantation patients suffered from initial-non-function and underwent re-transplantation. the one-year-survival rate was %, patients died within months after transplantation due to various reasons. patients were not referred for liver transplantation. of them never met transplantation criteria, improved by conventional therapy and could finally be discharged from hospital. the known reasons for liver failure in this group were mushroom poisoning ( ), paracetamol intoxication ( ) and fulminant hepatitis a ( ). patients suffering from fulminant hepatitis ( ) or intoxication ( ) were excluded from emergency liver transplantation for various contraindications. of these patients ( %) died despite conventional intensive care. we don't know if some of the patients in the transplantation group would have survived without transplantation, because whenever we decided on transplantation we could perform the operation within hours. but the good survival rate in the transplantation group ( %) the % recovery rate in the group, where there was no transplant-indication in our opinion and the fatal outcome ( % mortality) in patients with contraindications are an encouraging proof of a successful therapeutic strategy in acute liver failure. these results are based on a close cooperation between experienced transplant surgeons, hepatologists and intensive care doctors, using sophisticated laboratory and imaging techniques in a specialized center. introduction: during brain death patients suffer from multiple endocrinologic disturbances. one of the most important are those related with thyroidal axis. it is well described the euthyroid sick syndrome whose more frequent pattern consist of decreased triiodothyronine (t ), increased reverse t (rt ) with normal levels of tetraiodothyronine ( " ) and tsh, this lacking in " " levels lead to a change from aerobic to anaerobic metabolism which results in tissular damage. objective: .to study thyroidal pattern in brain death patients potential organ donors. .to avoid organ impairment by administration of t . .to study the hemodynamic and hormonal changes after the administration of t in these patients. material and methods:population: brain death patients of any etiology potential organ donors admitted to the intensive care unit. patients were classified in hemodynamically stable (group ) and unstable (group ). group received a bolus of . p.gr/kg. and a perfusion at a dose of - . p.gr]h of t . hormonal assays: total t (tt ), total " (tt ), tsh. fxee t (ft ), free " (ft ) and rt were determine at the moment of clinical brain death ( hrs) and in group two these assays were repeted at hours , and . results: patients ( male) with a mean age of years (range to yrs.) were studied. the clinical brain death was confirm later with other explorations (eeg, doppler). there were patients in group ( , %) and patients in group ( , %). hormonal pattern: at the moment of brain death tt was normal in cases ( , %) and decreased in i ( , %); tt was normal in patients ( , %) and decreased in ( , %); ft was normal in cases (i , %), decreased in ( , %); fl' was normal in patients ( , %) , decreased in ( , %) .rt was normal in cases ( , %) and increased in cases ( , %). there were no statistically significant differences in hormonal pattern between the two groups. only t levels at hours , and were significant in group . in the cases with ft decreased, the tt was normal in ( %) and decreased in ( %), tt was decreased in ( , %) and normal in ( , %), tsh was decreased in i ( , %), normal in ( , %) and increased in i( , %) and ft decreased in ( , %) and normal in ( , %) and rt was normal in ( , %) and increased in ( , %). there were no statistically significant differences in cardiac index, vascular resistances and pulmonary shunt before and after the administration ef t . conclusions: . the hormonal pattern most often find in brain death patients was: normal tt , decreased tt , normal tsh, decreased ft , normal fr and normal rt . . there were discrepancies in the values of ft and tt . there were no statistically significant differences in hemodynamic and pulmonary parameters. objectives: magnetic resonance angiographie (mra), a non-invasive procedure, provides flow-related information additionly to the anatomy of the vascular system. measurement of signal intensity and edge detection of vessel structures permits to calculate blood flow velocity and vascular diameters. we examined whether cerebral hemodynamic changes by altering the arterial pressure of carbon dioxid (pace ) could be detected by mra. methods: following institutional approval and informed consent, mechanically ventilated patients without elevated intracraltial pressure underwent mra with defined periods of hyper-, hypo-and normoventilation (pace : , , mmhg; arterial blood gas probes; avl). mra was performed with a . tesla magnetom (vision, siemens). two different mra techniques were used: a conventional time-of-flight- d-angiography (tr: ms; te: ms; fl: deg; slab: mm) for vessel diameter detection and a flash- d-gradient-echo-sequence (tr: ms; te: ms; fl: dog) for measurements of blood flow velocity. an axial view parallel to the ac-pc-iine (anteriorposterior-commissur-line) was used for repeated imaging of identical regions of interest toi) of the proximal part of the internal carotid (ica) and middle cerebral artery (mca) as well as of peripheral branches of the mca and the posterior cerebral artery (pca). results: changes of pace correlated with changing signal intensities, whereby under hyperventilation a decrease of , % (p . ) and under hypoventilation an increase of . % (p . ) was observed compared with normoventilation. blood pressures were stable throughout the whole study period, pace dependent changes in vessel diameters were more pronounced in peripheral branches of mca and pca. a change from normo-to hyperventilation produced a decrease in proximal vessel diameter of - . % (p _< . ) and in peripheral diameter of - . % (p _< , ). a change from normo-to hypoventilation produced an increase in proximal diameter of + . % (p < . ) and of + . % (p -< . ) in peripheral diameter. conclusions: pace related changes of cerebral vessel diameter can be easily detected by mra without injecting a contrast agent. the results confirm that co -reactivity is more pronounced in peripheral cerebral vessels, which are subjected to greater changes in diameter than major basal arteries. hyperventilation leads to a decrease and hypoventilation to an increase in signal intensity thus reflecting the corresponding changes in blood flow velocity, intensive care unit (icu) of "kat" hospital, athens, greece, ob!ective$; the value of bronchoscopy in pulmonary atelectasis of icu patients is under question the presence of an air bronchogram sign in xrays, which is considered as evidence of central bronchus patency, is referred in several studies as a negative criterion for bronchoscopy, whereas its absence as a positive one. it is also referred that air bronchogram sign correlates with delayed resolution of atelectasis, probably because of obstruction of many periferal airways (not central). the purpose of this prospective study was the evaluation of the air bronchogram sign on frontal chest film as a negative criterion for bronchoscopy and as criterion of delayed resolution of atetectasis, methods: icu patients with atelectasis were studied prospectively. they underwent bronchoscopy, bronchoscopic findings, presense of air bronchogram sign, and outcome of atelectasis were recorded, correlations were made, between: ) bronchoscopic potency of airways and air bronchogram sign } resolution time of atelectasis and broncoscopic potency of airways. ) resolution time'of atelectasis and air bronchogram sign, methods of statistical analysis were the t-student test and the chi square test, results:the patients were , men women , seventeen patients had atelectasis of whole lung, of upper lobe, and of lower lobe. ten patients had atelectasis in right and in left lung. eight from patients had air bronchogram sign in x-ray, there was no statistical correlation between air bronchogram sign and bronchoscopic potency of airways [ from patients with air bronchogram sign ( %) and from without air bronchogram sign ( %), had bronchoscopic potency of airways, p> . ], resolution time of atelectasis didn't correlate statistically with bronchoscopic potency of airways (mean resolution time in patients with bronchoscopic potency , days and in bronchoscopically closed bronchi , days, p> , ). there was also not a statistical correlation between resolution time of atelectasis and air bronchogram sign (mean resolution time in patients with air bronchogram sign , days, and without air bronchogram sign , days. p> ). conclusion~i; the presense of an air bronchogram sign in x-ray of icu patients with atelectasis, does not coexist obligatorily with bronchoscopic patency of airways and cannot be used as a negative criterion for bronchoscopy, neither as a criterion of delayed resolution of atelectasis. th. wertgen chest sonography (cs) is routinely used in our department to examine icu patients with clinical symptoms of pulmonary embolism, pneumonia, pleural effusion or unclear chest pain. we perform cs with a sector transducer ( . mhz) and a linear transducer ( . mhz) using acuson xp/ c. the sonographic signs of pulmonary embolism and infarction are most well demarcated, mainly wedge shaped and triangular pleural based lesions, more roughly structured, observed with a hyperechoic reflex in the center corresponding to the bronchitic (fig. ) . pneumonia is characterized by homogenously hypoechoic, wedge shaped parenchymal lesions, containing air or fluid bronchograms; they move with respiration (fig. ) . pleural effusions are spaces of various echogenicities, from anechoic to homogeneously echogenic, which may contain floating strands or complex septa, located between visceral and parietal pleuras (fig. ) . from march to april we did examinations by cs in icu patients ( male, female; age from - ). patients examinations pulmonary embolism pneumonia pleural effusion us-guided thoracic punctions were performed in patients. in two patients we found pneumonia or pleural effusion caused by a lung carcinoma. another two patients showed a normal cs (diagnosis: inflammation of the gall bladder, inflammation of the myocardium). conclusion: cs is a very useful method for icu patients with chest diseases. it takes less time and is less expensive than ctand sometimes of a higher diagnostic value than x-ray. last but not least cs is invaluable for the icu patient, because the examination is done save and quickly at bed side and the results of cs are very helpful in diagnoses and treatment. results : inter-observer reliability was evaluated as an % concordance. results of the tee classification were : class : n = ( %) ; class : n = ( %) ; class : n = ( %) ; class : n = ( %) class : n = ( %). therapeutic implications of tee in class patients were : cardiac surgery in patients (two cases of acute mitral regurgitation, two valvular abscesses and one hematoma compressing the left atrium), discontinuation of peep in one ventilated patient with an atrial septal defect, weaning of mechanical ventilation in one patient with an atrial septal defect, prescription of antimicrobial therapy in patients with endocarditis and prescription of anticoagulant therapy in patients with left atrial thrombus. the only noteworthy complication was a case of spontaneously resolving supraventrieular tachycardia. conclusion : tee is safe and well tolerated, and is useful in the management of icu patients with shock, unexplained and severe hypoxemia or suspected endecarditis. the aim of this study was to determine whether ultrasound guidance can help interns to improve the results of jugular vein access in icu. methods : in a prospective and randomized study, we compared, in patients admitted to the icu, an ultrasound-guided method (ultrasound group : patients) with an external landmark guided technique (control group : patients). all jugular vein accesses were performed by young interns with an experience of < procedures. results : internal jugular cannulatian vein was aci~ieved in all patients in the ultrasound group and in patients ( p.cent) in the control group (p < . ). average access time was longer in the control group ( • sec. vs • see. ; p = . ) and puncture of the carotid artery occurred in patients in each group (p = . ). patients ( p.cent) in the ultrasound group and patients ( p.cent) ia the control group (p < . ) were cannulated in rain. or less. the cannula was therefore unabie to be inserted within minutes in patients in the control group, with failure of eannulation in of these patients ( p.cent). failure was due to thrombosis (n = ), small calibre of the internal jugular vein (< ram) (n = ), abnormal vascular relations (n = ) or cervical irridation (n = ). among the primary failures of cannulation, an internal jugular vein catheter was able to be inserted in cases by an experienced physician on the side initially selected and with ultrasound guidance in cases. the catheter was inserted into the contralateral internal jugular vein under ultrasound guidance in the remaining cases. jugular cannulation was obtained at the first attempt in p.cent in the control group and p.cent in the ultrasound group. conclusion : ultrasound guidance improved the success rate of jugular vein cannulation by inexperienced operators in icu patients. when the internal jugular vein has not been successfully eannulated within minutes by the external landmark guided technique, the authors recommend the use of the ultrasound guidance. in the majority of cases right atrial or ventricular thrombi represent pulmonary emboli in transit. these may be fatal in patients (pts) treated conservatively with anticoagulation only. in literature the incidence of right heart thrombi in pts with proven pulmonary embolism (pe) is said to be in the range of - %. extremely mobile, long, worm-shaped masses in the right heart cavities carry an especially high early thrombus-related mortality rate which ranges from - %. current therapeutic strategies favour fibrinolytic therapy with consecutive anticoagulation. we report five cases ( male, i female, - years) of right heart and pulmonary thromboembolism. in these pts diagnosis and regression of thromboemboli following systemic intravenous lysis therapy with recombinant tissue-type plasminogen activator (rt-pa) was documented by transesophageal echocardiography (tee). a submassive pe occured in pts, a massive pe in pts. one patient (pt) had a cardiac arrest. in all cases tee clearly identified the extensive thrombns formation in the right-sided cavities of the heart and in the central pulmonary artery in cases. all pts were treated with mg rt-pa, pts in a front-loaded regimen over minutes, pt over minutes, and, due to the life threatening situation, in one case a bolus injection as ultima ratio was performed with no intracerebral bleeding complication. regression of thromboembolic masses after fibrinolytic therapy was demonstrated by transthoracic and transesophageal echocardingraphy after to hours. all pts survived and were put on coumadine, pt developed an intracerebral bleeding with persistent hemiplegia. conclusions: the use of thrombolytic therapy is highly efficacious for the therapy of pts with pe and concomitant right or ventricular thrombus formation. transthoracic and especially transesophageal echocardiography are powerful bed-side diagnostic tools for the immediate diagnosis and follow-up of successful treatment in this life-threatening condition. although widely used, catheterisation of the femoral vein in the groin using "landmark" technique is frequently complicated by accidental arterial puncture. suboptimal hygiene and patient discomfort are also associated with this technique. with regard to these last two factors cannulation of the femoral vein - cm below the inguinal ligament would seem an attractive alternative. as "landmark" technique is not possible for the cannulation of the femoral vein in this part of the thigh, ultrasound was used to locate the vessel and the results of this technique were evaluated. methods: a portable compact ultrasound device (site rite,dymax corp.) featuring a . mhz transducer (ultrasound depth - cm) fitted with a needle guide and a cm screen was used by residents with no previous experience in ultrasound guided cannulation. patients consisted of a surgical icu population. results: in patients catheters were introduced.in cases more than one ( - ) attempt was made and in patients the procedure was unsuccesfull due to the fact that the vessel was situated out of reach of the ultrasound (vessel depth > - cm), during the procedures one accidental arterial punction was registered. the catheters remained in situ for a mean of days (range - ) and were used for volume suppletion, medication, parenteral nutrition and haemodialysis.co-ionisation rates compared to those of subclavian catheters in our icu. in the first patients cases of asymptomatic thrombosis of the femoral vein were seer on ct-scans performed for other indications, in the following patients duplex scanning performed after removal of the catheter yielded another cases of asymptomatic femoral vein thrombosis. conclusions: ultrasound guided femoral vein catheterisation - cm below the inguinal ligament is a safe and simple technique that can easily be performed by residents without prior experience. the incidence and impact of thrombo-embolic complications associated with this technique are still subject to further investigation. objectives: to estimate the cost of antibiotherapy (ab-cost) in a multidisciplinary -bed greek icu and to correlate ab-cost with total cost of drugs and consumables and with patient's outcome, severity of illness and type of admission. methods: prospective data from consecutive patients admitted to the icu from / / to / / were studied. a tick chart was designed to record all drugs, materials and consumables regularly used for icu patients, but did not include low price drugs and consumables, which are provided from hospital's pharmacy as stock and were included in a fixed icu cost calculated for a month period. the chart also contained demographic details and data necessary for the calculation of several illness severity scoring systems. obiectives: over years evaluate the necessary efforts and expenses to implement a cis in the routine of a -bed stcu. methods: in june a commercially available, unix-based cis was installed on a -bed surgical icu. the goal was a paperless documentation at the bedside. after more than years clinical experience two aspects were investigated: what effort is necessary to install and support a cis, and what is the benefit for patients and personnel on the icu? results: the installation and support of a full-fledged cis requires a considerable effort: (a) the conceptual framework for the cis has to be defined. this includes the definition of documentation standards, as well as nursing and therapeutic standards, which is the essential basis for the configuration of any cis. (b) configuring a cis, i.e. "fine-tuning" it to the user's specific needs, is always a laborious task. moreover, constant maintenance is necessary. these tasks require the following personnel: experienced health care professionals for defining the conceptual framework, - trained health care professionals for configuration, system administrator. on a single icu ( - beds) these are not considered full-time jobs. (c) training is best done employing the "train-the-trainers" approach. (d) beside the necessary amount of man power and money to install and purchase a cis, administrative and mis support is needed, especially when interfaces to the hospital and laboratory information systems have to be set up. in general, a cis needs the commitment of all people involved. without a really professional approach with a longterm goal any major cis can turn into an unnecessary but inevitable night mare. after years clinical use and a thorough implementation of a cis on a major sicu it can be said that full-fledged cis offers an opportunity to dramatically improve the working environment on an icu. moreover, it adds to patient safety, quality of care and cost efficiency in one of the most advanced and expensive areas of medicine. conclusion: a major investment in man power and money is necessary to install and maintain a full-fledged cis. a sincere professional commitment to the goals of a cis is necessary. in exchange, a well configured and well maintained cis dramatically improves the quality of therapy and care on the icu. even return of investment and financial profitability of a cis seem feasible todayl from the clinical perspective it appears that the users themselves are the central determinant whether a cis makes a dream come tree or turns into a night mare. objectives: to establish a relationship between the activities of the staff and the occurrence of auditory alarms on the i. c.u. ard to evaluate confusion between auditory alarms. methods: laboratory based studies which investigated aspects of confusion between alarms in current use on the i. c. u. the observational studies were conducted over an month period and examined the frequency and duration of alarms together with the concurrent activites being undertaken by staff on the unit. the laboratory based studies showed that there were enduring confusions between the alarms on various items of medical equipment, for example a ventilator alarm and an e. c. g. monitor alarm. the results of the observation studies demonstrated that alarms are activated when specific activities are being undertaken by staff. sounds could be used in future recommendations for alarms on medical equipment. suggestions are also discussed for improving and rationalising auditory warnings in the i. c. u. obiectives: we investigated inferior petrosal sinus (ips), the lowest affluent to jugular bulb (jb), as a possible source of contamination of samples in jb for monitoring oxyhemogiobin saturation (sjbo ). pulling back the catheter the oxyhemoglobin saturation usually rises indicating extracerebral contamination (jakobs en met al: j cereb blood flow metab ; : ). methods: the study was carried out on patients undergoing ips sampling to differentiate cushing disease from ectopic acth syndrome and to lateralize any resulting pituitary lesion. we studied the value of oxyhemogiobkn saturation high in jb (sjbo ), at ips (sipso ) and at mid jugular vein ( th cervical vertebra) (smj ) bilaterally. results: we found significant differences between right sjbo and both right sipso (p= . ) and right smjo ( p= , ) and between left sjbo and both left sipso (p= . ) and left smjo (p= . ) we did not fred any difference bilaterally. objectives: we studied various methods of receiving and editing of clinical datas in critically ill patients (different ethiology). patients were investigated in regional intensive care center. methods : the following datas were studied : anamnesis, status praesens objectivus ( organs and systems ) ,. clinical and biochemical markers of critical condition , datas of eeg ,rheography . the medical information complex contained : channel electroencephalograph, -channel roencephalograph, ad-converter ( analog inputs, bit resolution, k hz), ibm dx , software includes set of routines for spectral eeg analysis, eeg-mapping, correlative analysis, and brain bloodstream reg-monitoring (written in turbo pascal . ), expert programs for estimation objective and humoral patient status (written in clipper . ) and statistics. there were used following programme-language instruments : borland c++ . , nantucket clipper . , ca-clipper tools ii. as the methods of statistical processing of dates were used: t-students criterion , fisher criterion, methods of correlation analisis, calculation of the regression levels, dispersion analysis, results : there was created the optimal structure of hard and sofware complex of search steady objective regularity in dynamic of critically ill patients condition. conclusion : the created system allowed to value effectiveness of intensive care and give us new opportunities in study pathogenesis of systems disorders in critical condition . over a five year period a patient data management system has been installed which allows individualised patient data to be accurately collected. using this data a costing system has been developed which ascribes costs thus: . direct costs -drugs, fluids, consumables, interventions. these are ascribed to individual patients, according to data collected from the pdms. . indirect costs -energy, depreciation, admm costs, maintenance etc. these are summed for the year and ascribed as an overhead per patient day. n.b staffcusts contain art element of both cost types the aim is to make as many costs as possibie 'direct', hence 'activity costs' have been calculated winch comprise staff time, drugs and consumables -these are direct costs. these costs of patient care are then searnlessly integrated into the financial and budget management of the icu environment. it was found that by calculating costs in this manner % of the total cost of icu are captured within the 'direct' element, and so are able to be ascribed to individual patients. this is much more accurate than simply dividing the total costs of ~cu by the number of patient days. temporal costs (variations during patient stay) and cross sectional costs (cost differences between admitting specialities) were also noted with interest. results of the initial analysis of data captured by the system will be presented. little is known about the resource costs (not simply cash costs) of icu. even less is known about individual patient costs, with previous estimates of these costs varying widely. however, if cost effectiveness studies are to be undertaken accurate calculation of individual, group and total icu cost is an essential, prerequisite, which, via this system of costing, is now achievable. information about intensive care of cancer patients is limited in the literature, despite the increasing use of such facilities in oncology over the two last decades. in order to determine if and how critical care facilities can be used specifically for these patients, we performed a world-wide inquiry in anticancer centers selecting the hospitals by using the international directory of cancer institutes and organizations. we mailed a questionnaire to centers and we received responses ( . %). there was at least one uncological (i.e. with > % of cancer patients) icu in (% % an -year old woman with graves disease presents with sore throat, vomiting, diarrhea, sinus tachycardia at /minute and a temperature of ~ several weeks before, treatment with propylthiouraeil had been stopped (rash and fever) and replaced by methimazole and ledide prior to a minor surgery. however, both drugs were discontinued by the patient two weeks before admission. shortly after arrival in hospital, patient's condition progressed to respiratory failure (upper airway edema), delirium and shock requiring icu admission, intubation and resuscitation with fluids and vasopressors. white blood count was /mm ~ with neutrophils. patient's hemodynamic data showed initial hyperdynamic profile followed by low output state with decreased sv ( %) (n - %) and cardiac index ( , ) (n , - ). echocardiogram confirmed cardiac chambers dilation as previously described in thyroid storm. lithium carbonate, corticosteroids, antibiotics and beta-blocker perfusion were given. plasmapheresis was started. free t& (n= , - pmo/l) went from , to , after the first two pheresis. after a remarkable clinical recovery, sub-total thyroideetomy was done i days after admission. in life-threatening thyroid storm, plasmapheresis is a very effective therapy when anti-thyroid drugs are counterindicated. purpose: to compare the reliability of prognostic indexes in crhically iu patients admitted in an intesive care unit (icu) who had acute renal failure (arfi and were treated with different dialytic techniques. material and methods: patients were included in a prospective study from june to november . patients presented arf defined by creatinin serum leve(s greater than pmol/l and previous normal levels. patients were divided in three groups. group i (control) : patients with arf who did not receive substitutive techniques. group ih patients under intermittent hemodialysis (hd) or peritoneal dialysis (pd). group ii : patients under continuous hemodiafiltrstion (hf). the statistical analysis was chi-square test and analysis of variance. results: the table shows the results we obtained, we did not find any significant difference betwen the two groups of patients undergoing dialysis. d(fferences were observed only between group i and the other groups as shown below. we did not find any significant association between the theoretical mortality predicted and the observed mortality according to saps in the three groups. due to exposure to a wide variety of unpleasant stimuli, for example, tracheal suctioning, venipuneture and physiotherapy, most pataents admitted to the icu will require some form of sedation. this review will describe the suggested properties of an ideal sedative agent for use in the icu and review the current limitations of some of the available agents from this perspactive. methods used to quantify the level of sedation, such as the ramsay score, glasgow coma score, newcastle sedation score and visual analogue scores, and their deficiencies will be examined. consideration will be given to defining the optimal level of sedation and the circumstances under which sedation might be varied over the icu course will be discussed. preliminary results from an ongoing study examining the role of light versus heavy sedation and ischaemia in a cardiac surgical icu population will be presented. the pharmacceconomics of icu sedation will be briefly addressed. finally, the role that sedation may play in increasing morbidity, pastieuiarly nosocomial pneumonia, in the icu will be discussed. objectives : therapy cost(tc) in icu patients is a substantial component of total hospital care cost. estimation of tc during this year, partitioning to various groups of drugs used and attempt to minimise it, were considered practically useful. methods : in collaboration with the hospital pharmacy we were able to have a complete report of au drugs used for icu patients (including enteral and parenteral nutrition). mean apache ii severity score upon admission was . and mean length of tcu stay was . days. price per drug unit and cost per group of drugs were also available drugs were divided into two groups: antibiotics ( ) cardiovascular drugs ( ), gastrointestinal system drugs ( ), enteral and parenteral nutrition ( ), respiratory system drugs ( ), sedative, analgesics and paralysing agents ( ), parenteral solutions with electrolytes, vitamins and trace elements ( ), anti-inflammatory agents ( ), protein substitutes and immunomodulation agents ( ), anticoagulative agents ( ). antibiotics were further subdivided into those "freely" prescribed (a) and those whose prescription and administration requires filling of a relevant form (b). results : !) tc for icu patients/day was . drs ($ ). total tc/patient was . drs ($ . . ). ii) partitioning total tc per group of drugs reveals : ( ) %, ( ) . %, ( ) . %, ( ) . %, ( ) . %, ( ) . %, ( ) . %, ( ) . %, ( ) . %, ( ) . %. t ) concerning antibiotics which consist the major cost component, group a and group b contributed by . % and . % to the total icu tc respectively. group b were administered to . % of all icu patients. conclusions : i) for the above studied patient population antibiotics consist almost half of total tc followed by protein substitutes and immunomodulation agents. ii) if tc control could be attempted in the icu, prescription of beth groups must be reviewed. appropriate treatment should be prescribed and readily provided to any patient. clinical significance of routine protein substitution, currently controversial, should be re-evaluated. new antibiotics (third & fourth generation cephalosporins, quinolones, carbaponems) should be prescribed on the basis of strict diagnostic procedures using modern technology available. rationalisetion of antibiotic therapy will lead to cost control, redistribution of icu expenses and substantial contribution to infection policy in our country. objectives: i -to investigate the clinic efficiency of the monitoring of the rso cerebral, in relationship to the stroke prevention, in patient undergoing carotid surgery. -to determinate the variations of the rso during the different surgical and anesthetic procedures in these patients methods: ten patients undergoing carotid endarterectomy. precise neurological exploration previously to the surgery and in the immediate postoperative period. angiography evaluation to the extend of carotid artery disease. invasive blood pressure, ecg, pulse-oximetry ( pso ) and rso were collected previousty to the induction of anesthesia. the premedication was administered intravenously -midazolam ( mcgr/kg) and fentanyl (i rncgr/kg) -. thiopental ( mg/kg),fentanyl ( mcgr/kg) and atracnrium ( , mg/kg) have been used for induction of anesthesia. co te is monitoring al~er the orotraqueal intubation ! the anesthetic maintenance is accomplished with lsofluorane ( , - , %) and bolus of atracurium and fentanyh the surgical procedure is standard (without arterial shunt during the carotid cross-clamping). we register each minutes: blood pressure, cardiac frequency, pso , co te and rso . the rso cerebral variate in relation with: the anesthetic induction, blood ~ressure, co te, cross-ulampping carotid and with the modifications of the head position. the maximum decrease of rso cerebral was in relation with the :ross-clampping carotid ( minimal value: ). no patient had neurologic complications and postoperative stroke after carotid endarterectomy were not observed. objectives: there are more than anesthesia in chelyabinsk emergency hospital every year. to % patients of it emergency anesthesia is applied. more than patients have ishemie heart disease (ihd), hypertansion (hp) and previos miocardial infarction (pmi). more than % of all patients are old patients (op). the resalts deep noninvasive bioimpedance monitoring (nbm) in surgical patients have been studied by us. methods: our nbm system "kentavr" includes parameters of cardiac and vessels function. it is realised by monitors in operation theatres and computer network. moreover we are able to examine surgery patients before anesthesia and perioperatively by using special computers system for cardiovascular reflex control by fast fourie transform (fft) of parameters simultaneously. results: pathients extremly needed peryoperative monitoring of hemodinamics. from these patients more % had stroke volume (sv) less than ml, n -co less than . /mim/m , % -ejection fraction (ef) less than n and % -puls bioimpedans microvessels (pbm) less than morn. patient had intensive care in special department. out of died. comparing with survived with these patients before operation hr was larger, sv, co,ef, pbm and puls bioimpedance aortha was smaller. much more of these patients were with ihd, pmi, hd, op. even with survived patients these parameters decreased the towards the end of operation. surgery patients had different variability of basic hemodinamical parameters with common tendency to increase power amplitude in low frequency by fft. conclusions: using of bioimpedanee noninvasive parameters allows to have criteria for corrections (infusies, vasodilatators, inotrops and others) and then us the final goal, to have more sucssesful surgery. with survived patients was perioperatively and postoperatively care more intensive. obiectives: the aim of the study was to compare the phi with the hemodynamically derived tissue oxygenation indexes as: oxygen delivery (do ), oxygen consumption (vo ), cardiac index (el), and arteriovenous difference in oxygen [(a-v)do ]. methods: patients ( males and females) with major trauma or major abdominal surgery were studied. on admission, a nasogastric tube allowing phi measurement was introduced and a pulmonary artery catheter was inserted for optimal hemodynamic management. each phi measurement was accompanied with a complete hemodynamic study comprising systemic and pulmonary artery pressures, blood gases, and cardiac output measurements with the thermodilution method. derived parameters vo , do , ci, (a-v)do were measured according to the standard formula. hemodynamic parameters were opt• as soon as possible with fluids, inotrepes, and vasopressors according to repetitive hemodynamic measurements. all patients were under mechanical ventilation. after hemodynamic stabilisation phi and hemodynamic measurements were repeated every eight hours, during a -hour study period. a total number of measurements were obtained and compared. statistics: results are presented as means + sd, correlations were performed between phi and the hemodynamically derived oxygenation parameters. a p< . value was considered as significant. results: mean values were phi= . + . , do = + , vo = + , c. = . + . , (a-v)do = . + . . no correlation was found between phi and do , phi and vo , phi and c.i, phi and (a-v)do . on the contrary in patients phi remained below . for more than hours despite adequate hemodynamically derived tissue oxygenation parameters. mortality in this group of patients was very high ( %). conclusion: no correlation was found between phi and the hemodynamically derived tissue oxygenation parameters our data suggest that phi is a better oxygenation indicator than the hemodynamically derived tissue oxygenation parameters, because it is closely related to the patient's outcome. objectives: the pathogenesis of septic shock and multiorgan failure is believed to be related to tissue hypoxia of the gastrointestinal tract. therefore new monitoring techniques, preferably organ specific, are required to establish the adequacy of tissue oxygenation. peep is used to reduce pulmonary shunt volume and improve blood oxygenation, but is accused to impair splanchnic perfusion. we studied mucosal oxygenation and perfusion on the capillary level in the stomach and the duodenum. methods: we used the erlangen microlightguide spectrophotometer (empho ll) together with a specifically designed fibre probe (bodenseewerk ger~tetechnik, berlingen) in combination with a standard gastroscope. measurements were performed on ventilated, traumatized patients (ages - years), with no evidence of shock or severe infection, after informed consent was obtained from the relatives. all patients were hemodynamically stable without inotropic support. an area of cm was analysed in the gastric corpus, the antrum and in the duodenum. in three patients we simultaneously measured the muc sal blood flow using a laser doppler flowmeter ( objectives: to investigate the influence of hb-o affinity in the monitoring of svo~ during improvement of cardiac index (ci) in cardiogenic shock. design: to state whether changes in svo: were associated in changes in actual pso (p~ ) and standard p~ (ps st) consecutive measurements of artero-venous bga, before an.d after therapy-induced changes in ci, were evaluated in patients (mean age -* y) suffering from cardiogenie shock, all under mechanical ventilation in psv modality. methods: together the hemodynamic measures, m~xed venous samples were analysed at ~ c using the abl radiometer for po , pco: and ph, and the osm radiometer for hbo %, hbco% and methb%. psost (i.e. the p~ at ph= . , pco:= mmhg and temperature at ~ c) was calculated automatically by the instruments on mixed venous blood as was the ps "in vivo" (i.e. the pso at the patient's value of ph, pco and temperature), using siggaard-andersen's computerizated algorithm. mean time between paired measurements was . -* . houm. the data were compared by anova test for linear regression and t-test for paired samples. results: a dose linear relationship was found between svo and oxygen extraction ratio (oer), r= . ,p= . . the improvement of ci ( . -* . to . + . l/min/m , p< . ) induced a significant increase in svo~ ( . -* . to . • . %, p<. ). a significant decrease in p ( . • . to . • . mmhg, p< . ) without any significant change in p~ st ( . • . to . • . mmhg, p=ns) was also found. these data show that either oer or the shift to the left of the oxygen dissociation curve account for increase in svo occurring with restoration of systemic blood flow. the program is intended to help the intensive care unit interne providing him with a practical tool when making decisions concerning patients in a critical condition. in his daily practice in intensive care unit, in this case the interne of the unit, uses this program for each patient as follows: on the first stage of data collection he should complete the following modules: ( )personal data ( )patient's pathology ( ) laboratory and~ monitor lug data ( )drugs prescribed or toxic elements ingested. in this way, the system allows optionally the consult with a computerized data base about the drugs prescribed, standardized parameters and techinques performed by the central laboratory. ( )reference to an antibiotics guide regarding becterian sensitivety in our unit, whitch ee checked every six month ( ) access to de questionnaired apache ii to load up new data. ( ) statistcs about patient's admission and discharge. results: once all data collection is finished the system performs the followin duties: ( )detailed drugs interactions, including toxic elements ( )diagnosis starting from the clinical, laboratory and monitoring data. in some cases, it also establishes therapeutic strategies, e.g. a coagulopathy ( ) give the l~narmacological incompatibilities between the drugs p~escribed and %he diagnosis established, and ( )perform dosage adjustments based upon the personal and pathological data. objeatve: to assess the power of diseri~,~ion ofa multiperpose severity score (sai~) when applied to subgroups ofpatieals (pta) according to their lemg~ of ~ay (los) in icu. design: in order to compute the saps probability, a model derived fi~m legible regression was developed. meaumree of calibration (goodmem..of.fit statistics) end discrimination (roc cm've and relative area under the cm've) were adopted in develotammtul asd validation set. the whole databue was ~ati~ed in five gronps reeked on los as follows: los = days, los = - days, los = - da~, los = - days, los > day~. area under the carve (auc) was ud~ninted for each ro~. s~ing: imlimlcus. patents: of ~ pts comec~ively admired ~ a period of three yeet~ ( ) ( ) ( ) , a total of was i~leded in this study. pts without saps, p~ yolmger them yearn, p~ with los shorter ~ hom'~ were excluded from this maly~is. iaterventinns: nose mema'onm~ end result: the logistic model developed gave good remits in terns of calibration md discrimin~on, both in developmental set (do.s g : . , p > . ; auc = . i- . ) and in validation ~t (g.o.g g : . , p > . ; auc = . ..+ . ). auc of each grottp showed a loss in di~zimination (i.e., prediaton) closely related with los, being . i- . in pts with los = days el . ~. ia tm with los > da~ (figure). following the present guidelines of integral management, in order to achieve optimization of sanitary resources and better use of facilities, we feel that the setting up of objetives is a key factor in the continuous process of improvement of quality care. postsurgical intensive care services maintain an interdepent relationship with other hospital services. within the general plan of the hospital it's of the utmost importance to delegate autonomy to the various depertments and service units in determining and achieving objetives. it's also necessary to establish mechanism for coordination of the activities in order to assure the succes of the program. the objetives cannot be improvised, they must be carried out in a specific manner in the following stages: .-analysis of the present situation (starting point). where are we?. defining objetives and making explicit the activities and methods to achieve them is to anticipate the future; it is of the utmost importance to comunicate said plans to all whom affect by encouraging them to attain the desired results. in the present paper we intend to show the guidelines to follow in carrying out a course of objetives. introduction:we presents results related to the quality of life (qol)of critical patients, from paeec project data. material and methods: the paeec project is a multicentre study define the type of patients cared for in spanish icus, and the therapeutic activity provided. ninety-five icus from spain are taking part. this study analyzes the qol of critical patients prior to their icu admission.for the evaluation of qol a questionnaire designed by our team for critical patients was used, with items grouped in sub-scales: physiological functions ( items); functional capacity ( items) and subjective aspects ( items). qol is classified in levels: normality ( points); slight deterioration ( - points);moderate deterioration ( - points); significant deterioration (>i points). the we present results related to therapeutic activity in critical patients and their age, from the paeec project. material and methods: the paeec project is a multicentre study to define the type of patients in spanish icus, and the therapeutic activity provided. ninetyfive icus from spain are participating. this study analyzes therapeutic activity in the first hours as evaluated by tiss, and related factors. results: the sample was , patients, sge . ~ . years. severity by apache ii system was . • points. the tiss score was . • points, distributed as follows: i (<i points): %; ii (i - points): %; iii ( - points): %; iv (> points): %.there is a positive correlation between the level of therapeutic activity and severity by apache ii (r = . , p < . ), and a very weak but negative correlation between tiss and age (r = - . , p < . ), so that an increase in age corresponds to a lower level of therapeutic activity.patients the multivariate analysis of the relationship between tiss and age took into account: severity, existence of previous history, need for mechanical ventilation, size of hospital, diagnosis and mortality. it indicated that there continued to be a relationship between therapeutic activity and age, so that as age increased, therapeutic activity diminished. conclusions: therapeutic activity performed on critical patients is less in the oldest patients, in whom excessively aggressive procedures are limited. a relational data base management system in the icu. c. kotsavassiloglou*, d.matamis, g. dadoudis, j. kioumis, d. riggos. icu dep., g. papanicolaou gen. hosp., exohl, thessaloniki, and * a' neurological clinic of aristotelian university, thessaloniki, greece. objectives: the introduction of the information technology in the i. c. u seems to be unavoidable because of the large amount of produced data and the need for their systematic analysis. such an information system should be a) easy to use, b) friendly to the user, c) powerful and d) modular. on that basis, we created a patient data management system (pdms) according to the expectations of the medical staff of an eighteen bed multidisciplinary icu. methods: we selected paradox for windows v . for the implementation of a relational data base because this program meets the above mentioned criteria. informations regarding the patients include a) demographic data, b) previous medical history, c)diseases upon admission, d)complications during hospitalization and e) outcome data. the diseases' registration consists of items classified in categories upon the principal system affected. specific informations about the need and duration of mechanical ventilation, nutrition, renal replacement, right heart catheterization and icp monitoring are also available. an extension was added concerning icu infections and related informations about antibiotic-resistant pathogens. all icu pathogens can be matched to their resistance or sensitivity and cost of antibiotics. the program can perform queries and various statistical analyses based on complex criteria. new modules can be added later according to the future needs and remarks of the users. results: the program was well accepted by the medical staff and patients were registered as a test. the first analysis of the data related a) observed mortality versus the apache ii predicted mortality, b) mortality according to the age, gender, pathology aud duration of icu stay and c) pathology upon admission and icu related complications. conclusions: the long term use of this pdms can be an efficacious research tool. it can be used in retrospective or prospective studies by addition of necessary modules. the first data analysis revealed the iack of an international diseases' classification system. the development of a worldwide common classification system is essential for the compatibility of the data analysis among various icus. this will allow the realization of multicenter trials on a large scale. s. nanas= n. sphiris, a. precates, a. lymberis, m. pirounaki, and ch. roussos dept. of critical care, university of athens, athens, greece the complexity of the cases submitted to an icu, the variety of underline disease, tbe severity, as well as the large number of substances administered to each patient constitute obvious the need of support with an easy available dss. this system will assure the safety of the administered treatment will help to adjust the dose according to the situation of each patient and it will screen for possible interaction and incompatibilities between the administered drugs. the goal of the present effort is the design and development of a software system acting as a decision support tool to physicians of icu. the application is organised around a relation database management system (rdbms) that consist of: a) all available substances ( . ), b) all generic names of medications available in our country for each substance, c) incompatibilities ( . cases) and d) interactions with other substances ( . cases). the following figure shows the structure of the rdbms. y ta~ortato~ [ c~rs using the stored parameters for each patient the dose and the rate of administration of selected substances will be possible to calculate. the continuous monitoring of the treatment for each patient supports the medical staff to make the necessary changes of the prescriptions. the application is currently developing in wireless pen based computer systems which place patients at the centre of "islands of information" located throughout icu. in conclusion this dss is a powerful and useful tool for icu staff because it provides without additionai work to the routine of daily practice, the currently available information for each order concerning drug interaction and incompatibilities as well as treatment monitoring is to obsea~ among critically ill pfdieats, stdjdivided following the diagn~s at the adn~ssio~ the diffmeax:es in the ~ and oxyplx~efic l~mmems bawe~ strvwors [s] and non sumvors ins] and to test the pc~'bih'ty to have soar survival criteria, as earliest as tx~able. method~ :we made a ~ study on consexa~e ~ilically ill paliffas, subdivided in series following the diastases at the admission: medical pafiea~ ( s and ns), surgical patients ( s and ns), a~d poliwauntas ( s and ns). follow up was done at d,.ays from the admission in ice. all the patienls were ramitored with a ~ c~eter and laeno:lymmi. "c and o .x.xyphorefic txuamaers va:~e couected at fin~es (t): at fiae ~draission (t ), at x~ars from t (t ), at (f ), (y ), (t ), % (t ) and horus from t cf ). in~,h ~ies, for ~y ~ a all the lin'~ n~an and sandaid d~viation was ~ tx~h for s and for ns. th~ betw~ s and ns tl~ roeaas of ~h porarneter ~e ccmpared tt~ng t-lest and p < . w~ considered ska~ significant in each series in the t wheae the mast significative diffemx:as ~goeamd bet~en s and ns, we made a txedictive criterion, asamting as predictive indices for stnvival the i:r values, higher or lower than flae treans of the ~rar~ers of au flae patients, axx)rdhlg to those ones t~iatistically diff~'e~ betw~m s and ns. fhmlly xse co:weatxt onaong the series the nrametees of the st~rs with the analysis of variance, to daserve the lxjsable differealt irea~ of sty hflices, following the diagn~s of admission: :nedkal, angical patient or poll~tam results: we c~ld not find ~ predictive criterion for politraonaas, perhaps ixx:ause of the few ntanber of l~fients. for high ri~ saw~cal patieras the following criterion at t has a sensitivi .ly of ~ ,and a ~ecificity of . %: sv > . nffmin/n~, map> mmhg, pmap< nmalqg cvp<i nunhg will nmah& lvswi> g m/m , sxo > ~ do > mlhnin/m , o er< %. for lx~dical l~tienls at t the following criteric~a has a ser~tivi.ty of % and a ~zificity of . ~ cvp< . mn~g, sao > %, s,g) > ~ vo i< ml/nfin/m , o er< %, shunt< % survlvops' data of the series ~ signitic~atly differenl~ both for the t~mody~nic a~ for fl~e ox rphomfic lxlmn~s; moreover we ~ that the vatt~ of hemodynamic mad ox.~ho~tic indices were higher in politrautms. conclus'ions: acx~ording to the fftffe~mt patho!o~es, the ~ rnelabo~c needs are diffeten~ so that it is juslified to mash ~ the~alceutic goals, following the type oflmthology. hen~ we foru~d for high ~k mrgical pmka~ and for medical patier~s assme, ff mllslied, a good prognosis while, if n [ ntljsfled~ the plinsliclioil ofdl~tth is no[ g(ioct finally, ab~ high iis~ supgical palieaats, according to what other atmhors say, txatws sh ~'n~ers ' therapeutic goalsvvould seem inadeqt~te, bec~jse they need a gear physiologic and themtx~ic elth~ in rdation to the rretabolic needs. figure ) . thus, the smaller european nations had a greater participation than ~e larger ones, with the exception of norway. a similar result was evidenced for contributions to intensive care medicine (figure ). these findings can be explained by different submission policies and language banners. however, there was no significant correlation with the gross national product of each country. conclusion: we conclude that the smaller european countries generally contribute more to international intensive care journals than the larger ones. objectives: to evaluate the agreement between a new and three old methods measuring ctp and to assess their reproducibility. methods: we studied patients ventilated with a siemens c respirator. we measured ctp by dividing the tidal volume with the increase in airway pressure (paw), either with the respirator setting used (ca) or with a fixed setting (cf). by modifing the inspiratory time (ti) without changing inspiratory flow, we were able to deliver two series of inflations ( , ,... ml) before and after curarisation of the patient. the same volumes were also inflated in paralysed patients with a super syringe. at the end of each inflation a plateau of sec was performed and paw was recorded. the above three sets of pressure-volume (pv) points were used to reconstruct the corresponding pv-curves (( , c , c the new method for ctp measurement without a super-syringe had the best reproducibility in paralysed patients and gave similar results without curarisation in the majority of them. however, agreement between the methods tested was unacceptable for clinical purposes. further investigation is required in order to improve the accuracy of ctp measurement in icu patients. m kunert, r.sorgenicht, l.scheuble, k.emmerich, h.g ker med.clinic b (dept.of cardiology) i heart center of wuppertal/university witten-herdecke,germany objective to determine the accuracy of activated partial thromboplastin time (apl-l) and activated clotting time (act) studies when samples are drawn through heparinized central venous catheters (cvc). methods a total sample of paired act/p't-/" values was analysed in patients ( m., f., + y.) for monitoring heparin therapy.all patients had a cvc (certofix trio,braun,frg) in the internal jugular vein receiving a continous infusion of . u heparin via the central catheter.act (hr-act, hemotec,usa) and ap'i-f (neothromtin, behring,frg) samples were drawn from the cvc using the double syringe technique (removing and discarding ml blood before drawing the sample). these blood samples were compared to act/ap'cf blood samples obtained by venipuncture (v.fem.) at the same time, act values were analysed directly in the intensive care unit (icu),api-i samples were measured in the hospital laboratory within minutes. results ac-i -~ pi-f~ cact/~pi r = , ) cvc samples + + . v.femoralis samples " + + p-value n.s. n.s. conclusion there is no difference in heparin anticoagulation studies drawn from heparinized central venous catheters compared to those obtained by femoral venipuncture,withdrawing ml blood prior to obtaining the blood specimen is a safe way for eliminating heparin contamination.not only the aptt test but also the act test is a useful method for heparin anticoagulation assessment in the icu. objectives: evaluation of the delicate balance between filter-coagulation and patient-hemorrhage using heparin as anticoagulant in continuous renal replacement procedures. methods: from january through august , we studied filter surviva[ and hemorrhagic complications during filter periods in critically d[ patients, treated with continuous arterio-venous hemo(dia)filtration, with special emphasis on the heparin dose, concurrent use of coumarins, systemic activated partial thromboplastin tirne(aptr), platelet count, mean arterial bloodpressure and the type of filter used. results: filters ( %) were disconnected because of coagulation. mean survival of multiflow an filters was twofold shorter compared to survival of fh gambm filters. a total of hemorrhagic complications occurred of which three patients died at aptt values of respectively , and seconds. after adjustment for mean arterial bloodpressure, platelet count and the type of the filter, the risk for filter-coagulation decreased % (relative risk . , %c . - . ) for each ten seconds increase in aptt. the risk for patient-hemorrhage increased % (relative risk . , %ci . - . ) at an aptt-increase of ten seconds. the occurrence of filter-coagulation and patienthemorrhage was not correlated with the administered dose of heparin. concurrent use of cournarines had a positive effect on filter-survival, without increasing the overall incidence rate of patient-hemorrhage. conclusions: the systemic apt]" is a good predictor of the risk for filtercoagulation and patient-hemorrhage. heparine therapy seems optimal at an aptt between and seconds, although one should realize that fatal hemorrhagic complications still can occur. objectives: the alterations in vascular tone which are primarily regulated by adreno-sympathetic tone(ast) are compensatory responses in hemorrhagic patients. this study was designed to evaluate the correlation between vascular tone and ast in patients with hemorrhage, methods: the vascular tone was expressed by volume elastic modulus (ev) that is defined as; ev = ap/(av/v) (ap; the arterial pulse pressure, av/v; the volume change ratio). ev was measured using a non-invasive transmittance infrared photoelectric plethysmography (tipp) and a volume oscillometric sphygmomanometer . we prospectively studied patients with hemorrhage. the initial ev measurement was performed on arrival and repeated for a hours duration. as a parameters of ast, serum concentrations of adrenalin (ad), noradrenalin (nor), plasma renin activity(pra) were measured simultaneously. we analyzed the correlation of ev and conventional parameters to ast by multivariate statistical analysis. results: ev values at transmural pressure mmhg on admission and hours later were respectively . + . mmhg, . +_ . mmhg (mean + sd). systolic pressure(pas) and serum hormones on arrival and hours later were respectively, pas; . _+ . , + . mmhg, ad; . _+ . , . _+ . ng/ml, nor; . _+ . , . + . ng/ml, pra; . _+ . , . _+ . ng/ml/hr. the ev values correlated significantly with ad (r= . , p= . , n= ), nor (r= . , p= . , n= ), pra (r= . , p= . , n= ). by multivariate statistical analysis, ev correlated more significantly with ad and nor and pra (p= . ) than the conventional parameters such as pas, heart rate and pulse pressure. conclusions: the alterations of ev correlates closely with ast. the compensatory mechanism in hemorrhagic patients can be detected noninvasively by ev monitoring. obiectives and method: autologous oxygenator blood was processed at the end of cardiopulmonary bypass (cpb) by either hemofiltration (hf , , m , fresenius) or by cell washing with a onntinous autologous transfusion system (cats, fresenius). prospectively the blood of patients for each group was processed and then retransfused intravenously to the patient. besides, volume and time requirements, standard hematologic chemistry, coagulation and complement activation were measured. results (mean values for oxygenator blood at the end of cpb, and results of concentrate after processing by filtration or washing): both processing techniques show excellent hemoconcentration of the diluted cpb blood with a good transfusion effect for the patient. filtration retains all plasma proteins and large molecular weight plasma bound waste products. in contrast, cell washing with cats significantly depletes plasma proteins and waste products. the newely developped cats machine gives eonsisinnt laboratory result in a fully automatic continuous processing mode. in conclusion, both filtration and washing are effective for processing cpb blood. filtra tion yields a highly concentrated whole blood, whereas cats washing produces a high quality autologous erythrocyte concentrate. soluble fibrin has during the last years gained interest as a marker for the activation of the coagulation in connection with various clinical conditions, e.g. disseminated intravascular coagulation, deep venous thrombosis and myocardial infarction. elevated levels of soluble fibrin in plasma can be detected by the chromogenic assay coaset fibrin monomer, relying on the ability of fibrin to enhance the tpa-catalyzed conversion of plasminogen to ,plasmin. using this test, it has been shown that the level of soluble fibrin can be correlated to severeness of illness in critically ill intensive care unit patients. a revision of the coaset fibrin monomer kit has now been made and the new product, coatest soluble fibrin, is considerably more convenient to handle and gives higher resolution at low fibrin levels. the test is performed by the addition of a buffer dilution of the plasma sample to a microstrip well containing the colyophilized mixture of tpa, plasminogen and the plasmin specific cbromogenic substrate s- . the reaction is allowed to proceed at,. room temperature for minutes before discontinuation. the absorbance at nm, measured in a microplate reader, is proportional to the content of soluble fibrin in the sample. the assay is carefully standardized and calibration curves are provided in the kit. the convenient and rapid assay procedure makes the coatest soluble fibrin test well suited for single test analysis in acute situations. objectives : blood coagulation abnormalities have been reported in the systemic blood of patients with cerebral lesions. the physiopathology of such events is not yet completely understood. we compare the coagulation profile of blood from the right jugular bulb with systemic blood of patients with head injury. methods: we studied patients, who were admitted to our neurosurgical intensive care unit between january and march with head injury and no other associated pathology (age - yrs), a glasgow coma score <= g, no abnormality in baseline coagulation profile and no history of coagulopaties. the patients did not undergo angiography. a one-way gauge certofix catheter was inserted through the right internal jugular vein up to the jugular bulb. an identical catheter was inserted through a subclavian vein. blood was sampled from either catheter (a=atrial; j=jugular) - hours after trauma (t ) and t hours later (t the inddence dpontolx'rative thmmhi~e and haumord~gic complieatiom were assessed in padents treated with indobefen, heparin calcine caeca), low mollecolar weight heparin (lmwh) (f.nosheparin) and undergoing hemodiludun, blood predeposhing, intra mad postoperative blood saving. ]'he indolmfon tempota~.norks platelet aggregation through ,,elective inhibition of the cyclatygenasis and thus atacbldonicadd( ).tbe n'mimum effect occurs after hours from the fast administration and is still present after hours. ~- patients, mean age --- yrs., weight --- kg were studied. ( . %) were male and ( . %) female. onderwent hip prosthesis ( previously plate and screw removal) hip revim'un ( stem, cop and stem + cop), tutal knee prosthesis, in the st anaesthesidogy depl from - to - - . as for antithromboembolic ptephylam, apart from hemodihitiun pts were with treated indobufen ndo), with heparin ealdum caeca) and with low mo!lecular weight hepam (lwr, ). as the slightest clinical and/or imtmmental suspidon of deep vein thrombosis (dv'i') or polmonary umbolism(pe), a phlebogram or sdndgram were respectively carried out. -the inddence of homologom transhisiom was significandy lower (p= . l) in the padeats treated with indobufen ( . ) compared .'ith heca ( . %). the con~gency table shows statistical signifleance for the use of heca in patients with vein deficiency in the lower limbs, past dvr and/or pe, coronary heart disease (cdh'), while there is no correlation for renal, cardiac or liver defidency, obesity, systemic hypertemion, atrhythmy, diabetes, chronic bronchitis and rheumatoid arthritis. by comparing the postoperative cumplications with the risk factors, there ks a highly significant correlation (p= . l) between cdh and thrombotic and humord~agic complieatiom (pe, death, he~atoma, die use of hum_ologous blood). thee data show that hep~in, preferred in patients with c'dh, roost likely for leagal-tuedical reasons, did not have the de~'ed effect. conclusions -the stastisfical aar~ais shows ~nifieanfly different efflea~ (pro . ) between the therapies (see table) : it can be seen that in patients undergoing autotramfusiun and hemedihidon, indobufen produo~ a lower incidence of haemotrhagic complieatiens compared to heca and lmwh and is more effective in the prevention d ~c complications at clinical e~idence. the duration of i~toperadve hospital stay is signi~cantlylonger for patients transfused with homologous red ceils and treated with hec, .a ( . -+ . days) and lmwh ( . +- a days) compared with indo(ll. _+ a days). one of the main causes for postoperative complications in major orthopaedic surgery is postopemtive bleeding with local effects in the operation site (hematomata, pain and delayed mobilization) and/or systemic and subsequent cardiodrculamry repercussions that are sometimes severe. the aim of this study is to assess the possibility to apply a new system of monitoring, control and saving postopemtive blood loss from the drainage. the bt recovery dideco (marandola, modena-italy) ~ used since it is the only apparatus capable of doing this. the apparatus consists of a pressure transducer, adjustable from - a + mmhg, which activates a peristaltic pump connected m drainage robes. the bt recovery display shows hourly bleeding in the first hours, total bleeding, time passed since the start of monito~g and subsequent salvage and the aspimtioo pressure on the drainage robes; the latter is inserted at - mmhg and then modified according to bleeding/minute, g bt recovery also has an alarm that sounds automatically if.' blood loss is more than ml/hour; air is in the circuit; the batteries are running low. materials and methods: pts were studied ( m and ~), aged . -+ .lyears, basal hemoglobin . -+ (range . - . )g/all, treated from st january, to mst december, in the st service of anesthesia and intensive care unit of our hospital. the patients underwent the following surgical treatment: total hip revision ( pts), cup revision (~ipts), stem revision ( pts), total knee revision ( pts). the average dumtion of the operations was -+ min. intranpemtive monitoring and blood salvage was applied to all patients. genera! anesthesia was used on pts. and integrated (epidural analgesia + light general) on the remaining t . anttthromboembolic prophylaxis consisted of external pressure bandage, isovolemic hemodilution with iodobufen in ( . %)pts., calalc heparin in ( . %)pts., low molecular weight heparin in ( . %)pts.; pt did not give a predepoalt of blood, gave unit, pts units, pts units, pts units. the data obtained was statistically analysed using contingency tables and anova. results: average intmop salvage was -+ ml, average postop salvage was -+ mi the average intra+postop +- ml. average postop loss was -+ ml. the global incidence of postop complications was: h~natomata . %, dvt . %, pulmonary thromboembolism , , myocardiac ischemia . %, acute myocardic infarction . %, respiratory deflciecy . %, arrhythmia %, cystitis . % there were nn complications in . % of pts. postop bleeding over ml in under minutes (with bleeding alarm activation) occurred in pts ( . %). this sta~tically correlates only with the type of operation performed (more frequently in total hip revision p= . ) and with a significant decrease (p~ . ) in the pruthrombic activity detected about hours after the operation. this bleeding, also made the alarm sound, calling the attention of staff who could act accordingly, by making the drainage pressure positive and incre~sthg the tension of the external pressure bandage. conclusions postop monitoring, control and blood loss salvage combined with predepoalting and intmop salvage has enabled allogenic transfusions in % of cases to be avoided in operations with high postop blood loss like hip or knee revision. the usefulness of the system can be seen by the fact that in the patients with so much bleeding to set off the alarm, there was no significant difference in the incidence of allotransfusions and complications. references )borghi b., bassi a., de simone n., laguardia am., fonnaro g. an injury of the brain may result in various disorders of hemostasis caused by the release of • into the circulation through a damaged blood-brain bar tier. disseminated intravascular coagulation(dic) is one of these disorders. it is a freguent but relatively rare ly diagnosed complication of subaraohnoidal haemorrhage. the aim of this study was to evaluate some parameters of both blood coagulation and fibrynolisis in patients with sah.in addition one wanted to find out wh~ther potential changes correlated with the pa• condition in the acute phase of sah and whether they influenced the course of this disease. patients with sah were studied. in of them sah was due to closed eraniocerebral injury and in the rema ining resulted from vascular malformation. the following parameters were evaluated:the prothrombine time,the activated partial thromboplastin time, the thrombine time,level of factor v,fibrinogen degrada tion products and fibrin monomers. the results let us show the presence of oic in patients with closed craniocerebral injury and in with vas. cular malformation despite the lack of clinical symptoms the tests in posttraumatic patients and in patients from second group showed incomplete dic.on admission patients with such changes in measured parameters were in poor condition.the course of the disease and the effe cts of treatment were also worse in these patients. the results showed ihal in patients with sah complex disorders of both coagulation and fibrynolisis occur, and they depend on clinical condition of the patient. they also influence the course of the disease. methods : charts of all patients admitted with d.i.c. over a ten year period ( - ) were reviewed. diagnosis of dic was based on the association of fibrinogen < g/ -platelets < / -fpd > ~tg/ml in the hours of the admission. results : patients -mean age + y -saps +_ -gestanional age _+ weeks -the two first conditions associated with d.i.c. were placental abruption ( %) and preeclampsia or eclampsia ( , %). bleeding episode was present in pts ( %) and surgical treatment has always been necessary. pts ( %) were given packed red ceils ( + u) and fresh frozen plasma ( + u). patients were given platelets packs. heparin was never administered. pts required mechanical ventilation and two patients hemodialysis. all the patients survived. correction of prothrombin time (p.t.) and fibrinogen (f) was quick (p.t. at t h ~ % -f at t h , + , g/i). but platelets count remained low (plat. at t h + / ) -no difference was observed in patients who received platelets. conclusion : prognosis of critically ill o.p. is good. blood loss is the main complication. correction of hypovolemia and anemia with concomitant surgical treatment are essential. the administration of coagulation factors or platelets is still under discussion. objectives: to evaluate the effects of antithrombin iii i at-iii) and a protease inhibitor, gabexate mesilate foy), on the coagulation and fibrinolysis in disseminated intravascular coagulation (dic). methods: after the approval of our institution and consent from patient's family, patients with a dic score ( , japan) more than points (dic or having a risk for dic) entered this study. they were randomly divided into two groups, foy (i- mg/kg/h for days or more) treated group and no foy group, each of patients. platelet count (plt), fibrinogen (fen), at-iii fibrin degradation product (fdp), d-dimer (do), fibrin monomer (fm), thrombin-antithrombin complex (tat), plasmin-plasmin inhibitor complex (pic), and prothrombin time ratio (ptr) were measured before the start of treatment (at admission) and i, , and days after the admission. at-iii at units for days was administered if the at-iii at admission was less than %. finally the patients were divided into four groups: group a, foy (+) and the at-iii ~ %; group b, foy (+) and the at-iii < %" group c, foy (-) and the at-iii %; group d, foy (~) anffthe at-iii < %, each of patients, to match the patients for backsrounds. all parameters, dic score and survival rate in a month following treatment were compared among the four groups. results: the at-iii and plt from day to were significantly higher in groups a and c than in groups b and d. the fdp, dd, tat, and pic after treatment decreased significantly from the baselines in groups a and c but not in groups b and d. the fgn and fm were not significantly different among the four groups. the ptr decreased in groups c and d but increased in group b. the dic score decreased significantly in groups a and c than in groups b and d. survival rates were %, %, % and % in groups a, b, c and d, respectively, although not significantly different. conclusions: in patients with dic or a risk for dic, foy had no expected effects but at-iii had suppressive effects on the coagulation and fibrinolysis mechanisms. a prognostic factor ? carbon monoxyde intoxication is a classical complication of inhalation injury. carbon monoxyda is also physiologically produced during the heme metabolism: heme is conversed to bi]irubin by the hemeoxygenase which is an intracellular stress protein. icu patients (pts) were studied prospectively for apache ii score and carboxyhemnglobin (hbco) arterial level to assess if hbco level could be correlated with the severity of the pts. objective: to evaluate a new technique of non-surgical tracheotomy. patients: adults, mean age years and children, mean age months ( me.- yrs). method: through a needle inserted in the trachea, a guide wire is retmgradely pushed out of the mouth and attached to a special device formed by a flexible plastic cone with pointed metal tip joined to an armoured tracheal cannula. this device is then pulled back through the oral cavity, larynx and trachea, and outwards across the neck wall by applying traction on the wire with one hand and counterpressure on the neck wall with the fingers of the operator's other hand. when the cone and / of the eannula have emerged, the cannula is cut off from the cone, straightened perpendicular to the skin, rotated and advanced caudally to its final position. results: endoscopic control facilitates and improves the safety of all manoeuvres. the pointed cone easily pierces the tissues, and the cannula is extracted without difficulty since it has the same outer diameter as the cone. tissue adherence around the cannula is absolute thus preventing local inflammation. the time in apnea required for dilation and cannula placement does not exceed see., and it is well tolerated because within safety limits in patients hyperventilated with oxygen. only one case of bleeding occured in a patient on dialysis with severe coagulopathy. autoptic findings in subjects who died due to progression of primary disease showed a very regular stoma with an almost complete lack of hematic and flogistie infiltration in recent tracheotomies. .conclusions: translaryngeal tracheotomy (tlt), by virtue of its greater inherent safety and lower tissue trauma than percutaneous techniques, can also be carded out in infants and children, a severe test bench for any tracbeotomy technique. further specific indications are recently stemotomized patients, since tlt is associated with a low rate of infection, and short term tracheotomies after laryngeal surgery, to prevent obstructive complications. references: fantoni a., translaryngeal tracheotomy, apice, ed. gullo, trieste, , . background: inhalation of no has been shown to reverse hypoxic pulmonary vasoconstriction , to reduce pulmonary pressure in pulmonary hypertension of different origin and to improve gas exchange. in putmoflary embolism, pulmonary hypertension is caused by mechanical vascutar obstruction and by reactive vasoconstriction. the effects of inhaled no in putmonary embofism has been partiatly studied' the purpose of this study was to investigate and determine the effects of no inhalation on pulmonary hemodinamica and gas exchange in a hypoxic canine model of pulmonary embolism. methods: two groups of adult mongrel dogs were studied: group (control} dogs and group (no inhaled) dogs. both groups were anestesized with tiopental, mechanically normoventilated with an hypoxjc mixture of and n~ (f[q , ) and instrumented (swang-ganz catheter, femoral artery catheter) pulmonary embolism (pe) was induced by fisher's method s. no inhalation ( ppm) in group was started rain. pdor to pe and kept constant throughout the experiment. no inhaled concentration was analyzecf by chemiluminiscence technique. pulmonary artery pressure (pap), central venous pressure and sistemic arterial pressure were continuosly recorded. cardiac output, artedat po~ (pan ) and mixed venous po~ were measured in both groups under hypo)dr conditions, before pe and , , and rain. after pe. pulmonary vascular resistance (pvr) and gas exchange (pao fio:~ ratio), were calculate using standard formulas. data were process and analyzed with non pararnetdc test, and reported as mean -so and statistical significance was considered if p < , . : no produced an increase in arterial oxigenation (pao /fio~ ratio) and reduced pap before pe induction in group . after pe we found no significant difference with .respect to the time eour.se of pap, pvr and gas exchange between beth groups throughout the experiment. probably, the severe mechanical obstruction produced in pulmonary embolism masked the small effects of no inhaled. obiectives: blood volume measurement would be useful in critically ill patient management if it were easy to perform. this is not the ease and current methods are based on radiolabelled red cell dilution. inhalation and uptake of a known mass of carbon monoxide (co) gas and measurement of earboxyhaemoglobin increase can give results accurate enough for clinical use. this requires a rebreathing system providing oxygenation and carbon dioxide removal, yet complete retention of all carbon monoxide administer&l, and so most authors hand ventilate with a bag and waters soda-lime canister, adding oxygen as necessary. we aim to popularise this method by; i)design of an automatic co administration system driven by the itu ventilator and ii)writing of software for a portable computer to perform all necessary calculations method: we show the computer is use estimating the co dose required and later estimating the blood volume. we also show the new gas administration system. this is a fully closed circle attached to a "bag in bottle", driven by the ventilator. the novel feature is the mechanism by winch driving gas (set to % ) spills automatically into the circle, balancing o uptake by the patient, yet allowing no co loss. conclusions: this equipment is easy to use, reduces human error and allows optimum ventilator settings to remain. the operator merely administers the volume of co determined by the computer and takes blood on two occasions. carboxyhaemoglobin measurement is easy to perform, thus there is a cost saving also. with our modifications use of this technique may potentially become more widespread, the video demonstrates the method in use in our itu. - ( %) underwent conventional surgical therapeutics. " ( %) with resection of tracheal stenosis with end-to-end anastomosis(rts). i ( %) with broncoscopic dilatation. one patient died and the others still have stable patency(sp) without continued treatment. - ( , %) have received endoscopic laser ablation with or without calibration tubes. of them ( , %) are receiving continued endotracheal treatment until now. ( , %) have sp wihout continued treatment. -i ( , %) endoscopic laser therapeutic case turned to rts and is having sp. conclusion: conventional surgical aproach has been progressively replaced in our hospital by endoscopic laser ablation and silicone calibration tubes. this study suggests that these technics are effective and could be the elective treatment for iatrogenic stenosis. obiectives: hemorrhagic disorders due to thrombocytopenia and thrombocyiopathia remain one of the most serious complications during long-term extracorporeal membrane oxygenation (ecmo) in patients with severe acute respiratory distress ~drome (ards). in the presented study, nitric oxide (no), kwown as a potent endogenous platelet antiadhesive, disaggregating and antiaggregating compound, was evaluated for its possible antagonistic effect on platelet trapping when added to the gas compartment of membrane oxygenators (mo). meti~ods: two parallel separated extracorporeal circuits, consisting of heparin bonded hollow fiber oxygenators (minimax, medtronic, carmeda eioactive surface), tubing systems, low pressure reservoirs, and roller pumps were prepared. for each measurement, a pair of circuits was simultaneously filled blood from the same volunteer. low-heparinized fresh warm blood was obtained from four healthy volunteers, who had no drugs for at least two weeks. the gas inlets of both oxygenators received dry gas ( % oxxygen, % carbon dioxide, % nitrogen); gaseous no ( ppm) was added to the gas of one of the oxygenators (no-mo), whereas the other one (mo) was used as control. after minutes no gas was switched off, so that the no-mo received no more no, and no was added to the gas inlet of the membrane, which had no no before_ to assure iutracircnit volume stability, drawn blood for measurements was replaced with saline, and platelet counts were corrected for dilution by hemoglobin values. the mean of four platelet counts (coulter counter) of each timepoint (start, , , , , , , , and minutes) was used for statistical analysis (paired sample t-test). results: in the no-mo platelets remained at + , % (percentage of baseline value, mean -+ sd) until min. in contrast, platelets of the mo continuously decreased after start and were significantly lower after minutes ( , + , % vs _+ , %(p< . ); min. , -+ , %vs , _+ , %(p< . ); min. , _+ , % ( p < . ). after switching of no gas to the mo, further decrease of plateleta was stopped and platelets remained at , +_ , % until termination of circulation. platelets of the former no-mo decreased slightly after cessation of no gas to , _+ , %. conclusions: these data indicate that gaseous no significantly attenuates platelet trapping in hollow fiber oxygenators, when added to the gas compartment. this might be a new therapeutical approach for membrane oxygenator induced thrombocytopenia during long-term ecmd. objectives: nitric oxide (no) plays a pivotal role in regulation of vascular hemostasis. several studies elucidated the antiadhesive, antiaggregating, and disaggregating properties of endothelially synthesized no to platelets. additionally, agonist-induced no production in platelets by the l-arginine-no pathway was found as a negative feedback mechanism after platelet activation. although noplatelet interactions were intensively studied by several investigators, no data exist, about changes in platelet surface molecule expression in no-modulated platelets measured by flow cytometry using monoclonal antibodies (moabs). methods: p-selectin (alpha-granule-membrane protein, gmp- , cd p) and glycoproteiu (gp , lysosomal protein, cd ) are expressed only after platelet activation and degranulation. activation was quantified in thrombin ( . u/ml) and adp ( . ram) stimulated platelet rich plasma samples (prp). blood was obtained from healthy volunteers (n= ), who had no drugs for at least days. for evahiation of no-modulated activation, the spontaneously noreleasing compound sin-i ( . mm) ( -morpholino-syndonimin-hydrochlorid) was added in parallel prepared samples prior to the addition of agonist. platelet surface molecule expression was evaluated with moabs directed against cd a (gpilbliia, fibrinogen-receptor, phycoerythrin(pe)-conjugated), cd p (fitcconjugated), and cd (fitc). only cd a-positive signals were gated in sideangled light scatter, and assayed for activation marker expression (defined as percent of gated population). results: basal p-selectin expression was . + . %, and increased to . _+ . % after thrembin-activation, and to . + . % in adp-stimulated samples. addition of sin- attenuated p-selectin expression to . - - % in thrombin (p<. , two-tailed paired t-test), and . + . % (p<. ) in adpactivated platelets. basal gp expression was . _+ . % and increased to . + . % in thrombin, and to . _+ . % in adp-stimulated samples. with sin-l, gp expression decreased to _+ . % (p<. ) in thrombin, and . : . (p . ) in adp-stimulated samples. conclusions: these data implicate, that no leads to a significantly reduced activation of surface molecule expression in thrombin and adp-stimulated platelets. in addition, flow cytometry might be a useful tool for studying modulation of platelet activation by no or no-releasing compounds. introduction: acute cadmium poisoning is very rare. on initial presentation may mimic metal-fume fever, but acute inhalation cadmium toxicity may produce fatal chemical pneumonitis. case report: we present a case of acute fatal respiratory failure secondary to cadmium-fume irthalation. a year old patient was trasferred from another hospital with acute respiratory failure presumably due to pneumonia. the last days before he had had commom cold symptoms. he had been cutting with a welder during one hour without any respiratory protective measure. three hours after exposure he developed progressive dispnea and was admitted to hospital. with presumtive diagnosis of respiratory infection, antibiotics were begun, however be failed to improve. all microbiological studies were negative. chest x-ray showed bilateral diffuse infiltrates. on seventh day he needed intubation and mechanical ventilation and on th he was admitted to our icu. antibiotics were stopped and new microbiological studies were performed including brochoalveolar lavage and virologic studies. all results were negative. he developed progressive hipoxemia and hipercapmia and finally, multiorganic disfunction syndrome. he died days after exposure. the metal he had been working with was a % cadmium alleation. blood cadmilam concentration days after exposure was . mcg cd/g cr, and urine cadmium concentration was . mcg/l. on postmortem examination, tissue cadmium concentrations were: blood ng/ml, liver ng/g, kidney ng/g and lung ng/g. these values confirm that cadmium was the cause of the fatal respiratory illness in this patient. conclusion: this case evidences the considerable hazard of acute poisoning after inhalation of eadmium-fume and stresses the need of appropiated safety measures against metal-fume poisoning. aim : lactic acidosis is considered the hallmark of cyanide poisonirig. however, the relationship between plasma lactate and blood cyanide levels has not been determined. the aim of this study was to determine the significance of plasma lactate concentration (plc) during the course of cyanide poisonings. methods : the patients were included according to the clinical suspicion of pure cyanide poisoning at the time of presentation. fire victims were excluded. serial blood samples were collected before and after intravenous hydroxocobalamin (hoco). blood cyanide concentration (bcc) was measured colorimetrically. plc was measured enzymatically. results : patients were studied. on admission, plc ranged from . to mmol/l, and bcc from . to gmol/l. mean systolic blood pressure was • mm hg, mean arterial ph . • . , mean anion gap was . + . mmol/l and mean pao . • . kpa. three patients died. before antidotal treatment, there was a significant correlation between plc and arterial ph (p = . ), anion gap (p = . ) and bcc (p = . ) but not with heart rate, pao , paco and blood glucose, or blood pressure. during the whole course of the poisoning, a plc _> retool/ was a sensitive and specific indicator of a blood cyanide concentration > ~tmol/ . sustained catecholamine administration reduces the correlation coefficient. conclusion : baseline measurement of plc allows assessment of severity of acute cyanide poisoning. thereafter, plc may be used to assess the adequacy of antidotal treatment, more especially in patients not requiring sustained infusion of catecholamines. aim: the aim of this case report was [o study the correlation between the plasma lactate levels and several clinical, biological, and toxicological parameters serially measured during the course of a cyanide poisoning treated with a high dose of hydroxocobalamin. a -year-old male ingested potassium cyanide leading to cardiac arrest. cpr was performed prior to hospital arrival where the patient received g hydroxocobalamin. sbp rapidly returned to normal allowing withdrawal of epinephrine. the patient remained comatose and died from brain injury days after the ingestion. methods plasma lactate and blood cyanide levels were measured serially. blood cyanide levels were measured using a colorimetric method.~ plasma lactate levels were measured using an enzymatic method. for correlation spearman rank correlation test was used. results. initial plasma lactate and blood cyanide levels were mmol/l and gmol/l, respectively. there was no overall correlation between sbp and either blood cyanide or plasma lactate levels. similarly, there was no overall correlation between arterialvenous oxygen saturation difference with either blood cyanide or plasma lactate levels. in contrast there was a strong correlation between blood cyanide and plasma lactate levels (r= . , p< . ). the time-course of the blood cyanide concentrations was described by a mono-exponentiai decay (r = . ) with a blood half-life of . h. similarly, the time-course of plasma lactate levels was described by a mono-exponential decay (r = . ) with a blood half-life of . h. discussion. in this case of acute human poisoning, sbp was a much poorer indicator of continuing cyanide effect both before and after antidotal treatment, than was lactate production. this suggests a potential clinical role for following serial plasma lactate levels as a marker of the evolution of cyanide toxicity. aim : cyanide (cn) poisoning in fire victims is frequent and rapidly fatal. in a prospective study we tried to assess the clinical tolerance of a high dose of hydroxocobalamin (hoco) administered at the scene of the fire in fire victims suspected of cn poisoning. methods : inclusion criteria : soot in mouth or sputum ~ any degree of neurological impairment. exclusion criteria : children, pregnant women, burns of total surface body area > %, multiple trauma. protocol desigrl following examination and the collection of a blood sample in dry heparin, a g dose of hoco ( g in case of cardiovascular collapse) was administered intravenously over min. the systolic blood pressure was monitored before and after the administration of hoco, and one hour later. results : there were females and males. the mean blood cn concentration was • pmol/ . the mean blood carbon monoxide was . • . mmol/ . nineteen fire victims eventually died. among the non-cn-intoxicated patients (blood cn < ~mol/ ), there was no significant change in arterial blood pressure. in the cn-intoxicated patients (blood cn > gmol/ ) a significant increase in blood pressure was observed both immediately (p < . ) and hour later (p < . ) after the admistration of hoco. no allergic reactions were observed. conclusions : in fire victims with cyanide poisoning, the administration of a high dose of hydroxocobalamin was associated with an improvement in systolic blood pressure. hydroxocobalamin is well tolerated in fire victims without cn poisoning. objectives: tricyclic antidepressant (tca) overdose can lead to serious complications including cardiac arrhythmias [ ] . because of the known risk of early deterioration and the implication for management, emergent evaluation is essential. we determined the diagnostic usefulness of the electrocardiogram (ecg) in tca poisoning. methods: retrospective study of all patients with tca intoxication (pos. ,toxicology screening in urine and/or pos. history) in a -beduniversity hospital from through . the severity was graded with mild= no symptoms or agitation; medium= disorientation, somnolence, tachycardia, or convulsions; and sever~ coma, significant arrhythmias or death. we analysed the first ecg after admission with a special emphasis on qrs-and qtc-intervals and the terminal ms frontal plane qrs-vector (tqrs), which, was reported to lie typically between + and * + + • the best correlation with severity grade was found with qrs-and qtc-duration (p= . ), the tca-dose (p= . ) and hf (p= . ); tqrs did not correlate. patients died ( . %). conclusion: qrs-and qtc-prolongation in the admission ecg, and the reported dose of ingested drugs are useful predictors for severity of poisoning due to tricyclic antidepressants. we did not find additional benefit in determining the terminal ms frontal plane qrs-vector. objectives: since treatment of amphetamine poisoning is usually symptomatic and often associated with a fatal outcome, a search for specific drugs to help the amphetamine-intoxicated victim is sorely needed. methods: we report a case of a suicidal ingestion of large amounts of the amphetamine-derivative , -methylenedioxy-ethamphetamine (mdea) and heroin (diacetylmorphine) and present the hypothesis that the two drugs produce opposing clinical effects. results: a year old caucasian male was admitted to the emergency ward because of acute-onset confusion. at presentation, he was agitated and showed increased muscular rigidity. he had taken tablets of "eve" (mdea, approx. g) and g of "smack" (heroin) by oral route approximately h before admission. because of rapidly progressive tachypnea and exhaustion, the patient was intubated and ventilated. the serum concentration of "eve" on admission was ng/ml (lethal range - ng/ml). trace amounts of cocaine and substantial amounts of heroin ( ngtml; mean value in heroin-related deaths: ng/ml) were also found in the serum. the patient was successfully weaned from the ventilator by day and recovered without persistent neurobehavioral disturbance. despite high serum levels of both drugs, the patient did not present with the classic signs and symptoms normally seen during intoxication with these drugs. amphetamines in general, and mdea in particular, have opposite clinical effects to heroin or diacetylmorphine. none of these were however present in the case presented despite the high ingested doses and the serum levels in the lethal range. conclusions: the fascinating fact that, apart from the respiratory depression, none of the clinical signs reported after massive overdose with these two drugs were present, might be attributed to the opposite pharmacological effects of mdea and heroin. we believe that the patient unwittingly saved his own life by the oral coingestion of both mdea and heroin. our clinical data raise an interesting point about the pharmacological treatment of acute poisoning with amphetaminederivatives. introduction: the acute attack of aip still carries a significant risk of mortality of around %. a succesful outcome depends on early diagnosis, removal of pricipitating factors and provision of intensive supportive therapy. objectives: twenty one patients ( females, male) with documented aip were seen over a -year period in the university hospital. patient was in clinical remission and were with the acute attack of aip, among them with respiratory paralysis were required artificial lung ventilation and -assistant ventilation with peee pathologic treatment during the attack was normosany, adenil, androgenes, glueosa, riboxin parenteral and enteral nutrition via nasogastric tube. symtomatic treatment -pethidine, propranoton, antibiotics, bronchoscopia. methods: intermittent phasmapheresis was performed on patients. the following measurements were peformed: level of porphobilinogen (pbg) in the wire and delta-aminolevulinic acid in the blood. hematological and routine chemical evaluations, hepatic, hemodynamic and respiratory function. results: after plasmapheresis the median pbg excretion (normal range - mkg per/ kgr creatinine) fill from mkg on admission . mkg, then on - day raise to mkg and then during treatment with normosong and prasmapheresis lowest level was . mgk. fatalities occured in two females during attacks with proforma cerebral involvement and patients attained clinical remission. conclusion: after therapy with plasmapheresis normosong we found that there was consistently reduce the urinary excretion of pbg and shortening the duration of the acute attack. objectives: pigs has been reported to present with a higher pulmonary arterial pressure (ppa) and stronger pulmonary vascular reactivity than many other species, including man. aim of the present study was to compare pulmonary vascular impedance (pvz) before and after embolisation in weight-matched adult dogs and minipigs. methods: we investigated pvz spectra in anaesthetized and ventilated (fio . ) minipigs and dogs. after baseline measurements the animals were embolised with autologous blood clots to reach a ppa above mmhg. results: flow ( and ppa matched pvz data (mean-+sem) are shown in the table. [zo = hz impedance (z; {dyn.sec_em- }); zl = first harmonic z; zc = characteristic z; z phase = first harmonic phase a@e {radians}; fmin = frequency of pvz the first m{n~mam; *, f p at least < . between dog and minipig, and before v~. after embolisation respectively]. before case report: a -yr-o]d woman affected by legs recurrent thmmbophlebitis, was admired in medmine department for tach.~pnea, chest pain, tachycardia and cyanosis. before starting two-dimensional transesophageal echocardiography (tee) to confirm the suspicion of pulmonary embolism, she suddenly had ventricular fibrillation. resuscitation and defibrillation were readily performed. when sinus rhythm was reinstituted she was in superficial coma with preserved corneal and light reflexes: right hemiplegia, poor perfusion and h~posphygrma of the left arm. tee showed dilation of rigth ventricle (rv), incomplete occlusion of pulmonary arter~ (pal at it~ hifurcation, severe tigth-to-left shunt through a patent foramen ovate, paradoxical embolism with incomplete occlusion of left subclavian artery mechanically ventilated with vt= ml, rr= /mm, fio =l, the patient had ph= . , pao = mmhg and paco = . systemic bp was / mmhg and hr= b/min with low dose epinephrine ( . g/kg/min) a thrombolytic infusion (rtpa: mg/ h) through a peripheral vein was started tee imaging and clinical status hours later were unmodified. a new rtpa infusion was performed through the pulmonary hole of a swan-ganz catheter with the tip close to the embolus. one hour later pa pressure decreased from / mmhg to / mmhg, etco increased from to mmhg and sao improved from % to % three days later the parietal, spontaneously breathing and with normalized tee scans of rv and pa, was transferred to rehabilitation service to perform physical therapy. conclusions: massive pulmonary embolism in a patient with patent foremen ovale, paradoxical embolism and refractory hypoxaemia was unaffected by systemic rtpa infusion, while intrapulmonary rtpa administration dramatically improved gas-exchange, hemodinamics and the general conditions of the patient. the presence of a large rigth-to-left _atrial shunt and the rapid rtpa metabolism could likely explain the effectiveness of its intrapulmonary administration in front of failure of systemic thrombolysis. introduction. cardiogenic shock during massive pulmonary embolism (blpe) is due to an acute increase of right ventricle (rv) afterload and possibly rv ischemia causing a failure of rv pump function. the rec~;mmended therapeutic strategies are: xoiume augmentation ~n ~rder m }ncrease rv pre-h~ad, adrenergic drugs to increase t'ontractillly and maybe coronary perfusion, fibrinolytic drugs to delermine clot lysis. there have been several reports of noradrenaline (na) as a useful drug in this setting for its sluing ~z, but also ~, properties. case report.an obese },ears old woman was transferred to our icu for tetanus. she was given the usual antibiotic and immunoglobuline therapy. l'wo thoracic epidural catheters were put in place at different levels and replenished with marcaine qid. a continous infusion of sedation (diazepam § was started together with mechanical ventilation. curarization ~,as given occasionally. fraxiparine . /die was used for prophylaxis of thrombotic disease, on day th at . a.m. she started to be hypoxic (sa %), tach ,tardic l l(i b/rain.), her blood pressure(rp) dropped frum norma~ values to r mm/hg, the central venous pressure (cvp) raised [rom lb to mm/hg and the end tidal co was mm/hg lower than one hour before. the physical examination of the chest revealed a clear bilateral ventilation and the chest x-ray was normal apart from an elevation of the :tiaphragm as compared to the previous. an e.c.g. showed sinus tachycardia, right bundle branch block and a possible inferior necrosis (which was already present on admission). a trans-thoracic echozardiography was performed which showed "an acute overload of the right centricle wilh remarkable dilatation. tricuspidal regurgitation ++. paradoxical movement of septum. small left ventricle with normal wall kinetics". the cardiac enzymes were later shown to be normal. an acute massive pulmonary embolization was assumed m be present.. a bolus of streptokinase x i(i u. was given fonowed by a continous infusion . two liters of colloids were also given in a sh~rt time, two hours later the patient was still deeply hypotensive, hypoxemic and anurir(bp / mm/hg, cvs mm/hg, spo %) despite a cominnus infusion of dobutamine fag/kg/min and adrenaline . ~tg/kg/min. at this stage a bolus of aoradrenaline ,g was given followed by a cnntinous infusion of . !*g/kg/min. an immediate improvement of the hemodynamics was noticed and one hour later the bp was / mmhg, the cvp mm/hg, the sao % and a brisk diuresis started. the hemodynamics kept stable and weaning from vasoactive drugs was achieved within two days. one month iater the patient was discharged home in good conditions.. con c i u sio n.ne administration may help to restore rv coronary flow and ;~ump function during mpe. aeute putmonary t~omboembo~sm [ffe) cou be mamfeslated with either respiratory or cardiovascular syndromes or both. the arm of the study was to establish leading respn'atory symptoms, frequency and form of the roendganographic (rig) changes as well as blood gas disturbance degree in acute pte with dommam respiratory disease appearance. the study includes retrospeotive analysis of i pte patients (pts), males (average age , yrs) and .q females (average age , yrs). they were admitted at university, olinie" with suspection ofpleuropnlmonary disease, including pte. final diagnosis of pte was based o~ evident risk factors in , % of the eases (deep venous thrombosis, surgery, trauma, imobilisation, malignancy ere), acceptable clinical, rtg, sdntigraphic and laboratory findings, as well as deep veins examination by dopple~-sonographie and radioisotopic -~enogmphy. respiratory symptoms appeared in all cases: sudden pleural pain ( %), dyspnea ( %), hemoptysis ( %), cough ( %) with association of two or more symptoms in %. chest xrays findings were abnormal in % with diaphragmal elevation ( , ~ lung opaeilies ( , %), atelectasis ( , %), plemal effusion ( , %), main pulmonary brancah asimetry ( , ~ oligemia ( %), heart shadow changes ( , %) and pulmonary arteries "cut off' ( , %). the association of two or more abnormalities was found in , % while normal chest x-rot was found in ~ of the cases. hypoxemia with pao < , kpa was found in , % followed with hypocapnia and respiratory alealosis in , % in , % of the gas exchage analysis were within normal limits. among cardiovascular symptoms short syn~cpa appeared in i , %, ecg changes-st q t type in "~ , %. results show high frequency of positive ~g findings in pte pts that is opposite to oppinion that chest x-ray in acute fie is the most ofran normal. leading symptoms are pleural pain and dyspnea, while hemoptysis were found in a half of the study group. blood gas changes were present in two thirds of the cases. kakkar, in his classic work ,clearly demonstrated the efficiency of low doses of heparin in prevention of deep vein thrombosis (lancet : , ) .after this first study the application of heparin prophylaxis became more and more diffused until to be considered a routine in many surgical departement.actually application of blood saving technique induces postoperative hemodilution effect. in that condition prophylaxis routinely applied seems a nonsense and can be at risk for postoperative hemorrhage. methods: to analize this problem we compared patients arrived in our intensive care unit (i.c.u.) in. : (group a) with arrived in : (group b) .every patient was operated for major abdominal surgery.in each one we considered the hemoglobin (hb) value,hematocrit(hct), and coagulation pattern (c.p.) at the arrive in i.c.u. and hours later. the patients was also divided in those receiving heparin prophylaxis (i) from not treated patients (ii) results:the application of blood saving technique clearly appears from the hb and hct level wich have a mean value of , +/- , (hb) and +/- (hct) in group a while in group b mean value are , -/- , (hb) and +/- (hct).patients of group a (ii) are the only one where a pathologycal c.p. with statistical significance has been demonstrated.in this goup we got four cases of evidence of venous thrombosis and one of pulmonary embolism.in patients of group b(i) we encontered the incidence of two cases of severe hemorrhage despite the absence of statistical significance in c.p.modifications. oxygen desaturation during broncho-alveolar lavage: role of oxygen saturation monitoring in prevention of acute respiratory insufficiency g. galluccio, b. valeri, s.batzella, m. di lazzaro*, servizio di endoscopia toracica, ospedale forlanini, rome, italy * servizio die anestesia a rianimazione, osp. forlanini the broncho-alveolar iavage is a diagnostic procedure employed in interstitial diseases of the lung. it requests the introduction through the working channel of a fiberoptic bronchoscope, after occlusion of a segmentary bronchus, of aliquots of saline solution at c, subsequently gently reaspired, in order to remove cells and proteins from elf (endoalveolar lining fluid), which is related to interstitial medium. bronchoalveolar lavage induces deep effects on pulmonary function: -lowering of the alveolar surface of exchange; -shunt effect, depending on the perfusion of non-ventilated districts; -increased pulmonary arterial pressure, due to hypoxic vasoconstriction; -decrease of lung compliance. in this report the authors present the result of oxygen saturation monitoring in a group of patients with interstitial lung disease, who underwent diagnostic broncho-alveolar lavage. in most patients with severe interstitial involvement, the lavage performed without supplement of oxygen induced a severe fall in the oxygen saturation during the late phase of the procedure. if supplementary oxygen was delivered during bronchoscopy, since its beginning, only slight modifications of the curve were detected. in patients without thickening of interstitium, in whom the lavage was performed in order to obtain material for bacterial or cytologic examination, no modification of oxygen saturation was observed in standard procedure. as conclusion the authors strongly reccomend monitoring oxygen saturation in patients with radiologic evidence of interstitial involvement also in patients with no evidence of dyspnoea. g. galluccio, b.valeri, s.batzella, m. di lazzaro*, servizio di endoscopia toracica, ospedale forlanini, rome, italy * servizio die anestesia a rianimazione, osp. forlanini the treatment of choice in patients with alveolar proteinosis consists of pulmonary lavage. this procedure requests the introduction, through the working channel of a fiberoptic bronchoscope, segment by segment, of aliquots of saline solution at c, subsequently gently reaspired, in order to remove the proteins deposited in the alveolar spaces. the method is very similar to that used in bronchoalveolar iavage, a diagnostic procedure used to obtain cells and substances from elf (endoalveolar lining fluid), which is related to interstitial medium. as known, bronchoalveolar lavage induces oxygen desaturation, because of shunt effect. understandably, one lung lavage has remarkably more deep effects on pulmonary function than bronchoalveolar lavage, for the amount of fluid introduced, the length of the procedure and the conditions of controlaterai lung. in this report the authors present the result of oxygen saturation monitoring in a patient who underwent pulmonary lavage for alveolar proteinosis. in the lavage performed without supplement of oxygen a severe fall in the oxygen saturation was observed during the late phase of the procedure. if supplementary oxygen was delivered during bronchoscopy, since its beginning, only slight modifications of the curve were detected. as conclusion the authors strongly reccomend the subministration of supplementary oxygen in pulmonary lavages, also in patients with excellent respiratory conditions. a. b. dublisky prof., m. r. isaakjan ass., v. a. zasukha, s. m. vinichuk prof., v. p. tserty ass. prof., chair of anaesthesiology, resuccitation and medicine of catastrophes, neurology of ukrainian state medical university, kiev, ukraine. objectives: detection of plasmophoresis's influence of results in treatment of ishemic insult. methods: we ve investigate patients with ishemic insult, treated with reverse plasmopheresis in complex treatment. after primary infusive therapy we took ml of patients' blood and separated it within min with rotation frequensy of /rain. after separation of erythrocytes from plasma, the latter has been returned to patients. we made - procedures during - days. hemoglobin, hematokrit, time of blood coagulation were determinated. the brain blood flow in internal carotid arteries, regional volum brain blood flow and total brain biood flow were evaluated with tetrapotar chest rheography and tetrapolar rheoencephalography. obtained date were comparised with control group after traditional treatment. results: it was found that after reverse plasmopheresis the hemoglobin and hematokrit levels decreased significantly in studied patients' plasma (from + . g/l to _+ . g/ and from + . % to _+ . % respectively). the time of blood coagulation by lee-white has increased by - . times (up to - rain). the level of brain blood flow has been increased significantly after reverse plasmopheresis in comparison with control group. the following tests of brain blood flow have been increased: a) the total volume brain blood flow from . + . ml/min to . _+ . ml/min (p < . ); b) the regional brain blood flow from . _+ . ml/min to . + . ml/min (p < . ); c) the brain blood flow in internal carotid arteries from . _+ . ml/min to . + . ml/min (p < . ). conclusions: the use of reverse plasmopheresis in complex treatment of patients with ishemic insult aiiows to improve rheological blood patterns, helps to increase volume brain blood flow. it results in quicer reparation of neurological functions. objectives: a prospective evaluation of the efficacy of continuous infusion of verapamil in reducing the incidence of postoperative atrial fibrillation after pulmonary surgery. methods: a total of consecutive patients, on verapamil, on placebo was included after lobectomy or pneumouectomy. a loading bolus of verapamil ( mg over minutes) was followed by a rapid loading infusion ( . mg/min) for minutes and finally a maintenance infusion ( . rag/rain) for hours. results: a mean plasma level of verapamil of ng/ml was obtained only after more than hours. atrial fibrillation occurred in five out of patients who tolerated the verapamil infusion, and in out of patients on placebo (p = . ). verapamil infusion was not tolerated in patients because of hypotension or a heart rate of less than /min, within hours of the start of the therapy. when atrial fibrillation occurred, the ventricular response, mean _+ sd, was not significantly slower during verapamil infusion ( + ) compared to placebo ( + ). conclusions: because of its frequent side effects and the only modest efficacy verapamil should not be considered for prophylactic therapy of atrial fibrillation after pulmonary surgery, and is probably not a good first choice for slowing the heart rate in case of rapid ventricular response once atrial fibrillation has occurred in these patients. results: study of haemostasis in these patients has showed deep disturbances of blood coagulation. fibrogen level has reduced to . + . g/l, fibrinogen and/or fibrine degradation products concentration have enhanced to . _+ . g/l, monofibrin soluble complex concentration to . -+ . g/l, blood plasmin level was enhanced to . + . mmol/ , plasminogen proactivator level was also enhanced to . + . ram, plateletes aggregation has decreased to %. after plasmopheresis aggregation was decreased in . times. it has been connected with decrease of fibrin and/or fibrinogen degradation products level and level plasmin in . times, and plasminogtnt activator level in . times. at the same time we have observed increase in total antifibrinalitic activity of blood in . times. activity of activators plasmine and plasminogene proactivators has decreased in . times and in the same time activity of activation inhibitors and antiplasmines has increased in times. conclusions: plasmapheresis leads to considerable improvement of a general condition and reduction of the haemorrhagic syndrom's sings (controlling of gastrointestinal haemorrage, reduction of intensity of subcutaneons haematoma). evaluation of continuous cardiac output (cc ) monitoring based on thermodilution technique in critically ill patients. methods: cardiac output (co) was monitored continuously using a modified pulmonary artery (pa) catheter, on which a heating filament is located and by which energy is transmitted to the circulating blood. a microprocessor calculated co by a new algorithm. standard bolus thermodilution technique ( ml of ice-cold saline solution) was used to compare cc with intermittent bolus cardiac output (ic ) measurements. the following subgroups were prospectively studied: i. heart rate (hr) > beats/min, . cardiac output > i/min . cardiac output < . i/min, . rectal temperature > . ~ and . pa catheter was inserted for more than days. results: a total of pairs of ic and cc measurements were obtained from the patients. bias (ico measurement minus cc measurement) of all measurements were . • i/min and the % confidence limits (mean difference• were - . / . i/min. also in the subgroups, cc measurement agreed closely with ico measurement (c > i/min: bias= . • i/min; co < . i/min: bias=- . • i/mln). elevated temperature and prolonged lay-days of the pa catheter did influence agreement of cc measurement with ic measurement neither (> ~ bias= . • i/min). conclusions: monitoring of cc using a modified pulmonary artery catheter with a heated filament has proven to be accurate and precise also in the critically ill when compared with "standard" intermittent bolus thermodilution technique. this method enhances our armamentarium for more intensive monitoring of these patients under various circumstances. background: the number of patients who need coronary artery surgery was) grows every year. most of these surgical operations are with extrar eircuiation (ecc). since january , this surgery is made without ecc in selected patients in our hospital. this technique is exceptional in spain. this type of surgery has proved useful in patients requiring revascularization of the left anterior descending, eireunflex or right coronary artery (not for grafting the pos~tefio~r descending branch}. blethods and results: since , patients aged to years (mean years) underwent cas without ecc. the mortality in programmed surgery was %. no patient was reexplored for hemorrhage. the mean values of some clinics parameters v~ere: a) blood requeriments: units per patient, b) need of mechanical ~entilation: i , hours, c) postoperative bleeding: cc, d) days at icui , . we used the student % t test or fisber~s exact test to compare these results with the mean values of surgery with ecc: a) blood requeriments per patient (p< , ), b) need of mechanical ventilation: hours (p< , ), c) postoperative bleeding: cc (p< , ), d) days at icu: (p< , ), e) programmed surgery mortality: % (p< , ). conclusion: our limited experience shows that this surgery is an alternative in the treatment of coronary disease, especially for aged patients with associated pathology and in jehova's witness. the need of mechanical ventilation, days at icu, blood requeriments and morbi-mortality were fewer than surgery with ecc. to study the hemodynamic and antiarrhythmic influence of ace-inhibitor enalapril in acute myocardial infarction (mi). methods: holter ecg monitoring, heart rate variability analysis, echocardiography ( and l days after beginning of the treatment), stress-echocardiography and stress ecg ( - -th day after the onset of mi). enalapril was included into the treatment of pts with mi (study group), with normal or increased blood pressure, from the -st day of the disease. the data were compared with pts treated without enalapril (control group). results: silent ischemia during stress-test was registered in pts of the study group and of control group, the arrhythmia episodes during stress test -in and pts and episodes of silent nocturnal isehemia -in and pts correspondingly. enalapril importantly attenuated the hypertensi~re re~aetioh % stress test. in pts of the study group the number of perifocal hypokinesis zones decreased; in the control group it didn't change. the quantity of ventricular extrasystoles in the patients of the study group decreased by %; the heart rate variability indices improved as well; in the control group the character of ventrieulir arrhythmias, heart rate and its va]~i~bili%y didn't change significantly. conclusions: the inclusion of enalapril into the treatment of mi is a useful t ol to improve hemodynamie parameters and decrease the incidence of ventricular arrhythmias. objectives: to study left ventricular (lv) systolic function in the patients with acute myocardial infarction (ami) before and after peroral captopril test. methods: the original echocardiographic parameter of lv contractility, "coefficient of effective systolic function" (cesf), was proposed in the study. cesf is calculated from lv stroke volume (sv), obtained from doppler aortic flow in lv outflow tract and lv end-diastolic diameter (edd): cesf =sv/edd. the study included patients with ami, who had local lv dyskinesia and global lv systolic dysfunction (ef< %). besides cesf, the ejection fraction was calculated before and after administration of mg eaptopril (on the fifth day of ami) by methods of bullet and simpson. results: the dynamics of these parameters, as well as heart rate (hr) and mean blood pressure (bp), is shown in the tabte. before cal~topril ef (bullet) . • . ef (simpson) . introduction: the cold system is a monitoring system for measurement of right (copa) and left (coart) ventricular cardiac output, cardiac function index (cfi), fight ventricular ejection fraction crvef), fight ventricular cnddiastolic volume (rvedv), intrathoracic blood volume (!tbv), global enddiastolic volume (gedv), lung water (etv) and excretory liver function (pdr). patients and methods: pts have been monitored by the cold system. above mentioned parameters are measured by thermal dye dilution and a fiheroptic femoral artery catheter. copa, rvef and rvedv measurements additionally were compared to measurements by the baxter explorer. :::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: ;;;k;;;;i cov (%) explorer ! ! [ gedv, itbv and pdr showed a significant decrease dufing the first - h after the operation, cfi and rvef si~canfly improved after k wheras etv showed a i~ in the early postoperative phase and fell to normal ranges at h. comparison of cold/explorer m~ements sb wed good correlations. discussion: concerning m ~toring of ri,ght ventric~ar function cold and explorer can he seen as equal. rvef gives an ar report about the performance of the right ventricle without use o f echocardiography. measuring itbv and gedv ~ improve ~gement and con~ol of th.e volume status, monitoring etv helps preventing lung edema. pdr shows good corre|ati n to liver blood chemistry and is bedside avai|ab|e. thus the cold system offers additional parameters for comprehensive m~nitofing of pts. ~e~ ~c surgery. obiectives: to evaluate the influence of an a!'~ered cardiac function on the cardiovascular response to the increase in oxygen demand induced by an increase in core temperature. methods: this preliminary study included adult critica!ly ill patients monitored by arterial and pulmonary artery catheters in whom thermodilution cardiac index {ci) and arteria! and mixed-vef)ous blood gases measurements could be obtained before and after an acute change in core temperature of at least . ~ (max rain apartl the patients were separated in two groups according to their cardiac function: patients had an impaired cardiac function as defined by a history of cardiac disease and an ejection fraction below % and patients had normal cardiac function. results: individual data are shown in the figure. in contrast to the control group (continuous line) in which c! increased without changes in oxygen extraction ( er), the q er in patients with impaired cardiac function (dottled line) increased without changes in ci. conclusions: the increase in oxygen demand associated with changes in temperature is met by an increase in c! in patients with unaltered cardiac function and in an increase in o er in patients with altered cardiac function. temperature should be taken into account in the assessment of the adequacy of cardiac output in patients with impaired cardiac function. objectives: to define the hemedynamic and metabolic response to physical therapy(pt) in relation to the type/level of sedation and the cardiac status in icu patients. methods: we studied mechanically ventilated icu patients ( • years) in stable hemodynamic status (no change in vasoactive treatment for at least hours), separated in groups: group = deep sedation, cardiac dysfunction required dobutamine (n= )r group = deep sedation (barbiturates), unaltered cardiac function (h=lo), group = moderate sedation, altered cardiac function (h= ) and group = moderate sedation, unaltered cardiac function (n= ). complete hemodynamic data, arterial and mixed venous blood gases, respiratory gas analysis (metabolic cart ccm, medgraphics) were obtained at baseline ( x) and twice (q. min) during leg mobilization. data were analyzed by anova. calcium channel blockers were used in complex preoperative preparation of hypertensive surgical patients. patients were allotted to groups based on their hemodynamic profile: hypokinetic: ejection fraction (ef)< . , patients; eukinetic (ef> . ),i patients and hyperkinetic (ef> . ),i patients. the most noticable change in hemodynamics was in the hypokinetic group: ef and cardiac output (co) were significantly decreased (p< . ) while systolic arterial pressure (sap) (p< . ) and peripheral resistance (pr) (p< . ) were elevated. the results showed that in hypokinetic patients on nifedipine ef (p< . t) stroke volume (sv) (p< . l) and co (p< . ) were increased while pr(p< . t), sap(p< . ) and diastolic arterial pressure(p< . ) were decreased. eukinetic type patients also showed an increase in ef,albiet to a lesser extent,than in the hypokinetic group. increased sv and co(p< . ) were observed in eukinetic patients though this was to a lesser extent than in the hyperkinetic group. in the hyperkinetic group of patients nifedipine had no effect on the aforementioned parameters except for a decrease in sap(p< . i). nifedipine increased ef in all hypokinetic patients. comparative results show that isoptin was less effective than nifedipine in decreasing peripl~eral vascular resistance and had a depressive effect on the myocardium. it can be concluded that the action of calcium channel blockers normalizing the circulation in the hypertensive surgical patient depends on: the condition of myocardium, the patients hemodynamic profile and their pharmacological properties. they were most effective in the hypokinetic group. zalo/nthinos e., daniil z. zakynthinos s., armaganidis a., kotanidou a., nikolaou ch..,roussos ch. critical care department, university of.athens, evangelismos hospital, athens, greece. introduction : surgical is the optimal treatrnent for ioculated effusions and the preferable procedure when multiple bands are seen in the pericardial sac by echo. patients : palients, post cardiac surgery, uremic ( men, women) with large pericardial effusion and clinical or echocardiographic findings of tamponade or both. these particular patients displayed numerous linear echo-dense bands and s~'ands crossing the pericardial space (in one of them a ioculated effusion compressed the left ventricule). one had aptt increased, four were mechanically ventilated. technklue : a fr polyurethane catheter with end and multiple side holes over ga needle was echo-guided to the ideal site (fluid abundant and closest to the transducer). the catheter was attached to a close system with a heimlich valve for continuous drainage (pneumothorax kit). subcostal entry was selected in one patient and chest wall in five. the patient's position was changed every hour at least. (we believe that the small changes in the position of the catheter and the mechanical breaking of the bands in relation with the movement of the heart assist the pericardial fluid to remove). results : in all cases only a small quantity of fluid was withdrawn in the first minutes( - ml) with some clinical and echo-findings improvement. the fluid was bloody or serosanuginous with high protein content (ht= % ,protein , gr/dl) in all cases. in first hours the mean volume of fluid removed was ml ( to ml). in that period echo showed no residual fluid. the catheter remained within the pericardium to days .. no complications are mentioned. conclusion : cardiac tamponade due to hemorrhagic high protein pericardial effusion in uremic and postcardiac surgery patients,, as it is revealed by echo dense bands, can be faced by -d echo guided perieardiocentesis. a -fr polyurethane catheter with multiple side holes, attached to a heimlich valve was effective to evacuate the pericardial fluid. no catheter was occluded though heparin infusions were not used. multiple changes of the patient's position may be fundamental. this -d echo guided pericardiocentesis performed in in~nsive care unit seems to be useful , safe and quick technique. determining the best inotropic drug represents a very serious problems. the use of more selective and potential inotropic and vasodilatative drugs does not always lead to improvement of hemodynamic parameters in patients with low cardiac output syndrome. this paper presents patients with acbp who need an inotropie support after extracorporeal circulation in first hours. the patients were divided into dobutamin et dopamine groups. the heart rate (hr). mean sistemic arterial pressure [map), central venous pressure (cvp). and termodilution cardiac index (ci) were measured. the measurements were without using inotropic drugs, and then using them after rain, min, and finally with one hour rate, within first hours. the statistical analysis shows that both drugs lead to an increase in hr in the first hour of the application. the final effect of dobutamine is no change in hr, whereas the effect of dopanime is very significant increase in hr. thus. an absence of taehyeardie response selects the dobutamine as a better choice. backeround: pulmonary vascular eadothelium possesses major metabolic functions, which when altered contribute to the development of serious pathologies such as ards. one such function is the conversion of angiotensin i to angiotensin ii, catalyzed by angiotensin converting enzyme (ace), located on the luminal surface of the endothelial cells. ace activity has been extensively studied in animals in vivo, by means of indicator-dilution techniques, providing: i) under toxic conditions, an early index of lung injury, and it) under normal conditions, estimations of dynamically perfused capillary surface area (pcsa). objectives: to validate the use of these techniques in matt: i) for pulmonary endothelial function assessment, and it) for pcsa estimation. methods: ace activity was estimated in ten adult haman volunteers, with no pulmonary medical history and normal pulmonary artery pressures, undergoing cardiac catheterization for coronary artery disease assessment. single-pass traspulmonary hydrolysis of the specific ace substrate hbenzoyl-phe-ala-pro (bpap; p.ci) was measured by means of indicatordilution techniques, and expressed as %metabolism (%m) and v=-hi( -m). bpap was injected as a bolus i) into a main pulmonary artery, and it) inside the right atrium, to assess ace activity in one and both lungs. we also calculated a,~,/i~, an index of pcsa. pulmonary plasma flow (fv) was determined by thermodilution. fp in one lung was estimated as . xf v. results: similar values of %m ( . + . vs . • and v ( . • vs . • were observed in both and one lung respectively. a~k~ decreased from • ml/min (both ltmgs) to :~ (one lung). conclusions: i) pulmonary endothelial ace activity and thus pulmonary endothelial function may be assessed in humans by means of indicator-dilution techniques, it) our data denote homogeneous pulmonary capillary ace coneentratious and capillary transit times in both haman lungs, iii) the % reduction of a=~/k~ in one lung suggests that this procedure can be used to quantify pcsa in man. (supported by the fonds de la recherche en saute du quebec and the national health system of greece). objective: verify whether antioxidant activity is higher in reperfused than in no-reflow myocardium after i.v. thrombolysis for acute myocardial infarction (ami). methods: patients with ami were included. blood for estimation of catalase (cat), glutathione peroxidase (gpx) and mn-superoxide dismutase (sod) was drawn before initiation of i. the mechanism of myocardial cell defence against free radicals is probably identical in both reperfusion and no-reflow phenomena. therefore, antioxidants cannot be used as reperfusion markers. objectives_ to evaluate the precipitating factors of hypothermic phrenic nerve injury following cabg with lima. methods: fifty two consecutive patients ( females), with a mean age of + (mean +sd) years were studied. during the ischemic arrest time topical hypothermia was obtained in al~ patients wffh ice slush and no cardiac insulation pad was used. all patients received a lima graft, with or whithout additional vein grafts. supramaximai, bilateral phrenic nerve stimulation was performed percutaneously preoperatively and whithin hours postoperatively. square wave stimuli of . msec duration were applied at the posterior border of the sternomastoid muscle. the compound muscle action potential of the diaphragm was recorded, using surface electrodes on the anterior chest wall. the time interval from the application of stimulus to the onset of diaphragmatic activity, phrenic nerve conduction time (pnct), was measured. values exceeding . msec were considered as abnormal. besults: preoperatively, all patients had normal (mean+sd) pnct, . • msec for the left nerve and . • mseo for the right nerve. on the first postoperative day, right pnct was normal in atl patients ( . • msec) , whereas left pnct was normal in patients ( . • msec) and abnormal in patients (incidence . %). in patients the left phrenic nerve was inexcitable and in patient left pnct was prolonged ( . msec). comparing patients with normal and abnormal pnct there was no difference in age, gender, number of grafts used, aortic cross-clamp and bypass time. however, patients with abnormal pnct had a lower preoperative ejection fraction ( • vs • p= . ). moreover, in all of them lima was dissected from its origin ligating all upper arterial branches, which provide the blood supply to the left phrenic nerve, whereas in those with normal pnct the small vessels originating from the upper to cm of lima were preserved (p= . ). conclusiojel~ a hypoperfused left phrenic nerve seems to be more susceptible to hypothermic injury during cabg with a lima conduit. objectives: to test if necessary interventions on systemic vascular resistance (svr) along with preset pump flew (q) during cpb could adversely affect autoregulatory response and cause vo shifts. methods: we studied males ( - yrs) who underwent cpb for cardiac surgery. at o oesophageal temperature - c we set pump flow at . i.m~ .min - . when map was higher than mmhg we calculated vo by using fick equation. then we infused sodium nitropruaside (sn) to control map at - mmhg for min and we calculated vq . without changing the sn infusion rate we set q at . i.m' .min " . ten min later we measured vo . we took vo changes into consideration if greater than %. statistical analysis using students-t-test for paired data and analysis of variance was used as appropriate. results: depending on the biphasic vo response to sn infusion during low and high q we classified pts in four groups (table). i. vo increases with sn and increases further during high q unmasking hypoperfusion and supply dependency. ii. vo increases with sn but the addition of high q results in systemic shunt. iii. vo increase during high q proves that vasodilatation can turn flow insufficient. iv. vo does not change with any intervention. the small number of pts and the wide standard deviation did not allow any statistical significance. conclusions: cpb is an interesting model for the behavior of microcirculation. intervention on svr and q can improve or impair effective regional oxygen delivery, resulting in either better perfusion or systemic shunt. vo monitoring seems necessary during cpb. preoperative cardiovascular optimization (opt) to ci > . l/min/m , _< paop < mm hg,and svri __< mmhg/ll/min/m decreases cardiac events (events) and mortality (mort) in peripheral vascular surgery patients (pvs). objectives: to determine if opt to the same endpeints decreases events in patients undergoing abdominal aortic aneurysm repair (aaar) and to study the r predictive value in pvs patients. methods: aaar patients and pvs patients were admitted to the s cu monitored with e pa and arterial catheters and treated to achieve opt. patients underwent surgery independent of success of opt data included demograph cs, incremental risk factors, laboratory and hemodynamic data pre, intra, a~nd postoperatively events, and mort. events included arrhythmias requiring treatment or prolonging the sicu stay > hours, a st depression > !mm or t wave inversion, an acute mr defined by a new q wave > . sec or cpk-mb > %. results are presented as means _ -. sd. opt was achieved in of ( %) and in of ( %) in the pvs and aaar group, respectively. events did nat differ between groups of ( , %) and of ( , %) in the pvs and aaar group, respectively (p>o. ). mort was of ( %) and of ( . %) in the pvs and aaar group, respectively (p > . ), while there was no difference in endpoints of opt between patients with and with.out events in the aaar group, there was a significant difference in ci between patients with and without events in the pvs group. of note, of ( %) patients who developed events in the pvs group had a ci < . in contrast to of ( %)in the aaar group. the positive and negative predictive value were % and % in the pvs and % and % in the aaar group. conciusione: f. the endpoints of opt used for pvs patients cannot be ~sed to reduce events in aaar patients; . pvs patients who have net achieved opt are at extraordinary risk of perioperative events; . preoperative card ovascu ar opt in aaar patients makes no difference in cardiac related events, background : comparison of the right and left filling pressures (cvp/pcwp ratio) is considered as a useful diagnostic clue : the normal ratio is _< . ; ratio >_ . may suggest right ventricul~ infarction while equalization of the cvp and pewp is a classic sign of tamponade ( ). however after cardiac surgery, many conditions (diastolic dysfunction, pulmonary hypertension, positive pressure ventilation) are susceptible to modify the '*normal" cvp/pcwp ratio. material and method : we determined cvp/pewp ratio in consecutive patients (pts) after uncomplicated cardiac surgery ( coronary artery bypass grafts; valvular replacements) measurements were made before and after tracheal axtubation. results :cardiac index : . _+ . /minlm~; laotate: + rag/i; cvp range : - rnmhg; pewp range : - mmhg. mean cvp/pcwp ratio before extubation is . ( % confidence imerval : . - . ) and after extubation, . ( % confidence interval : . -. . ), (ns, paired t-test). in % of the pts, cvp was higher than pewp. there are no correlation between the cvp/pcwp ratio and c! before (r = - . ) and after extubation (r = - . ) nor between the cvp/pcwp ratio and mean pulmonary arterial pressure (mpap), before (r = . ) and after extubation (r = - . ), discussion : cardiac performance is adequate according to ci and lactate. however the cvp/pcwp ratio is markedly higher than the "normal" (_< . ) ratio. this difference is not related to mechanical ventilation because the ratio is similar before and after extubation, nor to pulmonary hypetaension because of absence of any correlation with mpap, post-cpb diastolic dysfunction of the right ventricle could be an alternative explanation. in this group of pts, increased cvp/pewp is not associated with any impairment of cardiac performance (absence of correlation with ci), conclusions : cvp/pcwp ratio as high as within a large range of cvp ( - mmhg) and pcwp ( - mmhg) may still be considered as normal after cardiac surgery. this emphasizes the limitations of the hemodynamic monitoring after cardiac surgery (in comparison with echographic technics). careful analysis of the morphology of the cvp and right ventricular pressure curves (x descent, y descent, dip-plateau) is mandatory rather than relying on the quantitative assessment alone. reference : ( ) ntensive care.-university hospital -m~laga (spaink introduction. fibrinolitic treatment (ft) permits the treatment of acute myocardial infarction (ami) addressing the etiology, thereby eading to mproved ventncular function and a marked reduction m mortality. the main clinical oroblem is the reduced time of application. delay in hospitalization, which can be from to minutes, is potentially the most avoidable delay. method. to reduce delays in hospitalization, the following was carried out in two chases. audit: analysis of the time lapse from onset of symptoms to start of ft. showed that during "(he period june to december , patients with chest paros were treated within a eriod varying from minutes to hours from onset of symtoms. ages ranged from to (average , ), oelng males and females. they were glved initial ecgs to determine st mcreases suggesting ami. median t~me for this orocedure was l m.. potentia ami patients were then admitted to the coronary unit, [)atients, under age with no contraindications received ft the median time apse from admission to corona-y care and administration of ft was minutes ( . ), -he total median delay was minutes ~ -i h. min,~ delays n start of this procedure are grouped as follows: extra-hosdita delays (from onset of symtoms to arrival at hospital) diagnostic delays (from hospital arrival to ecg). treatment delays (from diagnosis to ft). objectives: protocol of procedure to implement a fast-track method. a protoco was drawn up with the object of reducing diagnostic delays to -i minutes and treatment delays to less than i minutes results. following rmplementatlon of this protocol in january , fts were glven, with an over all average delay of minutes. this fast-track method did not reveal any inappropnate ft or any increase m complications, conclusions: detailed study of the various times taken for diagnosis ane treatment of ami patients, showed up weaknesses in the system and improvements througn the protocol based on performence orocedures which led to a % reduction in the start of ft background: the importance of the early use of thrombo!ytic agents in acute myocardial infarction (ami) is based in the better remaining ventrictjlar function and smaller mortality rate because of the greater reperfusion and sma!ler infarction size, therefore, it is very impodant to apply this treatment to the maximum number of patients without thrombolytic contraindicati n, and within the minimun period of time. the "thrombolytic fast track" implementation allows to optimize the time to administrate thrombelytic agents avoiding multiple delays~ methodology: we anal!ze the application of thromboly c agents to patients with suspect of ami from the begin!ng of september until the end of february . in this time there are two different periods, during the first months thrombolytic agent were admin!strated at intensive care unit (icu), and during the second period we carried out a protocol of quick detection and thrombolysis therapy in susceptible patients at the emergency room in order to reduce the time to treatment. ma!n results are shown in the faffewins de ay h=hours m=minutes the implementation of the fast track does not need supplementary personal or equipment but a protocelized approach and training of the personal involved the main problem detected was the usual attendance overload of the emergency department that makes difficult to follow many structurated actions. conclusions: pratocqlized changes in the management of ami can significantly reduce the detay in the administration ef thrombolytic agents. it is not necessary to eomplet the procedure iq the emergency department, as the use of bolus schedules allows to begin the treatment in this area and to transfer the patient to icu afterwards. elective cardiac surgery. b calvet, f ryckwaert, p trinh duc, p colson. anesthesia -reanimation, hopital arnaud de villeneuve, montpellier, france. obhectives: the study was aimed at analysing the incidence of renal dysfunction following cardiac surgery and its prognosis (acute renal failure, post-operative morbidity and mortality). methods: two hundred and thirty seven patients (aged from to ) were consecutively operated on for elective cardiac surgery and retrospectively included in the study. patients with preoperative infections and operated on in emergency were excluded. each patient had preoperative invasive cardiac investigation with angiography and calculated ejection fraction (ef). anaesthesia, cardiopulmonary bypass (cpb) and cardiac arrest management were similar in all patients. general body temperature was reduced to - ~ c. renal dysfunction was defined as a % increase from baseline of serum creatinine. demographic data, asa, treatments, pre-operative creaunine level, cpb and clamping (axc) times, intra and postoperative use of inotrope, serum lactate level before surgery, at the end of cpb, at the time of admission in intensive care unit (icu) and on post operative day one and apache score were compared in patients with or without renal dysfunction using anova test for repeated mesures and x when appropriate. data are expressed as mean +__sd. p value less than . was considered statistically significant. results: thirtytwo patients ( , %) suffered from renal dysfunction. age, serum lactate level at the end of cpb, at admission in icu, at pod and apache level at admission in icu, intra-operative use of inotropes were statistically different in patients with or without renal dysfunction (p< , ). mortality rate was statistically different in patients with or without renal dysfunction(~, , % and %, respectively, p= , ). incidence of acute renal failure following renal dysfunction was , % ( patients required hemodialysis). conclusions: although our cdteria for defining renal dysfunction were very sensitive, the incidence of renal dysfunction following elective cardiac surgery was lower than communly accepted in the litterature ( ). however renal dysfunction appeared significantly associated with a poor prognosis. reference: -settergren g, ohqvist g current opinion in anaesthesiology , : - r ; , tzelepis, g. , , late complications were observed in % of cannulations: local infection in (i, %), catheter displacement by the patient in cases ( , %), catheter displacement during nursing care in ( , %) and malfunction in cases ( , %). conclusions: central venous catheterizations are followed by immediate and late complications in almost the same percentage acute poisoning with amphetamines (mdea) and heroin: antagonistic effects between the two drugs methods: after institutional approval and informed consent, selected patients ( _+ years) undergoing peripheral vascular surgery (n= ) or carotid endarterectomy (n= ) were investigated. patients included had either documented cad (n= ) or two or more (n= ) dsk factors (age > years, smoking, diabetes meltitus, hypertension, hypercholesterolaemia > mg/dl). -lead ecg recordings were carded out preoperatively, on ardval in the postanaesthetic care unit, and h, h, h, and h postoperatively. ecg recordings were analysed by an independent blinded cardiologist for signs of pmi (new st segment depression > . mv and/or new t inversion). in addition results: of the patients investigated developed ecg-documented pmi, % occurdng in the immediate postoperative phase. troponin i levels > . ng/ml were found in of these patients thus, comparing a cardiac troponin i cut-off level of ng/ml with intermittent -lead ecg recordings, we found a sensitivity of % and a specificity of % methods: demographic, clinical and ecg data were analyzed. . % of patients were male; . % female. cad was the most common underlying cardiac disease ( . %) and . % underwent open heart surgery. % received proeainamide for supraventricular and % for ven~cular arrhythmias. % received a loading dose. maintenance was provided by iv route in . % and by po in . % ( . %sr end . % ir). . % of patients were obese right ventricular function following cardiopulmonary bypass: is important the mode of myocardial protection we underwent this study in order to examine its safety and usefulness in pts with trustable coronary conditions (unstable angina ua the mean age for group a was • years, for group b • years, and for group c • years. a history of previous myocardial infarction was present in pts of group a, in of group b and in of group c. three pts in group a, in group b and in group c had previous coronary artery bypass grafting. the median time between the onset of symptoms and a was days ( - ) for group a we used a continuous fixed intravenous a infusion at a dose of the sn was % in groups a and b, % in c, and sp % for group a, (fixed defects included) and % for groups b and c. there was no difference of side effects among groups: chest pain (i pt -group a, pts -group b, and pts -group c), transient hypotension ( pt -group c), headache ( pts, group c), dyspnea ( pt -group a), while st depression was seen in pts of group b and in pts in group c. the rate of a infusion was decreased to /kgr/min in one group b pt due to development of chest pain s five year follow up of humoral immunity in paced patients athens polyclinic hospital, department of cardiology athens, greece author index a abiad ch bertschat, e betbes blanch, l del nogal saez e -meneza nolla, j. nolla-salas pilz~ u puig de la bellacasa e scarpa, n. van de wetering objectives: only % of patients suffering from acute guillain-barr@ syndrome (gbs) respond promptly to established therapies like plasma exchange or intravenous immunoglobulines. in contrast to serum, cerebrospinal fluid (csf) of gbs and ctdp patients contains enriched portions of antiexcitatory factors(i) and cytokines ( ) able to induce pronounced conduction block ( ). to reduce or remove such pathologic factors we introduced a technique with direct access to the subarachnoid space. methods: with informed consent we lumbally inserted g catheters in gbs-and cidp -patients under sterile conditions. some of them had not responded very well to established therapies. - ml of csf were withdrawn and retransfused by a bidirectional pump (flofors) after passing newly developed filters (pall). daily filtrations with several cycles were performed ( - ml) over one week. results: the gbs patients improved after days (median) for one grade (according to the gbs-scale from the gbs study group) . the ventilator dependent patients were weaned after days (median). patients not at all treated before ( / ) responded better than patients that had been pretreated ( / ) with plasmaexchange or intravenous immunoglobulines. / cidp patients drew benefit from treatment, stabilized iongterm. conclusions: csf-filtration is a relatively save and well tolerated additional procedure. the costs are considerably lower ( / ) than those for plasmaexchange or intravenous immunoglobulines. references:( )wsrz aet al: csf and serum from patients with inflammatory polyradiculopathy have opposite effects on sodium channels. muscle nerve ( ) . ( ) clinical observations were made in patients admitted to the clinic. they were in coma associated with acute alcohol intoxication.standard evaluations (ecg-monitoring, electrocardiography, neuromonitoring, studies of acid-alkali condition, biochemical and toxicologic investigation of blood and urine) prior to and following the treatment conducted were undertaken in all the patients.to correct irreversible impairement of functions twofold laser blood irradiation by means of alok- apparatus, the exposure within minutes, was carried out.the data obtained confirm more rapid coma withdrawal of the patients, reconstruction of the heart and central nervous system electrophysiologic indeces, reliable reduction in complications compared with the control group. objective: to know the actual incidence of the critical illness polyneuropathy(cip). setting: fourteen intensive/critical care unit beds, in bed university hospital, covering . inhabitants (majority rural area). the icu patients are medical, surgical and coronary, excluded the neurotrauma and neurosurgical. design: a conseculive and prospective study. all the patients admitted during three months, from january lth to march th , were eligible (patients with admittance diagnosis of polyneuropathy were excluded ). methods: patients with apache ii score > , at the admission and six days after admissions were included into the study protocol. diagnosis of sepsis, mof, and all the drugs administered days before were recorded. a complete neurological exam, by a neurologist, in absence of ssdatives and muscles reliant ( th, ~ and th days after icu admittance) was made. we evaluated the nerve and muscles function with and electromyography study in all patients, at same days. in some paeents with cip we performed a nerve biopsy. results: from patients ( apache ii score: . ) admitted in the icu, ( . %) enter the study protocol. seven ( , %) had an axonal polyneuropathy(cip), three very severe. only four of the patients with cip had pathologic clinical exam. apache ii score: cip vs non-cip was . vs . . the incidence of cip by diagnosis (cip/diagnosis) was: sepsis, / and mof, / . conclusions: . -we think that it is necessary to define the "critically ill" for some score, before designing a study to know the incidence of this syndrome. . -we think that the incidence of the cip is lower that the latest papers say. objectives:acute pancreatitis(ap)is becoming a more important problem among the elderly as the population ages. the increasing presence of gallstone disease,as well as the use of certain drugs,may also contribute to the occurrence of pancreatitis. methods:all patients(> years)admitted to our medical department over an eight year period were included.pancreatitis was confirmed by biochemical tests and imaging techniques.scores were developed using ranson's criteria and a multiple organ system failure(mosf)index . overall, patients were evaluated; ( %)had pancreatitis of unknown etiology . results:( )patients with pancreatitis of ~nlqnown etiology were sicker and had greater morbidity( % vs %),mortality( % vs %),and longer hospital stays than p~tierf~ with pancreatitis of known cause.( )the best predicto~of severity and outcome was the mosf index and not ranson's criteria;the higher the score,the greater the associated disease,the worse the outcome.( )curlously,no difference existed in associated medical conditions between patierts withknown and ur ~own causes of pancreatitis. conclusions:greater organ dysfunction exists in patients with pancreatitis of unknown etiology, even though age and associated medical conditions do not differ . the application of the total enteral nutrition in the burns disease has minimized the complication rate and consequently increased the survival rate of children and adults. time of initiation, composition, duration and way of administration are very important in obtaining the optimum beneficial effect from the treatment and diminishing the complication rate and side effects. the above features will be discussed in view of our experience in cases. ta buckle?,, ra freebalm, c gomersall g joynt, r young. tg short. department of anaesthesia and intensive cm+e, prince of wales hospital. the chinese university of hong kong, shatin, hong kong introduction: gastric mucosal ph (phi) monitoring has been proposed as a relatively noninvasive index of the adequacy of aerobic metabolism in the gut. to examine the accuracy of gastric intramucosal pit measurements as a function of time and as a function of the catheter itself to determine whether the measurement error between catheters is clinically acceptable. patients with a gastric tonometer (trip tm, tonometrics, worcester. ma) insitu for > days were studied. following informed consent two new tonometers were inserted equidistantly & correct position was confirmed radiographically. measurements of intramucosal gastric ph were then performed over a hr period. eight -ten measurements were made in each of ten critically ill patients.percent differences between the two new catheters were . % ie at ph . _+ . ( % limits) and between old & new catheters were . %, ie ph j _+ . ( % limits). conclusions: the results suggest that the function of the tonometer deteriorates over time and that the absolute values of phi m~ not ~ufficiently accurate. however as a trend monitor phi may be useful in the clinical setting. despite a continuous decline both in li'equency and severity of gastro-intestinal stress-lesion/-bleeding (gisb) due to both improvement in preclinical support and in intensive care medicine, patients with cerebral lesion are still considered at high risk for developing gis . therefore the question arises, whether m> specific (}lsb-prophylaxis besides general and neurological intensive care, specific pharlnaeothcrapy or even the combination of two specific drugs reveals any protective efli~ct on frequency and severity of gisb.this pntspcclive randomized study has been perfornted in patients snfrering t'rttna head-injury/cerebral lesion and with a glasgow-coma-scale on admission (gcs:,)of < . according to randomization the patients have been grouped as tbllows: h analgesia/sedation (n= ); ih analgesiajsedation plus pirenzepine mg/day (n= ); .[ih anatgcsia/sedalkm plus sncraltate x [ g/day (n= ); iv: analgesidsedatkm plus pirenzcpine mghlay plus sucralfate x e/day (n= ). slalislical analysis has been performed by chl:*tt~sl. rank correlatinn and unpaired t-test; statistical significance has been set with p < . . / patients ( . %) developed gisb. although the mean gcs~-value (x -+ sd) did not reach significance between patients with and without gisb ( . + . vs . -+ . ). a significant inverse correlation between gcs:, and the incidence of gtsb (rs~ = . ) has been shown. the frequency of gisb among the groups is as follows: h . %; lh . %; llh . %; iv: . % (ch -~ = . ; not signilicant). no gisb-induced blood translusion or mortality, respectively, could be demonstrated. survival rate between the groups did not differ significantly (chi-" = . ; p= . ) and reached an overall-value of . %.drug-specific glsb-prophylaxis -administered either as monotherapy (pirenzepine, sueralfate) or in combination of these two specific-drugs -reveals no additional significant influence on the incidence of gisb in patients with cerebral lesion compared to no specific prophylaxis besides the general trauma-/disease-specific intensive care measures. critical care dpt, evangelismos hospital, athens university scho~" of medicine objectives: the correlation of longterm presence of nasogastric tube (ngt) to gastroesophageal reflux (ger) is still in question. in case of positive correlation, peg should represent an alternative to tube feeding in patients unable to be fed orally. therefore, we investigated: i) the correlation between ng and ger and ii) the effect of peg on ger. methods: a -h esophageal ph-metry was performed in patients in recumbent position at ~ who had a ngt for more than days and were on sucralfate for gastric mucosal protection. the tip of the ph-probe was lied cm over the esophagogasttie junction, confirmed by x-rays. patients who presented a percentage of ger-total (i.e. with a ph less or more than ) (ger-t) more than %, underwent ~t peg. the presence of a creseent-notch on the esophagogastric junction persisting on inspiration and the grade os endoseopic and histologic esophagitis (scale= - ) was noted. two ph-metrles repeated on h and on days post-peg were compared to the pre-peg one, with the followin~ parameters taken in consideration: i) % ger-t, ii) number of ger-total per hour (no/h ger-t) and iii) the duration that ph was less than (tph< ). in case ot ger persistence at the ph-metry on ?th day post-peg (group ii) another endoscopy was performed, while patients with reduced ger (group i) were considered as esophagifis-free.results: out of patients presented a ger-t> %. eleven out of group i group (n= ) i ( objectives: the aim of the present study was to compare the performance of a specially modified version of a photo-and magnetoacoustic (pa/ma) gas analyzer (br~)el & kjaer, denmark) with a conventional quadrupole mass spectrometer (ms) (innovision, denmark) in inert gas rebreathing (rb) tests such as determination of functional residual capacity (frc), pulmonary capillary blood flow (pcbf) and lung tissue volume (vtc). methods : from simultaneous readings of inert gas concentrations with the ms and the pa/ma analyzer during rb experiments a comparison was made of the pcbf, vtc and frc values. the rb tests were performed during rest and exercise ( , and w) in ten healthy subjects. results: the differences (mean +/-sd) between simultaneous estimates of rebreathing parameters were the following (pa/ma -ms) for pooled data, pcbf: . +/- . i/min, vtc: - +/- ml and frc: . +/- . liters. conclusions: smell but significant differences were found between the estimates of pcbf, vtc and frc using the ms and pa/ma, respectively. reference: p. clemensen, p. christensen, p. norsk, and j. gr~nlund. a modified photo-and magnetoacoustic multigas analyzer aplied in gas exchange measurements. j appl physiol ; : - . objectives: because transcranial doppler (tcd) has been proposed to explore cerebral co vasoreactivity in brain injury (stroke ; : - ), we compared this technique with the kety-schmidt reference method to assess cerebral vasoreactivity in comatose patients. methods: mechanically ventilated patients (age - yrs, glasgow - ) in coma due to acute brain injury were investigated during stepwise changes in paco ( , , , and mmhg) by increasing inspired pco . middle cerebral artery velocity (vm) was measured by tcd. after insertion of a catheter in the ipsilateral jugular bulb, cerebral blood flow (cbf) was determined by the kety-schmidt method, using the inhalation of % n through the inspiratory line of the ventilator. for each patient a cerebral co~ vasoreactivity index was calculated as the slope of linear relationship between vm or cbf and paco . objectives: after cardiac surgery the fluid shill, between interstitial and intravasal space may be marked. this is due either to the intraoperative volume loading by the extracorporeal circulation or the increased postoperative diuresis. therefore, infusion of a large amount &fluids is necessary during the first postoperative hours. it still remains unclear which of the substances at disposal is the best for this purpose. aim of the present study was to compare the different fluids with special regard to postoperative bleeding and rheological behaviour. methods: patients undergoing cabg-surgery were investigated and randomizedly distributed to three different groups of postoperative volume replacement to stabilize the mean arterial pressure at mm hg. . ringer's solution, . . % gelatine solution, . % hydroxyaethylstarch (mean m.w. . ). we evaluated the following parameters within intervals of min: arterial and central venous pressure, heart rate, postoperative bleeding, urinary output, volume replacement. results: there was no statistically significant difference between the groups with regard to urinary output and bleeding. in spite of larger amounts of fluids necessary in the ringer treated group patients of this group showed symptoms of hypovolemia. hematocrit was increased in the ringer patients. this was statistically significant. introduction: pulmonary wedge pressure (pcwp) and central venous pressure (cvp) are frequently used as parameters for cardiac preload, although it is known that both are poorly correlated to the cardiac index (ci). it has been claimed that intrathoracic blood volume (itbv) measured with the thermal dye dilution method reflects cardiac preload better than pcwp and cvp. we studied the correlation between itbv and ci in a mixed population of critically ill patients. methods: in consecutive patients ( sepsis/sirs, acute heart failure, ards, transjugular intrahepatic portosystemic shunt) monitored with a pulmonary artery catheter, itbv was measured on regular intervals using the pulsion cold z- system (pulsion, munich, germany). ci, pcwp, and cvp were recorded simultaneously. results: a total of ol measurements was made. pcwp and cvp did not correlate to ci, nor did apcwp or acvp correlate to aci. itbv was correlated to ci in a non-linear fashion (f - , df = , p < . , (figure) ). aitbv was correlated to ac in a linear fashion (r = . , f = , df = , p < .o ). a rapid and efficient circulatory support system may save a patient in cardiogenic shock. left heart bypass with percutaneous and transseptal placement of the aspiration canuia simplifies the circuit and avoids the need for an oxygenator. we assessed this preclinical set-up in anaesthetized pigs using a centrifugal pump with a f arterial catheter and a f left atrial aspiration line. animals were supported for two hours at a mean flow of . liter ( ' rpm), a mean hematocrit of % and low heparinisetion (act double baseline). hemodynamic and laboratory samples were taken at baseline (a), minutes (b), one hour ( pulmonary hypertension (ph) usually involves obliteration and loss of functional pulmonary microvasculature. the microvaseular endothelium normally acts as a major metabolic organ, converting angiotensin i to angiotensin ii via the angiotensin-converting ectoenzyme (ace). it is unknown whether the loss of functional vasculature and altered pulmonary blood flow seen in ph will affect lung ace metabolic activity. we therefore estimated pulmonary vascular ace activity in patients with ph of various causes: primary; post atrial septal defect closure (asd); chronic thromboembolic (te); anorexigen; iv drugs; collagen disease. single-pass transpulmonary hydrolysis of the specific ace substrate h-benzoyl-pbe-ala-pro (bpap) was measured and expressed as % metabolism (%me . we also calculated an index of peffused functional capillary surface area (amax/km). all patients with ph had an abnormality of %met or amax/km, or both. as compared to control humans (mean %met = . % _+ . % s.d.), the mean %met in ph patients was . % _+ %. the %met in ph patients correlated inversely with cardiac output (r= . ), possibly reflecting more complete bpap hydrolysis with longer pulmonary transit times. amax/km was markedly decreased in ph ( + ml/min) as compared to controls ( _+ ml]min), consistent with a significant loss of functional capillary surface area. patients with collagen disease, asd and anorexigen-induced ph had the most marked abnormalities. in conclusion, patients with pulmonary hypertension have decreased pulmonary endothelial angiotensin converting enzyme activity, likely due to a loss of functional or perfused pulmonary microvaseulature. supported by the funds de la recherche en same du quebec and the national health system of greece. objective: to investigate adrenocortical function in patients with ruptured aneurysm of the abdominal aorta (raaa). studies investigating adrenocortical insufficiency in critically ill patients report an incidence ranging from % to less than %. this may in part be explained by difference in methods used (single cortisol measurement vs short acth stimulation test) and populations studied (heterogenous groups of patients with great individual variation in underlying disease as well as duration and severity of illness). methods: we investigated the adrenocortical function in patients with (raaa).a short acth stimulation test (synacthen test; ug - acth iv) was performed at hrs within hrs of admission. plasma cortisol was measured before (cort basal) and after stimulation (cort stim). a plasma cortisol level > . umol\l before or after stimulation was considered normal, severity of illness was assessed using apache ii. results: of the patients investigated died and survived. mean cort basal in nonsurvivors was significantly (p< .o ) higher than in survivors; . (range . - . ) vs . (range . - , ). this difference between nonsurvivors and survivors was also present for cort stim but lacked significance; . (range . - . ) vs . (range . - . ). while patients showed a cort basal < . , no cort stim < . was found. there was no significant difference in mean age or apache ii score between survivors and nonsurvivors; vs and vs . conclusions: single plasma cortisol levels were inadequate to assess the adrenocortical function in the patients studied, judged by a short acth stimulation test, our investigation in patients with raaa showed no adrenocortical insufficiency. mortality in raaa is associated with elevated plasma cortisol levels. obiectives: mortality in acute myocardial infarction (ami) prinicipally depends on hemedynamic impairment. thus, patients (pts) with elevated pulmonary wedge pressure (pwp) present high in-hospital mortality. however, the complete right heart catheterization is laborious, so the central venous pressure (cvp) alone is frequently used to assess the severity of ami. the accuracy of cvp in estimating pts with ami was tested in this retrospective study. methods: pts. aged + years, admitted to our ccu from to with their first ami, were inctuded in this study. all had undergone right heart catheterization because of overt or suspected heart failure. swan-ganz catheters ( f, cm, abbott, il, usa) had been used, every treatment had been temporarily interrupted l h before the calheferization. based on ecg findings the pts were retrospectively divided into groups. in group a we included pts with anterior ami, in group b, pts with inferior ami, and in group c, pts with inferior and right ventricular ami. the initial values of cvp and pwp were considered for the linear regression of the pwp variable on cvp and p< . was accepted as statistically significant.results: in g~oup a, the cvp and pwp vaiues were + mmhg and _+ mmhg respectively. despite the signifanf correlation (p< . ) between the two variables, it was not possible fo predict the exact value of pwp based on cvp value, pts ( %) presented cvp> mrnhg and of these ( %) had pwp_> mmhg. in group , the cvp was _+ mmhg and the pwp, _+ mmhg. significant correlation (p< . ) between the two variables also existed, however it was impossible to predict the pwp value. pts ( %) had cvp> mmhg but only of these ( %) had pwp> mmhg, similar was the relation between cvp and pwp in group c (p< . ). cvp averaged + mmhg, and pwp, _+ mmhg. pts ( %) had cvp> mmhg and from these ( %) presented pwp> mmhg,conclusions: a single measurement of cvp in ami does not ensure an accurate assessment of pwp. because every pt with ami needs optimal values of pwp in order to prevent pulmonary congestion or manifestations of low preload, the significance of complete right heart catheterization becomes apparent. in patients (pts) with advanced hf the need and the prognosis for heart transplantation (ht) can be predicted from vo= max. indirect measure of functional capacity with the six-minute walk test can also predict smvival in moderate hf. to predict vos max from indirect astinmtions of functional capadty such as - ~q~/, pulmonary and heart function tests, and to assess the prediddve value of the above parameters in hf pts survival. we evaluated pts (age + yeats nyha class: ii, hi, iv) with hf for pit. they underwent a pmgmmive exercise test on cycle ergometer for vo max determination, a -mw, a right heart catheterization and a spirometry and dlco estimation. introduction: brain death causes myocardial impairment by mechanisms that are not well understood yet. the aim of this work was to assess the echocardiographic features found in these patients from the clinical onset of brain death to somatic death, methods: seven brain dead patients were studied (patients" relatives refused to allow them to be used as donors). mean age was . ( - ) years old. four of the patients were female, none of the patients had any history of cardiac disease. transthoracic echocardiogram (echo) and electrocardiogram (ecg) were obtained at the onset of clinical brain death and were repeated every hours until somatic death. we we detected severe diffuse hypokinesia (ef< %) in patients and mild hypokinesia in others (ef - %). systolic function was strictly normal in only patients. corrected qt interval (qtc) in ecg was . _+ . msec (normal range - msec) just before somatic death (b). conclusion: in patients with brain death we observed a significant increase of left ventricular mass due mainly to ivs "hypertrophy" without any important change in the dimensions of the left ventricle. to our knowledge, this finding has never been reported before and its importantance in heart transplantations may be of particular interest. predict right ventricular outcome. l. jacquet, r. dion, p. noirhomme. m. van dijck. m. goenen cardiothoracic intensive care unit, st-luc univ. hospital(ucl) we have registred: heart rate (hr), blood pressure (bp), pulmonary artery pressures (pap), central venous pressure (cvp), pulmonary capillary wedge pressure (pcwp), pulmonary and systemic vascular resistances (pvr, svr), right ventricle end-diastolic end end-systolic volume (redv, resv), right ejection fraction (ref), right sistolyc ventricular work (rsvw) and cardiac output (co) using a thermodilution thechnique and a microprocessor (model ref- ; baxter-edwards laboratory); duration of cpb and aortic clamping, and the requirements of haemodynamic support after cpb.results: in the c group an increase post-cpb of the fc ( + . + . , p < . ) was produced without significantly changes in the redv, resv, ref, rsvw neither co. in the w group, hr increased from . + . to . + . (p < . ); redv was reduced from . -+ to . _+ . (p < . ); resv was reduced from • . to + . (p < . ). there were not changes in the other haemodynamyc parameters. there was a trend (no significantly) to an increase of ref in the w group ( . + . |• . ) compared with the c"group ( • . ($ . • . ) post-cpb. the need for haemodynamic support was similar in both groups.conclusions: the warm, continuous, anterograde-retrogade myocardial protection has obtained a decrease of preload, hr, and a trend to an increase in the ref, making an improvement in the right ventricular global performance when is compared with the classic form of cold myocardial protection. objective: to evaluate the effect of dobutamine on gastric mucosal ph (phi) after coronaly artery bypass surgery. design: prospective study in a university hospital intensive care unit (icu). subjects: elective cardiac surgery patients. interventions: dobutamine was infused at ug/kg/min for hours immediately after admission to the icu. hemodynamics were measured every minute periods until hours and again hours after stopping dobutamine. results: there were no significant differences in mean gastric phi between the groups but mean phi decreased in both groups during the study period. oxygen delivery and consumption both increased during dobutamine infusion but decreased to the control group level after stopping the dobutamine infusion. lactate levels did not change. baseline objectives: the aim of the study was to evaluate the usefulness of a low dobutamine dose in conjunction with intraaortic balloon pumping and mechanical ventilation in cardiogenic shock. we studied patients . -+ t . years of age suffered of post infarction cardiogenic shock characterized by a systolic arterial pressure< mmhg, urine output< ml/h and mental confusion or purpueral signs of low output, non responded to dobutamine infusion up to pg/kg/min. all patients underwent mechanical assistance by the intra-aortic balloon pump (iabp). five patients were additionally placed on mechanical ventilation due to blood gases disturbances. the end points in our study were: reversion of cardiogenic shock, improvement of patients survival or both on the th post infarction day and months later. results: three patients refused iabp treatment and / survived on the th day. on the th day / supported by the iabp and / that underwent mechanical ventilation plus iabp were alive (p < . ). on the th month / supported by the iabp and / that underwent mechanical ventilation plus iabp were alive (p< . ). conclusions: in conclusion, the combined use of mechanical ventilation and iabp assistance in severe cardiogenic shock might improve survival. obiectives: the study was aimed at analysing predictive factors of swan ganz pulmonary catheter (pc) requiremen t during elective cardiac surgery according to the need of sustained inotropic support after surgery. methods: three hundred patients (aged from to ; females and males)were consecutively operated on for elective coronary artery bypass surgery (cabg, n= ), valvular replacement (vr, n= ), combination of both (vr-cabg, n= ), or others (n= ) and retrospectively included in the study. each patient had preoperative invasive cardiac investigation with calculated ejection fraction (ee). anaesthesia, cardiopulmonary bypass (cpb) and cardiac arrest managements were similar in all patients. pc requirement was estimated from the need of either dobutamine, adrenaline, dopamine or enoximone use during the first hours after cardiac surgery. demographic data, asa and nyha classifications, preoperative ef and treatments, type of surgery, cpb and aortic cross clamping (axc) times, and postoperative incidence of complications were compared in patients with or without inotropic support using either student's t test or x with continuity correction when appropriate. results: seventy hree patients ( . %) required inotropic support after surgery. axc .and cpb times, mean stay in icu were significantly longer in patients with inotropie support (p< . ). type of surgery, preoperative ef, and nyha classification are the first significant factors related to inotropic support (p< . ). most patients operated on for double-vr or vr=cabg required inotropic support ( and %, respectively). postoperative mortality was higher in patients receiving inotropic support ( , % vs , % 'overall mortality, p= . ). conclusions: since pc insertion is most.often justified because inotropes are required, these results suggest that elective rather than routine systemic pc insertion could be helped by considering several but selected preoperative factors. background: cardiovascular depression due to anaesthesia, old age and major gastrointestinal surgery is becoming an increasingly frequent challenge .to the anaesthesia-surgory team. deliberate preoperative manipulation of haemodynamics and oxygen transport parametres towards prede~t~mined optimal values may prove to be effective "in reducing morbidity ~nd mortality in high risk surgical patients,. a new concept of using conlimaous perioperative measurement of cardiac'output to obtain and maintain supranormal oxygen delivery (do i) is presented. methods: continuous measurement of cardiac output is a relatively new form of on-line monitoring, in which trains of impulses are emitted from a thermal filament mounted on a pulmonary artery catheter. computer software recognizes patterns generated by minute changes in blood temperature and ealoalates cardiac output every - seconds. cardiac output and mixed venous blood oxygen saturation are displayed graphically on line. in tins tm study cardiac output was measured continuously by vigilance cardiac outpu t compl/ter (baxter). preoperative haemodynamic optimization was performed with the goal of increa- sing do i to at least ml/min/m accordfing to shoemaker's algorithm . this was.done by infusing colloids (albumin or hydroxy ethyl starch (haes-steril| until the desired do was reached. infusion was stopped if cardiac output ceased to increase with infusion, if there were signs of pulmonary oedema or if wedge pressure reached mmhg. vasoactive or inotropic drugs were infused if the desired do was not reached by infusion alone. anaesthetic technique included continuous thoracic epidural and isoflourane anaesthesia. expected mol:bidity and mortality rates were calculated by the "possum" score aasing preoperative clinical and paradinical estimates of organ function as well as surgery characteristics . materials: asa group ill-iv patients with a mean age of years (range - ) and a mean weight of kg (range - )) scheduled for major abdominal surgery were included. results: patients were excluded because do i could not be raised at all. mean do i was increased from ml/min/m (range - ) to ml/min/m (range - ). mean volume of preoperativdy infused colloid was ml (range - ). during surgery ml (range ) of colloid was infused. mean length of surgery was minutes (range - ). mean blood loss was ml (range ). expected mortality and morbidity rates ("possum") were % and %, respectively, whereas patient follow up upon discharge or at death revealed mortality and morbidity rates of % and %, respectively. conclusion: based on experience from the present study, continuous measurement of cardiac output has proved to be a valuable tool for perioperative optimization of do in asa group ili and iv patients during major surgery. however further studies including a greater number of patients are necessary to confirm the promising preliminary findings. we studied the hemodyn~c effects of three different combinations of positiv inotropic .agents, vasodilators, diuretics and av-filtration (av) in patients (pts) with severe left heart faille (left veutrieul x filling pressure (lvfp) > mmhg) due to acute myocardial infarction. hemodynamic measurements (intravascular pressures (lvfp), thermodilution (cardiac index (ci)) were made before (control) and after each therapy. in furosemide (f) + d butamin (d) + nitroglycerin (ni) reduced lvfp and a small increase of ci occurred. in of these pts :(group a) nitroprusside (hip) instead of ni increased ci significantly, in the other pts adding of amrinone (a) resulted in a pronounced increase of ci. group c (n= ): the combination of ni and av reduced lvfp but did not increase ci which was achieved by av+d+ni. in order to optimize the treatment of acute heart failure a combination of inotropic agents, vasodilators, diuretics and av-filtration should he used guided by hemodynamic monitoring. arias jr, miragaya d, sandard, san pedro dm ~, herndndez d, valenzuela . objectives: to evaluate the variation in nomdrenaline (na) plasma concentrations in patients with acute myocardial infarction (am ) after thrombolytic therapy with noniltvasive reperfusion criteria (clinical, electrocardiographic and enzymatic), in relation to infarct size and location.methods: consecutive patiens with ami, from october , to february , , admitted within hours alter onset of symptoms, undergone successfull systemic thrombolysis. of them were anterior (group a) and inferior (group b) . noradrenaline plasma levels at (na ), (na ) and (na ) minutes after admission were compared with ck-peak plasma levels by linear regression. differences were tested for significance by student-t-test for paired and unpaired values. na plasma concentration was measured by high-presssure liquid chromatography. p< ns . ns means -sem (normal limit for our laboratory: na < / pg/ml; ck < u/i ) conclusions: . the na plasma levels at admission (nai) are more increased in anterior than inferior amis, probably in relation to infarct size. . the decrease in na is more evidence in amis with anterior location. . this decrease is probably due to the major efficacy of thrombolytic therapy in amis with anterior location. arias jd, miragaya (group b) , probably due to certain degree of t~cg'rfueion. . there is not significant variation in na in conventional treated ami (group c). v.suchanov, a.levit, p.trofimov, icu, regional hospital, ekaterinburg, russiaobjectives: our task was to improve the technique of preservation of platelet rich plasma. methods: patients scheduled for multiple cardiac valve replacement in were divided into two groups: group i ( patients) -without pp; group ii ( patients) -pp was performed preoperatively. the first pp was made ten days and the second - days before the operation. prp was preserved by cryoconservation. our technique of cryoconservation is distinguished by the speed of freezing ( - ~ and absence of dmso. this made it possible to preserve % functionally active platelets during days. the prp was transfused back after heparin neutralization. the hospital ethics committee approved the investigation.results: the blood loss through the st p. o. d. was significantly greatest in the group i ( _+ ml) and all the patients required transfusion of the donor blood ( + ml) whereas the blood loss in group ii was +_ ml and olny patients required the donor blood. the number of platelets on the st p.o.d, was _+ . /l (group i) and + . /l (group ii), p < . .conclusions: our technique of prp cryoconservation makes it possible to avoid the crystallization phase during freezing of prr thus the infusion of prp may improve hemostasis after open heart surgery and limit the use of the donor blood. in-hospital outcome of women suffering an ami is generally considered worse than that of men, but it is still debated whether female sex is per sea negative prognostic factor or is merely associated with other negative determinants of prognosis. the purpose of the present study is to evaluate the independence of the association between female sex and mortality (in the patients of the swiss centers) and in the patients randomized in the isis- trail mortality rate in women was . % ( / ) compared to . % ( / ) in men; in switzerland: in-hospital mortality for women was . % ( / ), for men . % ( / ).the table shows the results of isis- in terms of odds ratios and their % confidence intervals either after unadjusted analysis or after adjustment for age, known to be the major confounding variable when prognosis of women after myocardial infarction is considered, and for all the available clinical and epidemiological characteristics collected at trial entry: these observations suggest that there is a small but independent effect of female sex on short-term mortality after acute myocardial infarction. ( ) and bubble ( ) oxygenators a, ere used. anaesthesia was balanced and pts were extubated to hrs after cpb. pts were monitored with swan-ganz catheters (sgc) for hrs after cpb. at that time qs/qt was calculate( according to )be standard shunt equation. after the sgc had been removed, an estimated shunt was calculated. measurements of qs/qt were performed: before induction of anaesthesia ( ), after induction of anaesthesia (i[), mins after cpb (iii) (iv) and (v) hrs afiter cpb, rains after extubation (vi), hrs after cpb (v[ ) and on the nd, rd, th, th and tb postoperative day (pd) (viii, x, x, xi, xi , respectively). analysis of data was performed by two-way analysis of variance, p < . being regard as significant.results: the figure shows the values for qs/qt expressed as means + sd. there was a significant increase in qs/qt above b~setine throughoul the whole investigated period except on the th pd. qs/qt reached maximum at rains after extubation (vi). objectives: many stndies have shown advantages of membrane oxygenalors over ubbie type oxygenators. the aim of this study was to evaluate the influence of x 'genator type on pulmonary shunt (as/at) after coronary surgery. methods: patients (pts) gave their informed consent to the study which was approved by the university ttuman research committee. pts were divided into two groups: a (n = ) with a membrane o~genator and a (n = ) with a bubble oxygenalor used during cardiopulmonary bypass (cpb). ths were monitored with swan-ganz catheters (sgc) for hrs after cpb. at that tfme os/ot was calculated according to the standard shunt equation. alter the sgc had been removed, an estimated shunt was calculated..measurements of os/qt were performed: betore induction of anaesthesia (i), mins after extubation ( ), hrs alter cpb ( ) and on the nd, rd, th, th and th postoperative day (iv, v, vi, vii> viii, respectively). analysis of data was performed by one-way analysis of variance, p < . being regarded as significant.results: the figure shows the values for qs/qt expressed as means _+ sd. os/qt was significantly greater at rains after extubation (ii) in a group. the difl'ereuce between the two groups was no more significant from hrs after cpb (iii) to the end of the investigated period. ! i * p < a. s betw~n ~o~ conclusions: membrane ox 'genation during cpb is accomplished by reduction in blood cellular destruction and less alteration in blood. the results of our study show the influence of oxygenator type on value of qs/ot only after extubation ( to hrs after cpb). the difference in qs/qt disappeared his after cpb and since that time the oxygenator type had no influence on qs/qt. it may be of particular importance in patients with severe forms of cardiopulmonary disease who are at risk of higher postoperative morbidity and mortality. objectives: hypomagnesemia has been reported with a variable prevalence ( to % ) in icu patients. magnesium deficiency can induce a number of climcal symptoms (primarily cardiovascular and neuropsychiatric) but can also be clinically silent ( - % are asymptomadc), methods: we measured whole blood ionized magnesium (lmg++) in patients on admission to the icu, using a nova electrolyte analyzer (nova biomedical), containing an img++ electrode. blood was collected in syringes with dry heparin (radiometer qs ). normal range of img++ was found between . - . mmot/l (healthy volunteers). results: for the entire population, we found a % prevalence ( / ) of hypomagnesemia (figure ) . among the surgical patients, the prevalence was highest after cardiac surgery ( %) and after thoracic surgery ( %) and was lowest after neurosurgery ( %). hypomagnesemia was also common in patients after liver transplantation (lvtx) or with hepatic failure ( % for both groups). conclusion: our findings confirm that hypomagnesemia is common in acutely ill patients, especially in those after cardiothoracic surgery or those with liver disease. nevertheless. it is difficult to define the associated factors with sufficient specificity, so that measurements of img++ are warranted to diagnose hypomagnesemia. hepariu influences platelet function and may lead to thrombocytopenia called heparin-associated thrombocytopenia (hat) regardless of the dose and route of administration. additinnal venous and/or arterial thrombosis may lead to life-threatening complications. the incidence of so-calied heparin-associated thrombocytopenia and thrombosis (hatt) ranges between i- %. hatt is confirmed by a heparin induced platelet activation assay (hipa). results: from / to / consecutive patients of our icu were reviewed retrospectively. all patients were treated with heparim the incidence of hatt was % ( ). in all cases diagnosis was proven by a positive hipa. / patients died. in / hatt could be confirmed before severe thromboembolic complications occured. / patients developed a deep vein thrombosis (dvt), / dvt and pulmonary embolism (pe), / dvt, pe and arterial thrombosis (at) and / a dvt, pe~ at and a sinus thrombosis. conclusion: the incidence of hatt in a r series of pts. is %. presence of thrombocytopenia and thrombosis of the great 'vessels is associated with a significant mortality ( / ). computed tom graphy (ct) and transthoracic/transesophageal echocardiography (tte/tee) are important tools in diagnosing and monitoring the extent of cenlrai venous and arterial thrombosis. a. cabral md, m. shahla md c. meneses-oliveira md and jl vincenl md.phd. department of intensive care. erasme university hospital, brussels, belgium objective: to determine extreme hemodynanuc patterns in cardiogenic shock. although ~.~xdiogenic shock is characterized by a low cardiac index (ci), high systemic w~,scular resistance index (svri), and high cardiac filling pressures, some patients may develop art atypical pattern. we reviewed the hemodyuamic pattern of patients with cardiogenic shock, as defined by an initial ct below . l/rain/m: in the presence of myocardial dysfimction attributed to ischemic heart disease (n= ), heart failure (n= ), valvulopathy (n= ) or recent cardiac surgery (n= ). after exclusion of patients with concurrently suspected/documented infection, this study included patients, of whom ( . %) survived. treatment of shock included dopamine (n= ), dobutamine (n= ), norepinephrine (n= ) and epinephrine (n= ). patients with arterial hypertension (ah) and initially law plasnla renin activity (pra) had been studied. in all patient changes of arterial pressure (ap) after single administration of enap was studied. nypotensive reaction wiht deereasin e of average ap about - mm hg ayter single drug administration observed only in patients. ezap monotherapy accomplished during one week with mg daily dose. hypotensive effect observed in patients including ones which were susceptible to single enap administration. after that first stage of therapy all patints began to combinate enap with hypothyazid in dose of mg per day~ after week of treatment such drugs combination lead to veritable ap lowering in addition patients. in the remaining resistant to such drug combination patients was add corinfar in daily dose of mg. this new drug combination permits to lower ap in patients. subsequent discontinuation of enap administration to such patients aid not connected with increasing of again.therefore the most of the patients with ah and law pra( , %)did not susceptible to enap therapy and enap and hypothyazid combination. on the contrary-combination of corinfar with hipothyazid was effective in % patients with ah and low pra. methods: in patients with cardiogenic shock due to ischemic heart disease (n= ), heart failure (n= ) and valvulopathy (n= ), hemod aamic data including measures of intravascular pressures, cardiac output and mixed venous gases were collected at regular times intervals, at least times a da?. all measurements were obtamed in a relative steady state and in the absence of severe anemia or hypoxemia. treatment of shock included dobutamine (n= ), dopamine (n= ), norepinephrine (n=i ) and epinephrine (n= objective: based on our previous studies of the function of isolated liver grafts, this experimental protocol aims at developing a novel extracorporeal liver support circuit, with an incorporated pig liver. methods:the graft liver was obtained from pigs weighing - kg. under general anesthesia the aqimals underwent total hepatectomy,following cannulation of the portal vein, the infrarenal aorta and the infrahapatic vena cava and peffusion wit h it of heparinised r/l solution at ~ the circuit consisted of the graft liver connected to a fluid reservoir and a centrifuge pump. ten healthy pigs weighing - kgr were connected to the circuit as follows: the rt carotid artery was connected to the portal vein of the graft and the rt jugular vein was connected to the fluid reservoir, through the centrifuge pump. the fluid reservoir collected the outflow from the graft's suprahepatic inferior vena cava. the cystic duct of the graft was ligated and the bile.duct cannulated for bile collection and measurement. bridges were adapted to the circuit to bypass the graft liver when necessary, in cases of by pass blood perfusing the graft was oxygenated through a bubble oxygenator. mean total priming volume of the circuit was ml. temperature was maintained at ~ and portal vein pressure at ( - ) mmhg. the flow was . - . ml/gr of graft liver mass per minute. observation period was hours (t ). results: results of the hemadynamic and metabolic monitoring of the recipients [map (t = mmhg , t = mmhg), hr (t = , t = ), rap (t = mmhg , t = mmhg), pap (t = mmhg, t = mmhg), pcwp (t = mmhg, t = ~mhg), svr (t = dyn'sec/cm ' , t = dyn'seclcm~ pvr (t = dyn.sec/cm o, t = dyn.sec/cm ,'~), co (t = . t/min, t = . t/min), do (t = ml/min, t = . ml/min), vo (t = ml/min, t = ml/min), o er (t = . %, t = . % ), ph (to= . , t = . ), po (t = mmhg, t = mmhg), pco (t = mmhg, t = mmhg), pvo (t = mmhg, t = mmhg), svo (t = %, t = %), be, na, k, ca ++, lactate, osmolality, ast, alt, pt, aptt, revealed hemodynamic and metabolic stability of the animal. consumption, co production and tissue oxygenation of the graft were also studied. conclusion; the described circuit proved to be safe and well tolerated by healthy animals but its value for temporary liver support is currently being estimated, in a surgically induced experimental fulminant hepatic failure modal. introduction: prosthetic materials like silikone, dacron, teflon e.tc. produce auto immune responses and may even trigger clinical syndromes like scleroderma, sjogren, sle el.c. in our study we followed the evolution of humorial immunity parametrs for up to five years in a cohort of paced pts with implanted metallic and silicone materials. method: paced pts (mean age +- yrs) without clinical or laboratory findings of malignancy or immune disorders were included. we measured the immunoglobulins, the complement, the auto antibodies and the proteins involved in inflammatory reactions every months. the initial and final mean values are shown in the obiectives: hsp, a systemic leucocytoclastic vasculitis and anaphylactoid purpura can be accompanied by abdominal pain and life-threatening intestinal bleeding. recently we could disclose, that these patients develop severe fxiii-deficiency and immense haemorrhagic oedema of the intestinal wall. by the following case report we will demonstrate and discuss the importance of fxiiideficiency for pathogenesis, therapy and outcome in hsp. case report: a year old man developed typical skin manifestations of hsp following an episode of severe (biliary ?) pancreatitis and percutaneous draining of a pancreatic pseudocyst. two days later he had a paralytic "ileus with immense hemorrhagic wall-oedema and massive dilatation of the small bowel. he got fever up to . ~ and developed severe gastrointestinal haemorrhage (blood transfusions necessary). the coagulation data disclosed a severe fxhi-deficiency (activity %), whereas quickvalues, platelet count and atiii-level were found to be within the normal range. elastase was markedly elevated. substitution of fxiii to normal levels leeds to the cessation of bleeding symptoms and abdominal pain, later resulting in a restitutio ad integrum. conclusions: hsp with intestinal involvement is a life-threatening vasculitis, in which careful and frequent examinations of the coagulation system, especially of fxiii are necessary. detailed analysis of the coagulation data suggest, that the severe fxiiideficiency is due to a specific degradation by proteolytic enzymes (like elastase) as well as consumption within the immense haemorrhagic oedema of the intestinal wall. knowing these facts, even most severe cases of hsp with intestinal involvement can be successfully treated by substitution of fxih. a -year-old woman presented a year history of occasional self-limited episodes of weakness, generalized edema and o!!~aria. the immunologic testing showed no~nnai levels of complements, clq inhibitor, and serum chemistry values, between or during a attack, she was not treated. she was a~mitted to the hospital with symptoms including nausea, vomiting, weakness and ol!guria. on examination, the patient presented facial and g~neralized edema. the systolic blood pressure was mm hg, pulse beats/mir~ute, hematocrit . , seln~n protein /i, and se~um albumin q/l. an leg-kappa pa[apfotein was demostrated ( . g/l) and urine was neaative for puotein. c~'stalloid and colloid don't increased the blaod pressure but resulted in anasarca, with a total of ii lit[as of in~ravenous fluids. therapy wink flozen plasma, . units of clq inhibitor, cortlcosteroids, annihistwnines and antifibrinolytic agents was uns~iccessfull. the a~minist~ation of dopamine, norepineph~ne and epinephrine was inefective. the patient died at the bores, only a few cases have been reported, all had igg paraprotein, the pathophysio!o~] is urd~no~n% but is possible that the paraprotein may be zesponsib!e for the increased capillary pe~leabilityo despite efforts to res~scinate the patients during an acute attack, the syndrome is often fatal. the variable course of systemic uapiliary leak syndrome and the unpredictability and self-limited nature of attacks cloud assessment of therapeutic inte~-vention. the purpose of the present work is to provide some information about the nursing care and results from our experience in continous arteriovenus hemofiltration (cavh).cavh is an extracorporeal technique, especially applicable in the critically ill patients, for disturbances, and for the control of azotemia.we used this method in critically ill patients men and women ages from - who had sepsis -arf congestive heart failure postoperative multiple organ failure and polytrauma .this method was applied to these patients from to hours. % of the patients recovered completely their kidney function, % improved their kidney function and % died.we concluded therefore that this method was very effective for the critically ill patients to whom it was applied, but it requires excellent and continuous nursing care; under the above mentioned circumstances the method works effectivelly. an animal model with rats undergoing a dialysis procedure was designed to test the hypothesis that recovery from ischemic acute renal failure (airf) may be affected by the type of membrane used in hemodialysis. male sprague dawley rats were allocated to groups: in group i, (n= ) airf was inducted by bilateral renal artery clamping for rain. group h (n= ) rats underwent a sham procedure. in each group, rats were dialyzed twice ( th and th day) with either a cuprophan (cupro), a hemophan (hemo) or a pan (an ) minidialyscr or stayed nondialyzed (no hi)). renal function was monitored daily by measuring urea and creatinine values and by two single shot inulin clearances on the days following dialysis. additionally hemolytical activity of complement was determined. inulin clearance on day was reduced significantly but there was no difference in the degree of decrement in glomular filtration rate (gfr) between dialyzed and undialyzed rats, nor between the dialyzed animals with different membranes (gfr: no hi): . _+ . ; cupro: . _+ . ; hemo: . _+ . ; an : . _+ . ). the evaluation of renal function by day nine revealed significant recovery for all airf-groups compared to day (p< . ), irrespective of wether they underwent dialysis or not, or the type of dialysis membrane. complement activation could be detected in all dialyzed groups but no statistical differences between the animal groups dialyzed with different membranes were noticed. our findings refute the hypothesis that in airf exposure to complement-activating cellulosic membranes impairs the recovery of renal function in rats. changes patients: patients who underwent first cadaver kidney transplantation in our unit between january and december in were involved. the recipients were divided into groups: group i." non functioning graft (n= ); group ii: delayed graft function (n= ), group ili: good graft function (n= ). the grouping criteria were: a/haemodialysis in the fii~t postoperative days, b/diuresis in the i st postoperative day, c,' scram crcatininc difference between the st postoperative day and the preoperative level. all of the parameters were involved into the exarainatio, which we measllre in our every, day practice. results: the preoperative haematocrit level differed significantly between group i. ( . ) and croup ii. and iii. ( . and . , p< . ). intmo! emtive significant differences were found between the different groups in systolic blood pressure (group i. hgrmn, group ii. hgnnn, group iii. hgmm, p< . ), mean arterial pressure (group i. hgmm, vs. group ii. hgnun p< . , vs. group iii. hgmm p< . ), and pulse-amplitude and rate-pressure product too. the second warm ishaemic time in group iii. was significantly shorter than in the other two groups (group iii. inin. vs. group ii. rain. p< . , vs. group i. rain. p< . !). the rejection rate was higher in the first days in the patients with non-functioning grafts (group i. % and group ii. % vs. group iii. %) . the other examined parameters have not differed significantly. conclusion: according to our results the success of the kidney transplantation is mnitifactorial. the most important factors of this relationship are: the perioperative fluid-balance, the maintenance of adequate perfusion blood pressure during the operation, good surgical technique and immunological problems.